Cereals constitute between 60 and 70% of worldwide crop production, and rice, wheat, and maize are the three most important cereals. More than 500 million tons of each is produced annually worldwide. Approximately one third of the population depends on rice for over 50% of their caloric intake, and half of the population derive a significant proportion of their total calories from rice. The cereals have been evolving independently from a common ancestral species for 50-70 million years, but despite this long period of independent evolution, cereal genes and genomes display relatively high conservation. Comparisons of the physical as well as the genetic maps of the grass genomes have led to numerous reports describing conservation of gene order and orientation, or synteny, among the cereals. Due to the high degree of synteny, the grasses, including cereals, are often considered as a single genetic system. Despite the gene similarity and genome synteny, cereal genome sizes vary considerably. Rice has a much smaller genome size than the other major cereals, estimated at 420 Mbp, while sorghum, maize, barley, and wheat have genomes of 1,000, 3,000, 5,000, and 16,000 Mbp respectively. The genome size and predicted higher gene density of rice make it an attractive target for cereal gene discovery by genome sequencing.

Over the past several years, selected regions of the rice genome have been sequenced, and an international project (IRGSP) to generate a highly accurate completed sequence was established. In this report we describe the use of random fragment shotgun sequencing to discover rice genes, molecular markers, and mapped sequences for the association of candidate genes and the traits they control. The genome of rice was sequenced to over 6-fold coverage by a draft sequencing approach and assembled. The accumulated sequence represents more than 99% of the rice genome, and contains over 99% of the publicly available full-length rice protein sequences. Over 42,000 genes or gene fragments longer than 500 base pairs and 63,000 genes or gene fragments longer than 300 base pairs were predicted using an integrated gene prediction, sequence homology, and protein domain/motif identification strategy. More than 85% of the predicted Arabidopsis genes display significant homology to genes predicted in rice, and approximately one-third appear to be plant-specific. Greater than 98% of the publicly available proteins of maize, wheat, and barley were found with significant homology in the draft sequence assembly and more than 95% of translated cDNAs were found in the rice draft gene predictions. Approximately 40,000 simple-sequence repeats (microsatellites) were identified in the draft genome sequence. Synteny between the rice genome and other cereal genomes was found to be significant, whereas synteny between rice and Arabidopsis is restricted to short regions of the genome carrying 5 to 15% of the genes. No evidence for lateral DNA transfer was found in a comparative analysis of the rice and human genomes. The Syngenta draft sequence of rice provides a solid foundation for completing a high-accuracy sequence, enabling gene identification, facilitating physical and genetic mapping, and serving as a syntenic model for the other major cereal crops: maize, wheat, and barley. Details of the sequencing and analysis will be presented.

Huada

Bin Han

Rod Wing

Plants undergo developmental changes in response to light, which, referred to as photomorphogenesis, is largely mediated by the two classes of photosensory receptors, red/far red light receptors phytochromes and blue/UV-A light receptors cryptochromes. Arabidopsis has five phytochromes, phyA-phyE, and two cryptochromes, cry1 and cry2. These photoreceptors act concurrently in regulating stem elongation, leaf expansion, photoperiodic flowering,, and changes in gene expression. To understand how photoreceptors regulate these light responses and the molecular mechanism underlying photoreceptor signal transduction, we have taken both conventional molecular genetics approach and bioinformatics approach. We analyzed how different photoreceptors work together to regulate photoperiodic flowering. Our results demonstrated that both phytochromes and cryptochromes act as photoperiod sensors by alternating cellular levels of the photoreceptors. We have also investigated the initial photochemistry of cryptochromes and found that cryptochromes undergo blue light dependent phosphorylation. We demonstrated that the blue light-induced cryptochrome phosphorylation is associated with the function and regulation of the photoreceptors. Finally, we will discuss new light signaling genes identified using the bioinformatics approach.

The genetic relationships of 43 wheat varieties were analyzed with SSR markers. The materials used included 14 cornerstone breeding parents used before 1980 and another 29 other large-scale planted varieties currently in use in China. A total of 501 different alleles were amplified, including 166 alleles of the A genome, 174 of the B genome and 161 of the D genome. Data obtained were used to estimate genetic similarity using the DICE coefficient, and dendrograms were constructed using the UPGMA method. The dendrogram with 501 alleles successfully differentiated all the cornerstone breeding parents and the large-scale planted varieties, and the dendogram tree was basically consistent with the pedigrees of these varieties. The correlation coefficient between the genetic distance matrix of 501 alleles and that of 450 was 0.99. Correlation coefficients among random samples of alleles suggested that 350 to 400 alleles were needed to detect genetic relationships among common wheat varieties. Correlation coefficients of a genetic similarity matrix based on 580 and those of 501 and 400 random alleles were 0.96 and 0.94, respectively. However, there were marked differences between the matrix based on the 501 alleles and those based on markers located on A-, B- or D-genome independently. The correlation coefficients between the genetic distance matrix of 501 alleles and alleles within A, B or D genomes were 0.77, 0.76, and 0.67. The estimation of genetic similarity should be based on data from all genomes rather than any one genome.

Key words: Wheat; SSR; Genetic relationships; Genetic diversity

Almond comprises at least 26 species that form a distinct and easily recognized natural group in the world. It originated in Central and Western Asia and then extended to China, India, and Mediterranean region. Sweet almond (A. communis L. var. dulcis syn. Prunus dulcis Mill.) is one of the most important fruit nuts in the world.

EST-SSR is the simple sequence repeat derived from EST sequence. It occurs in transcribed region and stands for the functional gene, so they are valuable in comparative genetic analysis, mark—assistant breeding, and germplasm assessment.

From more than one thousand ESTs (857 from private database and 200 from public database) of almond and peach, 58 SSRs were screened and 26 SSR primer sets designed. Fifteen of the SSR primer sets produced easily identified products in 43 accessions of five almond species, Amygdalus communis L., A. mongolica (Maxim) Yü, A. ledebourianan Schleche, A. tangutica (Batal.) Korsh., and A. triloba (Lindl) Ricker, and 2 accessions of peach (A. persica L.) which were used as an outgroup. This cross-species transferability allowed us to construct the phylogenetic tree and the result was consistent with traditional classification. Some species-specific alleles were distinguished and they were successfully used to identify two hybrids between species in our studies.

These SSRs were also used to investigate the genetic diversity of common almond (Amygdalus communis L. var. dulcis syn. Prunus dulcis Mill.) germplasm in the world, including 20 native cultivars of China and 17 currently cultivated in Australia, USA and European countries. Number of alleles detected ranged from 1 to 11 in the 37 accessions (mean 3.0 alleles) and heterozygosity (He) ranged from 0 to 0.809 (mean 0.405). The cluster analysis showed that Chinese and other almond were classified into two independent groups, which is consistent with their geographic origin. These two groups were differentiated by some specific alleles, strongly suggesting that geographic isolation or founder effect contribute to the evolution of these specific alleles.

Genomic SSRs were further compared with the EST-SSRs in the present study. Using the 7 genomic SSR primer sets, thirty-seven alleles were detected with an average 5.4 alleles per locus in Chinese accessions and 39 alleles with an average 5.6 alleles per locus in others. The number of alleles detected per locus was significantly (P<0.01) different from that of EST-SSRs.

Up to our knowledge, this is the first report on the EST-derived SSR analysis of almond. The significance of this work in almond breeding and the conservation and evaluation of almond genetic resources was discussed.

Key words: almond; Amygdalus communis L.; expressed sequence tags (ESTs); SSR

Acknowledgement: This work was financially supported by the National Natural Science Foundation of China (grant No. 30170649) and the National Tenth Five-Year Program of Science and Techonology (grant No. 2001BA707B02) of China.

More than 1300 soybean cultivars (lines) were obtained from 1923 to 2001 in China, it would valuable to evaluate the genetic diversity and relationship of excellent cultivars and their ancestors. The objective of this study was to analyze the genetic diversity of excellent cultivars and provide instructions for parents selection, genetic differentiation and heterosis forecast. A set of 31 SSR core primers were used to analyze 12 excellent cultivars and their ancestors, and detected 212 alleles with average 6.84 alleles per locus. Average alleles (4.97) and genetic diversity index (PIC and Shannon, 0.73 and 1.29) of cultivars were less than those of ancestors (5.61, 0.74, 1.53). T test indicated that genetic diversity on 31 SSR loci in them reached un significant level. Therefore, genetic base for cultivars were narrower than that for ancestors. The main characteristics of cluster analysis indicated cultivars and ancestors distributed evenly in two groups and those with geographic origin and pedigree were clustered together. This result was as same as reported in the literatures. So less alleles could be used to illustrate genetic relationship of cross related varieties. Correlation analysis of contribution ratio by pedigree and genetic similarity coefficient reached significant level, it is suggested that for parents selection that genetic contribution ratio was needed to be calculated firstly and then genetic similarity coefficient was taken account of between parents in order to reduce task.

Key words: soybean; cultivar; ancestor; SSR; genetic diversity

Shanyou 63, a cross between Zhenshan 97 and Minghui 63, is an elite hybrid widely used in rice production in China. The general characteristics of this hybrid include high yielding and wide adaptability. In recent years, however, this hybrid has shown several major problems in rice production including: the loss of resistance to diseases such as bacterial blight (BB) and fungi blast, in addition to its relatively poor cooking and eating quality. There is also urgent need for improving its resistance to insects such as stem borers and planthopper, as they frequently cause damage to the crop. We have conducted a series of work in order to solve the above problems: (1) Xa21, a wide spectrum BB resistance gene, was introgressed to the restorer Minghui 63 by molecular marker aided selection to improve its resistance to bacterial blight. (2) A Bt d -endotoxin gene, that is toxic to stem borer, was transformed to Minghui 63 to improve the stem borer resistance of this hybrid. (3) Xa21 and the Bt gene were combined into a line under the background of Minghui 63. (4) The allele at the Wx locus from Minghui 63 was transferred to Zhenshan 97 to improve the cooking and eating quality of this hybrid, resulting in a new version Zhenshan 97 with medium amylose content (AC), soft gel consistency (GC) and high gelatinization temperature (GT), indicating significant improvement of the cooking and eating quality. (5) Pi1 and Pi2 were both broad spectrum and highly resistance genes to fungi blast races. These two genes were intergressed to Zhenshan 97 to improve the blast resistance by molecular marker-aided selection. (6) The Bt, Xa21 and wx genes were pyramided in one new hybrid by MAS, and the agronomy traits and the performance of the hybrid are the same as the control Shanyou 63, and the blast resistance genes are further pyramided in this hybrid rice. These results are showing great promise as improved hybrid in rice production.

Key words: Inbred line; Qi319; Southern corn rust; Resistance gene

An EST based microarray was used to profile genome expression underlining light control of Arabidopsis development. Qualitatively similar gene expression profiles were observed among seedlings grown in different light qualities, including far-red, red, and blue light, which are primarily mediated by phytochrome A, phytochrome B, and the cryptochromes. Further, light-dark transitions also triggered similar differential genome expression profiles. Most light treatments also result in distinct expression profiles in small fractions of genes examined. The similarly regulated genes in all light conditions were estimated to account about one third of the genome, with 3/5 and 2/5 up and down regulated by light respectively. Analysis of those light-regulated genes revealed over 28 cellular pathways that are coordinately regulated by light.Similar gene expression profiles were also observed among wild type seedlings grown in white light and multiple cop1 mutant alleles grown in the dark. Overexpression of a dominant negative acting N-terminus of COP1 protein (N282) in darkness triggered a genome expression profile similar to those by white light and the cop1 mutations. Further, similar but weaker gene expression regulation attributable to HY5 in the light is largely included within those regulated by COP1 in the dark. This genomic study supports the conclusion that COP1 acts as a repressor of photomorphogenesis by controlling the degradation of transcription factors and thus regulating their target gene expression. Thus light controls Arabidopsis development through coordinately regulating metabolic and regulatory pathways, and largely achieves its role by negatively regulate COP1 activity.

Qifa Zhang

Antisense gene silencing has been widely used to study functions of interesting genes. The technology is also useful for generation of mutant libraries by introducing antisense cDNA libraries into cells. We constructed a rice antisense cDNA library using a binary vector modified from pCAMBIA1302. About 1000 random clones from the library were used as a pool to transform a rice variety Zhonghua No.11 by Agrobacterium mediated method.

Totally, 104 hygromycin (Hm) resistant calli were obtained and more than 2000 transformed plantlets were regenerated from these calli. Preliminary screening of transgenic plants was carried out by soaking leaf segments in hygromycin B solution for 7d. Of 558 T₀ plants tested, 544 showed Hm resistant and 14 partial resistant to hygromycin B. PCR analysis of 148 Hm resistant plants with primers specific to HPT gene showed that all tested plants were positive. Fragments of antisense cDNA could be amplified from 103 of 108 Hm resistant plants.

Various types of phynotype variations were observed in T₀ plants such as: height (10-100 cm), fertility (0-90%), tillering ability (no tiller to more than 40 tillers), shape of grain, heading time, etc. Phynotype variations were also observed among plants regenerated from the same callus. Co-segregations of trait variations with Hm resistance for some mutant lines were observed. For example, a T1 population showed that the pollen fertility was co-segregated with Hm resistance. So we PCR-amplified the antisense cDNA fragment from the transgenic plant using specific primers flanking the vector cloning site, and sequenced the fragment. The sequence BLAST result showed that the amino acid sequence exists a conserved F-box motif. It is reported that F-box proteins are related to ubiquitin-dependent protein degradation, while many crucial biological processes in plants, such as hormone response, circadian rhythm , photomorphogenesis, stress response, flower development and senescence are governed by proteolysis. Further analysis of the mutant library is underway.

Expressed sequence tags (ESTs) were obtained from cDNA libraries that were made from soybean seedlings (Glycine max L., SMV resistant variety Kefeng 1) treated with salicylic acid (2.0 mmol/L). After removal of low-quality and shot sequences (less than 80 bp), 26,085 ESTs were assembled into 2,937 contigs and the rest, 7,536 ESTs, remained as singletons. Included also in our analysis are two other public data collections: (1) the NCBI collection, 527 contigs and 964 singletons assembled from 2,289 soybean ESTs from Gm-c1014 seedling cDNA library and (2) the Washington University collection, 11,969 contigs and 27,146 singletons from an assembly of 120,077 soybean ESTs. In comparison to these public data, 18.91 % of EST collection was novel and 24.79% had homologous sequences (E value 10^–10) in the GenBank non-redundant nucleotide and the Swiss-Prot protein databases. After classified into 14 functional categories, comparison of ESTs from our collection and from the NCBI collection showed the most significant difference in the proportion of ESTs involved in protein synthesis/processing and primary metabolism. Since salicylic acid plays key roles in signal transduction, disease-resistance, defense response and stress tolerance, we identified sixty candidate genes related to these pathways from our data set. We chose 103 cDNA clones for experimental verification using hybridization-based method. 8 and 4 genes were clearly un-regulated and strongly down-regulated by SA, respectively. We also identified 680 SSR and 823 SNP candidates. An average of GC content in ESTs was analyzed in soybean and Medicago truncatula.

Based on the expression statistics, we estimated that gene count of the soybean genome was about 45,000-50,000 or twice as much as that of Arabidopsis.

Richard D. Vierstra, Brian Downes, Jed Doelling, Adam Durski, Jennifer Gagne, Derek Gingerich Mina Kurepa, Jan Smalle, Allison Thompson, Joseph Walker and Peizhen Yan

Selective removal of intracellular proteins plays an integral role in the growth, development, and environmental adaptation of plants. One essential proteolytic pathway targets proteins for degradation by covalently tagging the proteins with one or more ubiquitins, a small highly conserved proteins. These ubiquitinated proteins are then selectively broken down by the 26S proteasome with the concomitant release of free ubiquitins. The complexity of the ubiquitin/26S proteasome pathway is best illustrated by estimates that >10% of plant proteins are targets and by our discovery of over 1300 genes in Arabidopsis thaliana that encode pathway components. Correct substrate recognition is accomplished by: (1) the coordinated action of ubiquitin-protein ligases (or E3s) that ligate ubiquitin to specific proteins; (2) enzymes that remove ubiquitins from various targets (UBPs); and by (3) factors within the 26S proteasome that detect ubiquitinated proteins before degradation. Structural diversity within the E3 families (~700 members in one family alone) is apparent that likely imparts much of this target specificity. To understand this diversity, we are analyzing the functions of various pathway components by reverse genetics, using Arabidopsis as model systems. Knockouts in E3s, UBPs and various 26S proteasome subunits have been generated. Phenotypic analyses show that the ubiquitin/26S proteasome pathway is essential to plants and has important roles in embryogenesis, meristem development, plant growth, hormone signaling, and various stress responses. Recently, six other polypeptides have been discovered that also serve as covalently modifiers of other plant proteins. One appears to have an essential role in nitrogen recycling and another involved in the heat stress response. The complexities of these pathways suggest that protein modification by other polypeptides is a common method of post-translational regulation in plants.

To discover genes essential for agronomic performances of crops the China Rice Functional Genomics Program (CRFGP) was initiated in 1999. The CRFGP was funded by the Ministry of Science and Technology of China through the National Basic Research Initiative and was expected to last for an initial period of five years. The mission of the CRFGP is to generate intellectual property related to functional genes and resources for improved crops as well as to train scientists working in the fields of functional genomics. Twenty research groups from the Chinese Academy of Sciences and several universities were organized to participate in the program, which focuses on the identification of genes related to flowering, plant architecture, fertility, reproduction, metabolic controls and stress responses in rice through a combinatorial approach based on genetics, molecular biology and functional genomics. The CRFGP is divided into a total of six projects: 1, Creation of a rice mutant library; 2, Gene profiling and identification by cDNA-microarray; 2, Development of a highly efficient gene cloning system based TAC (Transformation-competent Artificial Chromosome) vectors; 4, Functional genomics of rice transcriptional factors; 5, Development of a rice functional gene database; and 6, Research and development of transgenic rice lines. Some of the recent progress made by the CRFGP will be presented.

Jen Sheen

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling modules with essential regulatory functions in eukaryotes, including yeasts, worms, flies, frogs, mammals, and plants. Numerous studies have shown that plant MAPKs are activated by abiotic stresses, pathogens and pathogen-derived elicitors, and plant hormones. The Arabidopsis genome and EST sequencing projects have revealed large gene families encoding MAPKs and their immediate upstream regulators, MAPKKs and MAPKKKs. However, little is known about the constitution of plant MAPK cascades and the specific roles that particular MAPK cascade genes play in particular plant signal transduction pathways. We have developed a comprehensive approach based on genomic information, transient expression assays, and transgenic and genetic analyses to determine the function of all Arabidopsis MAPK cascade genes involved in essential plant signaling pathways. The transient nature of the protoplast systems allows direct functional analysis of plant genes at an unprecedented high throughput rate. The experimental approaches are especially powerful in unraveling the functions of genes that are difficult to tackle by traditional genetic and biochemical approaches due to redundancy, lethality or low levels of expression. The involvement of MAPK cascades in hormonal, stress and innate immune responses will be presented. Since the functions of MAPK cascades in plant signal transduction pathways are likely conserved, our studies using the Arabidopsis genome resources will have broad implications and applications in other plant species. The elucidation and manipulation of MAPK cascades in plants has revealed fundamentally important intracellular signaling processes and provided new tools for crop improvement in stress tolerance, disease resistance, and yield enhancement.

Jiayang Li

Plant shoot architecture is determined mainly by the formation pattern and growth habits of ateral shoots. Rice later shoots (tillers) are formed from axillary meristems that locate between a leaf and the shoot axis. Axillary meristems are developed from cells at the base of the subtending leaf, or from cells in the shoot axis just above the subtending leaf. To understand the rice tillering, we isolated and characterized a naturally occurred rice mutant, mon culm 1 (moc1). Compared to wild-type rice plants that normally have more than ten tillers, the moc1 mutant show no tillers due o lacking the axillary buds. MOC1 gene was cloned by a map-based approach. Sequence homology analysis indicates that it encodes a putative transcription factor, which is supported by the finding that MOC1-GFP fusion protein is a nuclear-localized protein. RNA in situ hybridization demonstrates that MOC1 is mainly expressed in the cells at the leaf axil where axillary buds will initiate. Transgenic rice plants that overexpress MOC1 are able to develop more than one tiller at a single axil, in contrast to a single tiller in the wild-type plants. We also found that the expression of OSH1, a rice homolog of maize Knotted1, requires the normal function of MOC1. Therefore, MOC1 may play a critical role in the formation of tiller buds by promoting the initiation of axillary meristems.

The semidwarfing gene in rice (sd-1) is one of the most important genes deployed in modern rice breeding, since it led to record yield increases in the 1960s and remains the predominant semidwarfing gene present in current rice cultivars. The phenotype of sd-1 is consistent with dwarfism resulting from a deficiency in gibberellin (GA) plant growth hormones and a partial block in GA biosynthesis. Recently, a GA 20-oxidase gene (Os20ox2) was isolated from the Sd-1 locus in three separate studies by using 1. map-based cloning, 2. degenerate PCR and 3. public rice genome sequence. Altered Os20ox2 gene sequences isolated from several independent mutants were shown to encode the sd-1 semidwarfing locus. The quantification of GAs in elongating stems by GC-MS showed that the initial substrate of GA 20-oxidase activity (GA₅₃) accumulated, while the content of the major product (GA₂₀), and of bioactive GA₁, was lower in semidwarf compared to tall lines. Different gene isolation approaches are discussed in the context of rapid progress made in rice genome sequencing. Using the Os20ox2 sequence we also describe the development of ‘perfect markers’ for two different sd-1 alleles.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae is a devastating disease in rice worldwide. The resistance gene Xa4 has been widely used in breeding programs and played an important role in protecting rice from this disease. Using 642 highly susceptible individuals and a random sample of 255 individuals from an F₂ population developed from a cross between IRBB4 and IR24, the Xa4 gene was genetically mapped to a region less than 1 cM. A contig map was constructed for the Xa4 region consisting of six non-redundant bacterial artificial chromosome (BAC) clones and spanning approximately 500 kb in length. Analysis of recombination events in the Xa4 region located the gene locus to one BAC, 3H8. Assay of the recombinants using the subclones of 3H8 in combination with sequence analysis further narrowed the Xa4 locus down to a 47 kb fragment. Candidate genes have been identified from this fragment which have been transformed into the rice cultivar Mudanjiang No. 8, resulting in a more than 100 transgenic plants. Preliminary tests by inoculation with the Xoo strain showed that several of the transgenic plants may be resistant to the strains tested. Further analysis is still in progress.

The Oryza sativa Root Architecture Associated 1 (OsRAA1) gene, has been characterized molecularly. OsRAA1 shows tissue specific expression in the shoot apical meristem, the elongation zone of root tip, steles of the branch zone and the young lateral root. OsRAA1 - encodes a 12.0 kDa protein that has 58% homology to the AtFPF1 in Arabidopsis. Constitutive expression of OsRAA1 under the control of maize ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root in rice, which are similar to the phenotypes of the plant treated with auxin. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. The transgenic plants have longer flag leaf cells than the wild type control. Northen blot analyses demonstrated that the OsRAA1 expression was induced by auxin. These data suggested that OsRAA1 might be involved in the pathway of Auxin/IAA signal transduction in establishing the root architecture in rice.

Key words: OsRAA1; auxin; Oryza sativa; root development

Iron is an essential mineral element of all organisms. In plants, iron deficiency is the most common nutritional limitation for growth because iron is almost exclusively present in its oxidized, low-soluble form Fe (III) which is not readily available to plants in soil. There are two efficient mechanisms to acquire iron from soil in higher plants (strategy I and strategy II). Characteristics of strategy I (all higher plants except the Gramineae) include increased solubilization of Fe³⁺ by extrusion of protons and organic acids from plant roots into the rhizosphere, chelation of Fe³⁺ with organic acids, reduction of the chelated Fe³⁺ to soluble Fe²⁺ at the root surface and subsequent transport of Fe²⁺ in the plant as well as morphological changes of roots, such as thickening of the subapical root zone, formation of root hairs and so on. The efficient iron uptake processes of strategy I are induced upon iron starvation.

In tomato, the iron-inefficient mutant T3238fer, which was originally identified from the boron-inefficient T3238 tomato, is a very informative mutant for study of molecular mechanism of strategy I. The mutant plants are not able to switch on the efficient iron uptake process of the strategy I upon iron-deficiency. Seedlings of the mutant exhibit severe chlorosis and die at an early stage unless they are supplied with Fe or grafted onto a wild-type rootstock (Brown et al., 1971). It was supposed that the FER gene might be a central regulatory gene controlling the whole iron deficiency responses and iron uptake in roots of the strategy I plants. Genetic analysis showed that the T3238fer is a monogenic, recessive trait located on chromosome 6 of tomato (Ling et al., 1996). To study the molecular regulation mechanism of iron uptake of strategy I plants, we have recently isolated the FER gene from tomato genome using map-based gene cloning approaches. The FER gene encodes a transcriptional regulator controlling iron uptake in roots of tomato. The further studying of this gene will give new insights into the understanding of the molecular regulation of the iron uptake mechanism of strategy I plants and form the basis for the manipulation of plants to generate high-yielding, iron-efficient or iron-rich crops to contribute towards the improvement of iron deficiency in humans which afflicts more than two thirds of the world population. Here, I will present the isolation and characterization of the FER gene.

In generation of M₁, there are 38% and 13% of seedling rate under 300 and 350 Gy while the origin with normally 75%, as well as 7510 and 2490 families to have been harvested respectively. Main visible injury on M₁ was physiologically semi-sterility or sterility. The variation of seed set was continuous seed set from zero to normal.

All 10000 families of M₂ were sowed. Some of them were no germination or germination but no seedlings for low seed set of 5%, or died at about 8% of no chlorophyll formation for albino or chlorina. Only 8940 families survived, and 24 single plants per family were transplanted.

After transplantation, the most mutation, occupied to the total amount of 5.95% and 6.65% in sowing and transplanting families, was the fertility in seed set lower than 50%. Dwarf mutants including tillering, small or big grain, malformed, semi-dwarf and other type were separated and occupied to the second at amount of 1.78% among 8940 families.

Different mutants by M₃ separation and selection were listed as following: bright green leaf, chlorosis caused by low temperature, faded green leaf, yellow green leaf, awn, gold hull, white hull, clustered spikelets, extra glume, long grain, minute grain, round kernel, triangular hull, shrunken endosperm, neck leaf, leafy head, long sterile lemmas, depressed palea, lax panicle, pendant panicle, sheathed panicle, short panicle, white panicle, accelerated overgrowth of internode, find culm, lazy growth habit, reduced culm number, drooping leaf, glabrous leaf and hull, narrow leaf, rolled leaf, brown leaf spot, spotted leaf, heading date, late heading, germinating in the panicle, male sterile, open hull male sterile, malformed semisterile, etc.

Some mutants unfound or report before were: brown hull spot, spotted hull, brown sheath spot, spotted sheath, increased culm number, brown pericarp, erect panicle, easier germination, leaf premature senility, necrotic hull, tipsy growth habit, etc.

Two traits to be induced in one family was only faded green leaf with dwarf. Majority was recessive mutation. The mutation frequency took place roughly for 5% for 8940 families.

* This research was supported by National 973 Project (authorized No: G19990116).

Two major genes for soybean cyst nematode resistance have been cloned and their function has been predicted. The soybean genome analysis is based on genome-wide genetic maps and studies focusing on individual genes. To gain better understanding of the structure of the soybean genome we have investigated several large contiguous regions of genomic DNA using a combination of genetic mapping, physical mapping of BAC clones, DNA sequence analysis and fluorescent in situ hybridization (FISH). For several of the markers employed in this study, homeologous BAC clones were identified that map to different chromosomes based on both FISH and genetic linkage analysis. At the DNA sequencing level, the BAC clones and their homeologous counterparts are more than 80% homologous. These data support published results suggesting that modern cultivated soybean is derived from an ancient tetraploid. In addition, we also found microsyntenies on the deduced protein level between soybean and Arabidopsis genomes.

Yaoguang Liu

Jiming Jiang

University of Wisconsin, Madison

In recent years, since founding the systemin, several polypeptide signal molecules (like CLV3 and SCR) were found to be present in the extracellular area. More important, it is also proved that they play a very important developmental role. So, extracellular area of plant cell (apoplast) had been revealed as a powerful signal source that could determine the fate of cell development. The further studies about ECBP21 may be benefited for elucidating the mechanism of extracellular CaM or enriching the studies of polypeptide signals in plant kingdom. This work laid a foundation for elucidating the functions of ECBP21 by means of molecular biology. Further studies are being under taken.

Acknowledgements: This work was supported by the National Key Basic Research Special Funds of China (Grant No.G19990117), the National Natural Science Foundation of China (Grant No. 30170476).

Qun Li, Yonghan Xu, Yongyou Zhu, Haichen Zhou, Zuhua He

*This work is partially supported by ‘863’ project: 2001AA222321.

To construct a plant artificial chromosomes and introduce it into plant cells would be an effective way to understand the essential elements necessary for maintaining chromosome function and structure in plants. The plant artificial chromosome may also be used as a new vector to introduce hundreds or thousands of Kb DNA segments into target plants. An artificial chromosome should consist of three elements at least, centromere, replication origin and telomere, which are available in Arabidopsis research society. Two YAC clones containing 180 bp repeat sequences in the centromere domain of Arabidopsis and BAC clones containing telomere sequence and ARS (automatic replication sequence) have been collected for constructing artificial chromosome. The telomere sequence in the YAC was confirmed by PCR amplified using telomere repeat primers and by sequencing for the PCR products. A 2000bp fragment of ARS was isolated from the BAC clone by ClaI digestion and subcloned into ClaI digested pBluescript.

The stratagem of constructing plant artificial chromosome is to integrate two plasmid vectors carrying necessary elements into the centromere contained YAC from right and left arms respectively. The left arm vector contained the telomere sequence, the ARS sequence, a kanMX4 gene of G418 selection marker and a truncated TRP1 gene. The right arm vector carries a telomere sequence, the ADE2 gene of selection marker, GFP and a truncated URA3 gene. Retrofitting YAC clones carrying the centromere segment with the constructed right and left arm vectors is being performed by homologous recombination in yeast. The artificial chromosome is going to be transferred into Arabidopsis or Brassica protoplast by liposome-mediated method later this year.

Allopolyploidy is a major force in the evolution of angiosperms. Nevertheless, the initial events that accompany allopolyploidization are not understood. Recent studies in several model plant systems, including Brassica, Arabidopsis and wheat, indicate that interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to rapid genomic changes such as sequences elimination, novel fragments, gene loss, silencing and activation. In order to further study the genetic and epigenetic changes during allopolyploid formation, we choose several sets of newly synthesized amphiploids with various Triticum and Aegilops species as parents, to study possible changes on representative EST sequences. In this report, we show that following allopolyploidization, rapid changes occurred in EST (Expressed Sequence Tag) sequences.

In the amphiploids between Triticum timophevii and Triticum monococcum, 41.6% EST sequences exhibited changed patterns. EST sequence changes were initiated and completed in different generations. Novel fragments and simultaneous disappearance of two parental fragments were not observed in this cross combination. In contrast, we found a lot of new fragments in the amphiploids between Triticum monococcum and Triticum tauschii. Novel fragments were also found in the F1 hybrids between barley and Elymus humidus. About 33.3% EST sequences detected changes in the F1 hybrids and amphiploids between Triticum asetivum cv. Chinese Spring and Secale cereal. We revealed three types of changes in this cross combination: disappearance of one parental hybridization fragment(s) in the amphiploids, disappearance of one parental hybridization fragment(s) in the amphiploids and some F1 plants, simultaneous disappearance and appearance of rye hybridization fragment(s) in the amphiploids. We also found disappearance of one parental hybridization fragment(s) in the F1 hybrid plant, but re-appearance in the amphiploids. The results indicate that most of the genomic changes occurred are at random. We compared the RFLP patterns between the newly amphiploids and Triticum zhukovskyi, T. urartu and T. monococcum using EST sequences as probes. It is concluded that T. zhukovskyi originated from hybridization of T. timopheevii with T. monococcum, but little divergence occurred during the evolution. On the other hand, strong divergence in the EST sequences was detected between T. urartu and T. monococcum.

1.It is possible to eastimate genetic relationships of Chinese soybean land races using SSR marker. The core SSR primers could be selected by distribution of the loci in soybean genome and gene diversity scores. The truely genetic relationships of soybean land races could be reflected using over 570 alelles.

2.Genetic relationship based on SSR data was not consistent with that determined by agronomic traits, more genetic information would be obtained from SSR data associated with agronomic traits.

3.Chinese soybean land races had three high genetic diversity of population, the first was consisted of North Spring planting soybean, Northeast Spring planting soybean and Huanghuai summer planting soybean. The second was the population of South Spring planting soybean and South Summer planting soybean, and South autumn planting soybean was another high genetic diversity population. Soybean land races might be several original places.

Key words: Soybean land races; genetic diversity; the level of gene diversity; Simple Sequence Repeats

Maximum lifespan of a species is relatively stable and is mainly determined genetically. It has been found frequently that a single gene mutation may extend or shorten the lifespan significantly in model species. A number of genes regulating lifespans have been cloned recently in model organisms (SCH1, SCH9, CPR1 and SIR2 in Yeast; AGE-1, DAF16, SIR2, CLK1, CLK2 and CLK3 in C.　C.　elegan INR and CHICO in Drosophila; SHC in mouse). Extensive studies have revealed that some common molecular pathways are involved in regulating lifespans in different species, particularly insulin/IGF-1 signaling pathway and generation of ROS and the corresponding response to oxidative stress. In model plant Arabidopsis, however, although mechanisms underlying regulation of flowering time and control of leaf senescence have been studied extensively and three pathways regulating flowering time have primarily been elucidated, little is known about genetic mechanisms involved in the extension of its lifespan. In this study, we have isolated and characterized three Arabidopsis mutants with extended lifespans (designated els1, els2, els3, respectively). Compared with wild-type, the els1 mutant was shorter in stature, with rotund and increased rosette leaves and sunken petioles, as well as reduced male fertility. Although its flowering time was almost identical to that of wild-type plants, the period from flowering to death was significantly prolonged. Its lifespan was consequently extended by 200%. The els2 mutant was significantly larger than wild-type plants, with increased size and number of rosette leaves. The mutant not only flowered later than normal, but also the duration of flowering time was significantly prolonged, which resulted in the extension of lifespan by 150%. The els3 mutant exhibited a similar morphology to wild-type, but its rosette leaves were larger and its plant height was higher. Its lifespan was significantly prolonged by 80%. Genetic analysis revealed that phenotypes of these els mutants were caused by a single recessive mutation. Chromosome mapping of these mutants with simple sequence length polymorphism (SSLP) markers indicated that the els1, els2 and els3 mutations were associated to three loci on two chromosomes: two loci near the markers AthBI2ob (els1) and ciw2 (els3) on chromosome 2, and one locus near the marker nga106 on chromosome 5　us near the marker nga106 on chromosome 5els2). No other loci related to late-flowering have been found in this area. These results suggest that the three loci may be novel and involved in the extension of lifespan in Arabidopsis.

Key words: Extended lifespan; els mutation; Mapping; Arabidopsis

Songjie Tian, Tianyu Wang, Yunsu Shi and Yu Li

The understanding of genetic relationships among crops and their wild relatives will facilitate the use of wild germplasm in breeding of cultivated crops. For maize, a wide range of methods, including morphological, cytological and molecular techniques, have been used to clarify the genetic relationships between maize and the putative closer relatives, i.e. teosinte and Tripsacum. However, many basic questions have not been answered yet.

AFLP markers were used to investigate the relationships among cultivated maize, teosinte, Tripsacum and Coix in the present study. Two Tripsacum accessions, five Coix accessions, two Zea diploperennis accessions, one Zea mays subsp. huehuetenangensis accession, three Zea luxurians accessions, two Zea perennis accessions, eight Zea mays subsp. mexicana accessions, nine Zea mays subsp. parviglumis accessions, and 28 Zea mays subsp. mays (cultivated maize) accessions were used in this study. Totally 641 scorable AFLP bands were obtained using 21 PstI/MseI primer combinations, among which 586 bands were polymorphic. Averagely, there were 30.5 bands for each primer combination. Cluster analysis based on UPGMA method revealed that Coix was the most distinct taxa from maize. Tripsacum was also quite different from maize. However, teosintes (including different taxa of Zea except cultivated maize) were most closely related to maize but with considerably high genetic diversity. The relationships among maize inbreds revealed by AFLP markers were basically consistent with the known pedigrees but with a few exceptions.

Key words: maize, teosinte, Tripsacum, Coix, genetic relationship

Du Jinyou, Tianyu Wang, Yunsu Shi and Yu Li

Genetic diversity analysis will help better preserve and use maize germplasm and assign specific accessions to heterotic groups which are of great importance in maize breeding programs, thus increasing the efficiency of germplasm use and improvement.

In this study, we analyzed the genetic diversity of some maize accessions with SSR and AFLP techniques to identify the genetic relationships among these germplasm. The major results of the present study were as follows.

The genetic diversity of 58 maize inbreed lines and one teosinte was analyzed based on SSR markers. Using 40 pairs of SSR primers, totally 259 polymorphic fragments were detected. The range of polymorphic information content (PIC) was 0.14-0.89, with the average of 0.60. The result of cluster analysis showed that the accessions assessed could be clustered into eight groups. This is partly in accordance with the previous classification based on conventional methods but with some differences.

The genetic diversity of 28 maize inbred lines and one teosinte accession was analyzed based on AFLP markers. Totally 535 polymorphic bands were produced with twelve AFLP primer combination (PstI/MseI). Percentage of polymorphic AFLP bands was 72%. In this study, AFLP technique was compared with SSR technique. It was suggested that both systems can be used to analyze the genetic diversity of maize inbred lines. The present study indicated that AFLP markers are more suitable for the assessment of genetic diversity in maize germplasm because of its high frequency of polymorphism.

The genetic diversity of 60 maize inbreed lines with different drought tolerance, which were collected from Europe, the America and China, was analyzed based on AFLP markers. Using 20 AFLP primers combinations, totally 487 polymorphic fragments were detected, with the average of 24.4 for each AFLP primer combination. The result of cluster analysis showed that the accessions assessed could be clustered into four groups which were basically consistent with the pedigrees.

Suitable molecular marker systems for genetic diversity assessment of maize germplasm were proposed according to the results of the present study. Possible relationships between genetic diversity and heterotic groupings were also explored.

Key words: Maize; Drought tolerance; SSR; AFLP; Genetic diversity

Brassica napus (AACC) is one of the most important oilseed crops in China as well as in the world. Special attentions had been paid to quality and yield improvement in Rapeseed breeding in last four decades. But the less polymorphism of B. napus restricted their application in crop improvement for short history. Create new-typed B. napus with alternative genomic combination would be valuable for rapeseed breeding.

It was revealed that B. napus, B. carinata (BBCC) and B. juncea (AABB) are amphidiploid coming from natural doubling of the hybrids of three Brassica diploids, B. campestris (AA), B. oleracea (CC), and B. nigra (BB) thousands of years ago. Divergent evolution and artificial selection has made the A and C genome in B. napus much different from the A genome in B. campestris and the C genome in B. carinata (BBCC) respectively. To distinguish such differentiation, A' and C' are used for the corresponding genomes in B. campestris and B. carinata separately. To combine the A' genome from B. campestris and the C' genome from B. carinata would lead to a new typed B. napus, A'A'C'C' created.

The hexaploid plants (A'A'B'B'C'C') were obtained through doubling the chromosome of interspecific hybrids between B. carinata (B'B'C'C') and B. campestris (A'A'). GISH and molecular markers revered the truly doubled hybrids. The A'A'B'B'C'C' hexaploid plants were pollinated with natural B. napus (AACC, 2n=38) to yield pentaploid seeds with A'AB'C'C genomic composition. GISH results confirmed the pentaploid plant with expected 8 B chromosome number. The expected abnormal meiosis behavior was observed in the A'AB'C'C plants. Hundreds of plants with 38 chromosomes have been obtained through the self-pollination of pentaploid plants. Some of the 38-chromosome plants were B chromosome free and had high rate of A' and C' in genomic composition with good agronomic characteristics. About 100 plants with variable A'C' composition were cross pollinated with natural B. napus (AACC) resulting 700 hybrids, some of them showed very strong biomass and yield heterosis.

To transfer the useful genes of the C₃-C₄ intermediate species Moricandia nitens into Brassica crops through chromosome homoeologous pairing and recombination, sexual crosses were made between somatic hybrid (M. nitens + B. oleracea) and B. napus, (M. nitens + B. rapa) and B. napus, and between (M. nitens + B. napus) and B. oleracea. Ninety-seven plants out of 232 progenies were trigenomic sesquitetraploids with the genomic composition of MCAA or MACC identified with the observation on their morphological performance, characterization of molecular markers and the chromosome counting. Thousands of seeds were yielded from crosses between MACC and B. rapa, and 98 B. rapa-liked individuals were obtained from 300 self-pollinated individual plants. To screen the recombinants with C₃-C₄ related characteristic is highlighted.

One of the main characteristics in C₃-C₄ plants is that the gene encoding the P subunit of glycine decarboxylase (gdcsp) specifically expressed in the vascular bundle sheath of leaf. To develop a STS marker specific for gdcsp of M. nitens would be helpful for screening the target recombination plants in those 98 individuals. Degenerated primers were designed to isolate the gdcsP fragment according the sequence information in Arabidopsis and pea. Two fragments, 2969 bp and 3252 bp, including a 1.3 kb and 1.6 kb upstream domain were obtained by PCR and Genome Walking PCR from M. nitens and B. napus respectively. RT-PCR results indicate that the region of gdcsp, enclosed by the cloned fragments, have the products of transcription. A STS marker specific to M. nitens was designed according to the sequence of cloned fragments. It is verified that the marker is correlated to the C₃-C₄ character, because the sesquitetraploids plants with the specific band have a little lower CO₂ compensation point than that have no band. Using this marker, only one plant has been screened out from those 98 individuals above, but without offspring.

The specific leaf anatomy (Kranz-like structure) in the bundle sheath cells is another main characteristic for C₃-C₄ plants. Leaf sections were observed under light microscope among the 98 plant lines. One plant from thousands of checked plants showed the kranz-like structure. The ultramicrostructure microscope investigation revealed the C₃-C₄ anatomy structure. However, the plant did not show any significant changes on the CO₂ compensation point comparing with their B. rapa parent.

It is clear that recombination of M/A chromosomes should be happened in the sesquitetraploid plants although the expected C₃-C₄ plants did obtained directly. An alternatively indirect way might be introduce some key C₃-C₄ specific genes to those recombinant B. rapa by genetic transformation. Towards the new target, the two 1.3 kb upstream fragments, which containing the promoter region were fused with GUS and GFP and constructed into the expression vector pCAMBIA 1303. The plant transformation works are in process.

Key words: C₃-C_{4 ;} Brassica _; recombination; gdcsp; STSs; Kranz-like.

The genetic diversity, molecular identification and phylogeny of tea plant (Camellia sinensis) and its 23 wild related species and varieties in section Thea genus Camellia were investigated.

Fifteen decamer oligonucleotide primers were selected from the 61 screened. A total of 102 polymorphic bands (6.8 polymorphism/primer) out of 107 reproducible products (7.1 fragments/primer) were amplified from the selected 15 primers, corresponding to 95.3% polymorphism. The relative polymorphic frequency ranged from 0.04 to 0.96. The general frequency was 0.30, varying from 0.16 to 0.60. It was lower than that of intraspecific of tea plant.

According to the presence and absence of unique makers, it was possible to identify 14 of the 24 species and varieties. The combination of band patterns obtained by 4 primers allowed identifying all the species and varieties studied.

Molecular phylogenetic dendrogram and principal coordinate analysis using SHMM method were constructed using UPGMA and Nei and Li’s similarity coefficient from pairwise comparisons of RAPD data between 24 species and varieties. Both in the dendrogram and principal coordinate analysis, the 24 species and varieties of section Thea could be divided into two groups, 5-locule and 3-locule ovary group, and further divided into 3 and 2 subgroups, respectively. Compared with the author’s previous morphological combination of section Thea, they were general consistent with each other. The genetic relationship and molecular evolutionary tendency of some species were discussed from the genetic distance.

RAPD markers not only generally confirmed previous morphological classification and evolution of section Thea in genus Camellia, but also provided reliable and robust method and evidence of the diversity, identification and phylogeny of tea plant and its related species.

To carry out the plant protection article and to protect breeder’s right, it needs that the new variety must has special characters that can distinguish it from other cultivars. The morphology traits, such as seeding growth habit, leaf type, plant height, spike type, grain size and color, disease resistance et al., have been used to identify cultivars for many years. However, since the limited number of morphology traits and as more cultivars receive protection, it become more difficult to distinguish new cultivars by using these characters. In addition, since the interchange of germplasms and elite parents among breeders, genetic background of developed cultivars will become more similar. This will further increase the difficulty of identification new varieties by using conventional traits alone. But molecular marker that is numberless can provide the unique DNA characters of a new variety. Thus it shows that use of genetic markers would solve these problems.

Four major kinds of molecular markers, RFLP, RAPD, ALFP and SSR, are often used in wheat. Since the limited number of polymorphism of RFLP and RAPD in wheat and patent protection of AFLP technique, the usage of these three kinds of markers is limited. Nevertheless, SSR is high polymorphism, co-dominant, locus-specific and even distributing in genome. Therefore, it was widely used to build genetic map and genetic diversity study in major crops like wheat, rice, maize and soybean.

In this report, to clarify the function of SSR molecular marker in wheat cultivars identification and to provide scientific basis for variety registration and protection, we applied non-denaturing polyacryamide gel system for SSR analysis. A set of 59 wheat SSR primers located on 21 wheat chromosomes was used to analyze molecular markers of the 79 winter wheat varieties that come from China, USA and Australia. A total of 224 alleles were detected by using the 59 SSR primers. The number of alleles per primer pair ranged from 2 to 12, with average of 3.7. The value of allelic polymorphism information content (PIC) ranged from 0.19 to 0.85, 0.57 per primer on average. Combined 12 primers together, all of the 79 wheat cultivars could be distinguished. The results from this study indicated that SSR markers play an important role in variety identification. The new electrophoresis system is more simple, faster, and more economical than the conventional electrophoresis system.

Key words: SSR markers; Common wheat; Variety identification

Perhaps all flowering plants have experienced one or more episodes of polyploidization at some time in their evolutionary history. Recent evidence indicates that this genome doubling may be accompanied by a variety of non-Mendelian phenomena, some of which operate during hybridization and polyploid formation while others manifest more gradually on an evolutionary timescale. Here we present our studies on these phenomena, drawing attention to recent paradigm shifts necessitated by new insights from model plant systems. Allopolyploid formation in some plant groups is associated with an unexplained and in some cases directed process of genomic alteration leading to non-additivity with respect to parental genomes. Novel intergenomic interactions become possible as a consequence of the merger of two previously isolated diploid genomes, variously leading to intergenomic colonization and/or homogenization of formerly diverged sequences. Several epigenetic processes may accompany nascent hybrids and allopolyploids, such as nucleolar dominance, gene silencing and mobile element activation. These myriad phenomena do not characterize all polyploid systems, and some newly formed allopolyploids appear to be genomically quiescent. Although a direct connection to adaptation remains to be established, the surprisingly diversity of genetic and epigenetic responses to wide hybridization and allopolyploid formation and their apparent high frequency suggest that non-Mendelian phenomena contribute directly to polyploid stabilization and diversification.

Key words: polyploidy; molecular evolution; genome duplication; evolution; epigenetics; speciation

AFLP is based on the selective amplification of a restricted number of genomic fragments. This technology can identify cultivars and specify relationships between closely related plant accessions. The genetic relationships among 16 cultivars of Iranian wheat were investigated using AFLP markers as discriminating characters. Resolved bands were scored for presence and absence in a binary matrix and relationship among cultivars calculated using simple matching coefficients. The results have assisted in the development of a dendogram suggesting genetic relationship among genotypes. Cluster analysis by UPGMA showed that 16 cultivars can be placed in two main groups with a similarity ranging from 0. 456 to 0.776. Variability observed had a narrow genetic base and it is necessary to expand the diversity of the wheat genetic base. With using AFLP, identification of diverse germplasm, selection of parents and estimating of heterosis at molecular level will be possible. Also our results indicated that A FLP technology can generate molecular markers that can be used reliably for DNA fingerprinting of important cultivars.

Key words: AFLP; Molecular markers; Polymorphism; Genetic fingerprinting; Iranian wheat.

Key word: soybean; core collection; sampling strategy

Key words: soybean; genetic diversity; core collection; SSR marker

Triticum compactum and common wheat constitute two of the six groups of the hexaploid wheats with an AABBDD genome. The genetic variations and genetic relationships had been analyzed within and between fourteen common wheat cultivars widely used in world wheat production and thirty two Triticum compactum varieties with 28 D-genome-specific SSR primers, two for each chromosome arm. All 28 D-genome-Specific SSR primers produced amplification products in 46 genotypes. In the 14 common wheat cultivars, a total of 65 alleles were detected on SSR analysis, a number of alleles per WMS ranged from 1 to 8 with an average 2.32; In the 32 Triticum compactum varieties, these 28 SSR indentified 82 alleles in the D genome with an average number of allele per WMS 2.93 (from 1 to 10). The polymorphisms in the D genome of microsatellite markers were found to be higher in Triticum compactum than in common wheat. Genetic distance (GD) between common wheat cultivars of the D genome and each of the investigated Triticum compactum varieties ranged from 0.1034 to 0.4828, the average distance 0.2717. Genetic distances within common wheat and within Triticum compactum were 0.2523 and 0.2773, respectively. The result that there were slight genetic diversity between common wheat and Triticum compactum, which provided useful information to broaden the genetic basis for wheat breeding.

Most Chinese saline lakes occur in the west and northeast. They belong to many types according to their chemical composition, such as Chloride type, Magnesium sulfate subtype, sodium sulfate type, carbonate type et al. Since the varied climate, geological position and chemical type of saline lakes, the halophile has much genetic diversity. The species of halophile studied mainly were halophilic bacteria, Artemia, D.salina, Spirulina et al. Most of them have different strains that have special characters. The genetic diversity of these halophilic organisms not only provides directly economical application, but also provides gene resource for gene engineering, especially for the salt tolerance. Many kinds of salt tolerance genes may be cloned from the halophilic organism, and used for plant transformation. The strategy and methods for cloning salt tolerance were discussed. These methods maily are difference display, map based cloning, transposon tagging, homology cloning, and gun-shot cloning, and gene chip et al.

Key words: plant salt tolerance; Genetic diversity; halophilic organism; Chinese saline lakes; genetic engineering

This study was conducted to test two hypotheses related to origin of cultivated barley (Hordeum vulgare, or HV). It was previously proposed that H. agriocrithon (HA) in Tibet was the progenitor of the cultivated barley in the Oriental region, but the difficulty with previous data is that HA did not possess the amount of genetic diversity to account for the genetic variation observed in HV of this region. The two-rowed wild barley H. spontaneum was proposed to be the progenitor of HV in the Occidental region, but the sharp contrast in the occurrence of the rDNA allele 104 was not congruent with such hypothesis. In this study, a total of 192 accessions constituting three samples, including 82 entries of HS from Tibet (sample 1), 57 accessions of HS from 10 countries (sample 2), and 59 accessions from 4 countries (sample 3) coincided with the major barley growing areas in the Middle-East were assayed for rDNA spacer-length variants. A total of 22 rDNA alv phenotypes were detected from which ten slvs were identified as alleles at two rDNA loci. Comparisons of the numbers of the rDNA phenotypes and alleles and the diversity indexes indicated that the HS sample from Tibet possessed the lowest level of genetic variation. Combined analysis of the data from this and previous studies indicated that the two wild forms together did not have the level of genetic variation to account for the amount of genetic diversity observed in the cultivated barley of this region. The 104 allele was again rare in the HS sample from the Occidental region, and in contrast this allele occurred at the highest frequency in HV of this region. Thus, the two gaps concerning the origin of cultivated barley still remain, after combing the information provided by this and previous studies. The implications of such findings remain to be investigated.

Key words: Hordeum vulgare L; Oriental; Occidental; Evolution; rDNA

In this study, using 96 SSR primers, 845 alleles were obtained in 80 accessions of autumn soybean (Glycine max (L.) Merr) from soybean core collection in China. The number of alleles per primer is 2 to 23 with a mean of 8.8 and polymorphism informative content is 0.141 to 0.938 with a mean of 0.738. 7.00 primers were averagely needed to identify all 80 accessions with the least range of 4 ~ 39 primers. More importantly, the criterion of selecting core primers to study DNA fingerprinting of soybean gemplasm was disussed. The representative, highly informative set of 60 core SSR primers provides a method to build fingerprint of Chinese soybean germplas.

Key words: Gene mapping; C-banding; Fluorescention in situ hybridization; Rye

Yongtao Yu, Yanchun Song, Yunsu Shi, Tianyu Wang, Yu Li

Maize production in China has been seriously affected by corn borer, especially Asia corn borer (Ostrinia furnacalis (Guenee)). Integrated pest management has been paid great attention to in China but it applies to only 20% of maize fields annually. That is, about 80% of the growing area (about 16 million ha) is open to this pest. If the annual rate of yield decrease were 10%, it implies that the maize production of 1.6 million ha would be lost.

From the economic and scientific view, breeding for corn borer resistance should be a priority in reducing the damage of this pest. Suppose that single-cross hybrids with moderate resistance could be planted on the half of these 16 million ha and the yield could be 2500 kg/ha, the total production of 20 million tons would increase. Since the 1970s, breeding for corn borer resistance has made progress but it has not been very effective because the inheritance of the Asia corn borer resistance which were believed to be controlled by quantitative trait loci (QTL) is complicated. Other reasons include: (1) results of evaluation for resistance under natural conditions are not very reliable due to the instability of climate; (2) progeny evaluation in the field must wait until the late stages of plant growth; (3) artificial infestation needs techniques to raise insects and is not easy to be operated by ordinary breeders. Molecular marker assisted selection (MAS) may be one of useful ways to raise breeding efficiency. Its prerequisite is tagging genes conferring traits of interest. As for the resistance to Asian corn borer, the genetic mechanism has not been very clear although that it is a quantitative trait has been known. Through genetic mapping of QTL controlling the resistance, marker-assisted selection and isolation of genes with major effect will become practical and easier.

The 120 F_2:3 families of “H21 ´ Mo17” were phenotyped in 2000 and 2001 by artificial infestation in terms of leaf feeding damage and stalk tunnel damage and genotyped using 71 SSR and AFLP markers. Three QTLs were identified for resistance to leaf feeding damage on chromosome 1, 2 and 5, and four QTLs were identified for number of holes on stalk on chromosome 1, 3 7 and 8. No QTLs were found for stalk channel length but three QTLs were identified for the channel length per hole on chromosome 1, 4 and 9. Besides, five, two and three QTLs were detected for plant height, leaf width and leaf angle, respectively. Presently, more markers are adding to the linkage map to accurately map QTLs conditioning the resistance. In addition, another mapping population “K36 ´ Zi 330” is using for QTL analysis for this important traits.

Key words: maize; Asian Corn Borer resistance; QTL mapping

Zhengqiang Ma, JizhongWu, Huilan Zhu, Zhongxin Kong, Feng Lin, Yigao Feng, Dajun Liu

Grh2, one of green rice leafhopper, N.cincticeps resistance genes was located on chromosome 11 of resistant variety DV85 (Indica). Taichung 65 is a Japonica cultivar with elite characters, but susceptible to green rice leafhopper. C189 and G1465 are two RFLP markers flanking Grh2 gene, which were transformed into CAPS markers this study. Both phenotypic selection and CAPS marker assistant selection were conducted in BC₆F₂ population derived from the cross of Taichung65 and DV85 in order to pick out the important breeding materials with Taichung65 background and resistance to green rice leafhopper. The linkage distance was calculated with the molecular and phenotypic data, meanwhile the effect of the selection method was analyzed. Since the experimental methods in this paper, including rude extraction of DNA, CAPS primer design, and the method of CAPS marker assistant selection, were characterized by fast, easy, and high effective to operate, it can be applied broadly to the selection for various agronomic traits.

Key words: BIL population; Ferrous iron toxicity tolerance; QTL analysis; Rice

Flax (Linum usitatissimum L.) is an important fiber and oil-producing crop. However, flax production has been restricted by some diseases, among them, flax rust caused by Melampsora lini Ehrenb.Lev. and flax wilt caused by Fusarium oxysporm f.sp.lini are two main diseases in flax. The most effective method to control these diseases is to screen and to breed resistant varieties. By identifying the molecular markers for resistant genes to flax rust and wilt, we hope to set up a procedure for maker-assisted selection in flax disease resistance breeding. The major results in this paper are as follows:

1. Identification of molecular markers for the rust resistance gene M4 in flax

2. Identification of molecular markers for the wilt resistance gene FuJ7(t) in flax

A cross between wilt resistant flax variety, Jinya 7, which was also bred by the author, and susceptible variety, Jinya 1, was made for mapping wilt resistance gene(s). Through inoculation test of its F₁ and F₂ progeny, it is proved that two dominant genes control the resistance of Jinya 7 to wilt. With 48 EcoRI/MseI primer combinations, AFLP analysis was performed on two parents and their F₂ resistance and susceptibility bulks. A total of about 3300 distinguishable bands were amplified, of which three were stable. The linkage analysis of the three polymorphic DNA fragments with the resistance gene(s) was tested in the F₂ segregating population between Jinya 7 and Jinya 1. The DNA fragment AG/CAG was found closely linked to one of the wilt resistant genes, with a genetic distance of 5.2 cM, which was tentatively named FuJ7(t). The cloned fragment AG/CAG was sequenced and then transformed to a SCAR marker, which can be used more conveniently in the identification and marker-assisted selection for gene FuJ7(t).

Key words: Flax or Linseed (Linum usitatissimum L.); Rust; Wilt; Resistant gene; Molecular

marker; RAPD; AFLP; SCAR

Wheat Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is one of the worldwide destructive disease of wheat. It causes both severe yield loss and decrease in grain quality. In addition, FHB contaminates cereal products with mycotoxin. Breeding wheat resistant to the disease is the preferable approach to minimize the damage caused by FHB. Sumai3 and its derivatives such as Ning7840 have been used as the sole resistance resource for genetic research and breeding application in many breeding programs. Resistance genes from Sumai3 and Ning7840 have been well characterized through molecular mapping. However, resistance in Wangshuibai, a Chinese landrace with high and stable scab resistance performance, is still a puzzle to be answered.

Powdery mildew (Pm), caused by Erysiphe graminis DM f. sp. tritici (Em. Marchal), is one of the most important diseases of common wheat worldwide. Among the 30 documented Pm genes, the recessive gene Pm5 was located on chromosome 7B. Up to now, six alleles were reported at the Pm5 locus. Wheat variety ‘Fuzhuang 30’ carries the gene Pm5e and has proven to be a valuable resistance source of powdery mildew for wheat breeding. Forty microsatellite markers, which were mapped on chromosome 7B of wheat in the ITMI population, were chosen for screening of polymorphism between two parents of the F₂ mapping population from the cross ‘Nongda 15’ (susceptible)/‘Fuzhuang 30’ (resistant). Microsatellite fragments were detected on an automated laser fluorescence (A.L.F.) sequencer and analysed using the computer program fragment analyser (Pharmacia). Linkage relationship between DNA markers and the gene Pm5e was established with MAPMAKER/Exp version 3.0b. LOD scores ³ 3.0 were used to develop the linkage map.

For the molecular mapping of the gene Pm5e in ‘Fuzhuang 30’, a total of 100 F₂-derived F₃ families from the cross between ‘Nongda 15’ and ‘Fuzhuang 30’ were tested with race no. 15 of powdery mildew. The observed segregation of 22 homozygous resistant, 55 heterozygous and 23 homozygous susceptible families fitted a 1:2:1 segregation ratio (c ² = 1.02, 0.50 < P < 0.60). Among the 40 microsatellite markers, 21 (52.5%) produced polymorphism between two parents, indicating that microsatellite markers revealed a high level of polymorphism in wheat cultivars. Seven microsatellite markers, namely Xgwm577, Xgwm783, Xgwm883, Xgwm984a, Xgwm1144 and Xgwm1267, were found to be linked to the gene Pm5e, of which two codominant markers Xgwm783 and Xgwm1267 were relatively close to Pm5e with linkage distance of 11.0 cM and 6.6 cM, respectively. It is possible to use the 136 bp-allele of Xgwm1267 in ‘Fuzhuang 30’ for marker-assisted selection during the wheat resistance breeding process for the facilitation of gene pyramiding. The gene Pm5e was mapped in the distal region of chromosome 7BL with the map distance of approximately 145 cM to the centromere and located between Xgwm1267 and the RAPD marker UBC405-628 which was linked to the gene Pm5e with the linkage distance of 12.6 cM in the previous study.

In addition, a microsatellite linkage map of chromosome 7B was constructed using the same mapping population. Nineteen microsatellite loci were mapped on chromosome 7B, nine on the short arm and ten on the long arm. This information will be very useful for further mapping of agronomically important genes of interest on chromosome 7B.

Annongs-1, a thermo-sensitive genic male sterile (TGMS) rice line, has a new TGMS gene. Genetic analysis indicated that the sterility of Annongs-1 was controlled by a single resessive gene which was named as tms5. In our previous study, tms5 was mapped on chromosome 2 with an F₂ population from the cross between Annongs-1 and Nanjing11. Recently, a RIL (recombinant inbred line) population from the same cross was developed and used for fine mapping the tms5 gene.

AFLP, RAPD, RFLP and SSR techniques combined with BSA (bulked segregant analysis) were used to screen molecular markers linked to the target gene. As a result, two AFLP markers (E:AA/M:CTG₄₀₀, E:AT/M:CAC₄₂₀), one RAPD marker (OPE-16₃₅₀₎), and four SSR markers (RM279, RM492, RM327, RM324) were linked with tms5 gene. The tms5.gene was located between Rm492 and E:AA/M:CTG₄₀₀ with a genetic distance of 5.4 cM and 3.0 cM respectively. Unfortunately, no RFLP markers show polymorphism between the two parents of our RIL population, although there are some RFLP markers located at very close loci in the JRGP map, such as C365 and G227,

In order to find markers more closely linked with tms5 gene, four SSR primer-pairs.(SSR1, SSR2, SSR3 and SSR4) were designed based on the nucleotide sequence of C365 and G227 found in GENEBANK. Then, these 4 SSR primers were used to amplify Annongs-1, Nanjing11 and the individuals of their RIL population. The result showed that SSR1₄₀₀ derived from C365 was closely linked to the tms5 gene with a genetic distance of 1.04 cM. A CAPS marker (CAP1₄₅₀) derived from a SSR product SSR4₇₅₀ linked to the tms5 gene with a genetic distance of 2.08 cM.

The BAC library of AnnongS-1 was screened using G227 and C365 as probes, and eight positive clones were obtained. A BAC contig covered 240 Kb in tms5 gene-encompassing region was constructed.

Key Word: Rice; TGMS gene; tms5; BAC; Mapping

This research was supported by Plant Transformation Project (J99-A-002)

Physiological and genetic variations exist richly in the cotton plants regenerated from somatic embryo. A stable homeotic variant (chv1) of cotton from regenerated plants was identified. Using light microscope and scanning electron microscope to investigate the morphological changes in chv1, the results revealed that all the floral organs in the chv1 plants converted into leaves, meanwhile the placenta and ovules bearing on the basal part in central leaf-like organs can be discerned. A flower of the variat consists of three to seven bracts, fourteen to thirty one leaf-like organs. Arrangement of the leaf-like organs is intermediate between spiral and whorl.

The floral organ mutant, chv1, may be a useful material in the research of cotton flower development and somaclonal variation. Firstly, The chv1 provide an evidence to support old theory in cotton, that flower organs are modified leaves. This theory is supported by the observation that triple mutations lacking all three of the ABC gene activities and make all flowers become leaf-like. Secondly, although the carpels of the chv1 have converted to leaf-like organs, ovules still emerged at the base of the organs, which normally bear on the axile placeta. This observation supports Angenent’s viewpoint that the ovule should be seen as another class of floral organs. Finally, the chv1 is a somatic variant, and the research of molecular mechanism of somatic variant is still in infancy. Further study on the genes involving the mutation of chv1 will help us to shed light on the mechanism of somatic variations.

Key words: cotton; floral organs; somatic mutation

Funded by the National Natural Science Foundation of China (Grant No. 30080001)

1.Construction of linkage maps

A population of 104 F₇-derived recombinant inbred lines developed by single seed descent from common wheat cross of Wangshuibai (resistant)/Alondra (susceptible) was used for this research. With JoinMap3.0, the integrated linkage maps were constructed using 292 polymorphic markers, including 143 SSRs, 145 AFLPs and 4 RAPDs. 189 markers could be mapped and they formed 19 linkage groups, covering a total genetic distance of 988 cM. For 17 linkage groups their chromosome identity could be determined based on the microsatellites map of wheat published by R?eder. Apart from chromosome 3D, 4B, 5D, 6D, the other chromosomes maps were obtained. About 30% polymorphic markers couldn’t be constructed to linkage maps. The major reason is the linkage maps didn’t cover the whole genome of wheat. Some markers have no sufficient linkage with the mapped markers. In order to get the complete linkage maps, more additional markers need to be used to saturate the maps.

2.Analysis of major QTL for wheat scab resistance

The scab resistance of individuals of Wangshuibai/Alondra was evaluated in the field of Nanjing and the greenhouse of Oklahoma State University. QTL of resistance for scab was firstly analyzed by Kruskal-Wallis Analysis, then Interval Mapping and MQM (multiple-QTL model) Mapping were carried out using MapQTL4.0 software. The analysis result was output using MapChart2.0. Kruskal-Wallis Analysis showed that BARC147, Xgwm493 and BARC102a linked closely to resistance for scab (p<0.005). Which indicated there is a QTL in this chromosome region. Interval Mapping also detected a prominent QTL on chromosome 3BS between BARC147 and BARC102a with LOD>2.8 in two places data. This QTL was as similar as the major QTL of Sumai3 and could explained 14.5% phenotypic variance. With the flank markers of QTL used as cofactor, MQM Mapping displayed this QTL was located between BARC147 and Xgwm493 with LOD >2.82. The genetic distance of this fragment is 10.5 cM.

Glu-B3 STS-PCR, Gli-B1 SSR and SEC-1b STS-PCR markers are used for identification of loci for LMW glutenin, gliadin and secalin, respectively. Results indicate that cultivars and advanced lines such as CA9632, Jinmai45, Lankao24, Yannong18, Jingdong8 and 39 doubled-haploid lines of Zhongyou9507/CA9632, both of Glu-B3 and Gli-B1loci are absent, but SEC-1b is presented. In addition, secalins are detected using PAGE and ELISA, and the results are identical to that detected by multiplex PCR. Furthermore, 28 F₂ plants of Zhongyou9507/CA9632 were tested, which shows that the multiplex PCR also can detect homogenous and heterogeneous plants carring 1BL/1RS translocation.

Key words: Common wheat; Glu-B3; Gli-B1; SEC-1b; 1BL/1RS translocation

Strong interspecific heterosis on biomass has been observed among F₁ plants (A'AC) between Brassica napus (AACC) and B. rapa (A'A'). However the triploid hybrid could not be utilized as commercial variety because of abnormal meiosis and poor seed production. If the A genome in the B. napus is substituted with the A' genome of B. rapa, a new-typed B. napus with A'A'CC genomic composition would be created. And the strong interspecific heterosis may be utilized in the intergenomic form of A'ACC produced from crosses between the new-typed B. napus (A'A'CC) and natural B. napus (AACC). A serial of new-typed B. napus (A'A'CC) lines were creased by backcrossing the F₁ (A'AC) plants to B. rapa and selfing the BCF₁ plants followed morphologically and cytologically identification and marker-assisted selection on the progenies.

Five natural B. napus were used as male parents to cross-pollinate to four new-typed B. napus (A'A'CC) lines (new ´ nature) and two natural B. napus varieties, which were used as parents to create the new typed B. napus (nature ´ nature). Thirty intergenomic hybrids were produced in NC design . The parents and F₁ hybrid plants were grown with a randomized complete block design with 3 replications in 2001. Biomass and seed yield were investigated. The mid-parent heterosis for biomass, seed yield and harvest index was 36.87%, 58.16% and 17.15%, respectively. General combining ability (GCA) effect of female and male for biomass and yield was significant or very significant. SCA effect was only significant in harvest index. These results indicated that some main genes with additive effect controlled the biomass and the seed yield. The correlation coefficient between the seed yield and the biomass reached to 96.59%, which revealed that the biomass heterosis in the interspecific hybrids had a real potential to be transformed to intergenomic heterosis of seed yield when the unbalance of chromosome pairing to be solved. The seed yield in the highest “new ´ nature” was significantly higher than that of “nature ´ nature” and commercial hybrids, 37% and 38% respectively. It proved not only the A'/A or A'/C intergenomic interaction, but also the commercial value of intergenomic hybrids.

Key word: new-typed Brassica napus; natural Brassica napus; intergenomic heterosis; GCA; genome interaction

MAPMAKER is one of the most widely used computer software package for constructing genetic linkage maps. However, the PC version, MAPMAKER 3.0 for PC, could not draw the genetic linkage maps that its Macintosh version, MAPMAKER 3.0 for Macintosh, was able to do. Especially in recent years, Macintosh computer is far more unpopular than PC. Most of the geneticists use PC to analyze their genetic linkage data. So new computer software to draw the same genetic linkage maps on PC as the MAPMAKER for Macintosh do on Macintosh have been crying for.

Microsoft Excel, one component of Microsoft Office package, is no doubt the most popular software in laboratory data processing. Microsoft Visual Basic for Applications (VBA) is one of the most powerful functions of Microsoft Excel. Using this program language, we can take creative control of Excel, including genetic linkage map construction, automatic data processing and more. In this paper, a Microsoft Excel macro called MapDraw is constructed to draw genetic linkage maps on PC computer based on given genetic linkage data. Use this software, you can freely construct beautiful genetic linkage map in Excel and freely edit and copy it to Word or other applications. This software and its help information is just a Microsoft Excel file named MapDraw.xls. You can freely copy this software from ftp://211.69.140.177 and the source code can be found in Excel’s Visual Basic Editor.

Keywords: Genetic linkage map construction; Microsoft Excel; MapDraw

Soybean is an important economical crop and main resource of protein. Soybean seeds contain at least three lipoxygenase isozymes lipoxygenase1, 2 and 3. These isozymes are responsible for the production of unpleasant grassy and beany flavors in soybean seeds that have limited the development of soybean products for human consumption. Treatments with heat, acid, alcohol or antioxidants have been employed to reduce the concentration of compounds that contribute to the undesirable flavors. An alternative solution to this oxidation problem would be to genetically eliminate lipoxygenase from seeds.

Developing seed lipoxygenase-free soybean cultivars with desirable agronomic and seed characteristics is the best solution. Aim of this study is using markers to reduce the number of backcrossing generations to obtain desirable lines for further backcross based on the lines with lacking of lipoxygenase. The targeted gene is detected by IEF-PAGE; using SSR markers to accelerate the recovery of the recurrent parent genome, to select the ideal lines, which not only have the desirable gene, but also have little difference with the genetic background of an elite variety called Ludou 4.

The main results are as follows:

51 pairs of SSR primers used in the research have specific alleles of ludou 4. On the soybean molecular linkage group, only H and L linkage groups have one SSR locus, the other 18 linkage groups have at least two SSR loci and the loci were evenly distributed.

A procedure was useful in marker-assisted background selection. That is, first use limited numbers of markers to screen all the individuals, eliminate the individuals not fit the requirements, then use more makers to screen the residuals. With this method, ideal individuals can be obtained with high efficiency.

A line lacking Lox-2 was got, which can be seen as a near-isogenic line of loudou 4 based on the background selection results. Candidate lines were chosen for further backcross with ludou 4, to accelerate the process of getting near-isogenic lines of loudou 4 lacking different types of lipoxygenase.

Genetic analyses of resistance to southern rust (caused by Puccinia polysora Underw.) was studied with mixed the major-gene and polygene inheritance model, using the high-resistant inbred line P₂₅ and the high-susceptible inbred line F₃₄₉ and their single crosses. Besides, by 174 F₂:₃ lines, a genetic linkage map of maize (Zea mays. L) was constructed, and the resistant gene was mapped. The main results as follow:

Gene tagging is the basis of marker-assisted selection and map-based cloning. Ma et al (1994) reported a co-segregating RFL marker xbcd1231of Pm4a, a dominant powdery mildew resistance gene of wheat that is still active in combating powdery mildew disease in many wheat-growing areas. xbcd1231 has been successfully used in marker-based breeding for powdery mildew resistance. So far, there is no better PCR-based marker available for this gene, which is needed to simplify the molecular genotyping process. Thus, we decided to convert xbcd1231 to a STS marker. To this goal end sequencing of BCD1231 was performed. PCR of the genomic DNA of Pm4a isogenic lines with a pair of STS primers, designed based on the sequence information, amplified a 1584 bp monomorphic fragment. However, nucleotide polymorphism of this amplified product between the two isogenic lines was discovered after four-cutter enzymes Hae III and Msp I were applied. A F2 population of 88 plants was surveyed with this STS marker and the polymorphism was co-segregating with the resistance, which is consistent with corresponding BCD1231 RFLP genotyping result. To identify more markers tightly linked to Pm4a, a group of SSR markers were also surveyed for polymorphism between the isogenic lines. One polymorphic SSR marker was found and was applied in genotyping the segregating population. Linkage analysis showed that it was 1.9 cM from Pm4a. These PCR-based markers will make the identification of Pm4a in breeding materials an easier process.

In this study we reanalyzed the QTLs underlying four yield traits for data of an F_2:3 population from a cross between two indica rice varieties, Zhenshan 97 and Minghui 63, and the results were compared with that of recombinant inbred line (RIL) population from the same cross. The analyses of data from both populations were conducted using composite interval mapping with LOD threshold 2.4. Totally, 33 QTLs were identified for the four traits in F_2:3 population. Of them, 19, 2 and 12 showed partial dominant, full dominant and over dominant effects. Twenty-five QTLs, distributed on 9 of the 12 chromosomes were detected for the four traits in RILs. Comparison of the QTLs detected in the two populations showed that 8 of the 19 QTLs showing partial dominance, 1 of the 2 QTLs showing full dominance and 1 of the 12 QTLs showing over dominance, detected in the F_2:3 were also detected in the RILs. Of the 10 QTLs detected in both populations, 1, 2, 3 and 4 QTLs affected yield per plant, tillers per plant, grains per panicle and grain weight, respectively. The results also clearly showed that the QTLs for the traits with high heritabilities were more likely to be detected in both populations, as were the QTLs with large additive effects. Three QTLs with pleiotropic effects observed on chromosome 3, 5 and 7 were also detected in both populations. The above results implied that QTLs with large effects could be detected across generations and could be used as the targets to improve quantitative traits using marker assistant selection.

Maize dwarf mosaic is an important widespread virus disease of maize (Zea mays L.) in the world, causing significant losses in grain and forage yield in susceptible genotypes. It has also become a major concern in China, where it is mainly caused by sugarcane mosaic virus (SCMV), formerly also denoted as maize dwarf mosaic virus strain B (MDMV-B). For ecological and economical reasons, the cultivation of resistant maize varieties is the most efficient method to control the virus disease. However, information regarding the genetic basis of resistance to SCMV in Chinese maize germplasm is still lacking.

Key words: Genetic linkage map; SCMV; SSR markers; QTL

Key words: Maize, Tagging and Mapping, Disease resistant gene, Rpi1, Rfg1.

* This research was supported by NSF project of China (39893350)

Introduction: Maize dwarf mosaic virus is one of the devastating and widespread viral diseases in the world. It has been becoming economically important in many areas of China. The breeding and cultivation of resistant varieties is the basic and most important way to prevent the yield losses caused by the pathogen. The resistant materials and their inheritance pay an important role in the efficiency of resistant breeding. Many reports showed that both major genes and polygene have involved in the resistance to the virus. Only a few genes have been identified in the resistant materials in the world. An elite inbred line, Siyi, has been newly screened with resistance genes to the virus. Two complementary dominant genes have been identificated on chromosome 3 and 6 by microsatellite markers respectively, which is a basic work for the marker assistant selection and map based cloning of the resistance genes.

Materials and methods: The parents, F₁, F₂ and backcrosses from the combination Siyi×Mo17 were planted to study the inheritance of the resistance to maize dwarf mosaic virus, and F₂ progenies were used to map the new resistance genes.

Discussion: There is a resistance gene family to MDMV nearing to the centromere of chromosome 6, in which a few resistance genes, Mdm1 in Pa405, a major recessive resistance gene in Huangzaosi, and a major QTL in FAP1360A, were identified. One resistance gene on chromosome 6 in Siyi was located on the region. There is the other resistance gene family to MDMV on chromosome 3, in which a few resistance genes, the other major QTL in FAP1360a, the resistance gene on chromosome 3 in Siyi, were identified.

The near isogenic lines for wheat powdery mildew resistance were evaluated with the AFLP technique. Greater genetic variance was detected in donor parents of the near isogenic lines, and good genetic consistency was found in the NILs. Characteristic bands of the resistance gene to powdery mildew Pm21 and Pm13 were found, which could be used in the identification of the disease resistance genes and the near isogenic lines.

As important genetic stocks for the study of gene mapping, tagging and gene cloning, the near isogenic lines (NILs) were attached importance in recent years. Powdery mildew of wheat, which is caused by the fungus of Erysiphe graminis DC, has been a serious disease in China as well as many wheat production areas in the world. To date, thirty genes conferring powdery mildew resistance have been found in wheat. In our laboratory, with the recurrent parents Bainong 3217 and Beijing 837, several NILs for powdery mildew resistance have been bred. These NILs have been backcross after five to seven generations, but the similarity to the recurrent parent and the consistency in the NILs have not been evaluated in molecular level.

In our study, the background of eight NILs with the respective resistance genes Pm4b, Pm2/Pm6, Pm12, Pm13, Pm16, Pm21 and two unknown resistance genes were evaluated using the AFLP technique. All the NILs were bred with the recurrent parent (RP) Bainong3217. Twelve combinations of primer were used, and in total 791 bands were detected, in which 339 were polymorphic. The average polymorphic bands rate amounted to 42.86 percent, and the genetic diversity coefficent averaged to 0.4086. But the genetic diversity mainly focused among the parents (resistance donor). In total, 325 polymorphic bands were found among the parents, accounting for 41.34 percent of the total. Only 62 polymorphic bands were detected in the NILs, accounting for 8.04 percent. The genetic diversity of the donors was 0.6900, whereas the genetic diversity of the NILs was 0.090. Therefore, it could be concluded that greater consistency had attained in the NILs.

The Jaccard genetic similarity coefficients between the NILs and the RP were calculated. The average similarity between the NILs and the RP averaged to 0.96. The NIL1 with Pm4b showed the greatest similarity to the RP (0.9848), and the genetic similarity among the selected lines amounted to 0.9932. The second was NIL5 with Pm16, with the similarity 0.9815 to the RP, and the similarity among the selected lines was 0.9932. The NIL3 with Pm12, although it was backcrossed after 7 generations, showed less similarity to the RP (0.9683). So NIL1 with Pm4b, NIL5 with Pm16, and NIL2 with Pm2/Pm6 were evaluated as better NILs for further study.

In the donor parents, Hongquanmang, the accession from T. aesvitum, owned the most similarity to the NILs, in the next place were Mardler and VPM, an accession of T.aestivum and a translocation line from common wheat and wild relatives. Line31, R43, R1A, which were the translocation lines from common wheat and wild relatives, owned less similarity to the NILs. Am6, which came from the dibiploid of the durum accession DR147 and the accession Ae39 from Aegilops squarrosa L., showed greatest genetic difference to NILs. So it was obvious that AFLP could reveal the correct genetic relation of the NILs and their donors.

After analysis all the bands of the NILs, the RP and the donors, we found two bands, M-CGA/P-ACT-500 and M-CGA/P-ACC-300, were specific for NIL6 with Pm21 and its donor R43. After further analysis in the F₂ segregating population, we found that the bands were present only in the resistant plants, and they could be seen as the markers of Pm21. Another band M-CTG/P-AGC-270, was found specific for NIL4 with Pm13 and its donor R1A, a translocation line from T. aestivum and Agilops longissima.. These bands would be useful in the identification of the NILs and map cloning of the resistance genes.

Gossypium bickii, a wild allodiploid cotton originating in Oceania, has the feature of delaying the forming of oil glands in seeds, which means that there is no gland or no gossypol in seeds before germination. By hybridizing wild diploid bickii (G₁G₁) as paternal plant with cultivated diploid (G.arboreum) [A₂A₂] as maternal plant in 1980, We obtained an allodiploid (A₂G₁), whose chromosomes were then doubled and a fertile allotetraploid (A₂A₂G₁G₁)(2n=4x=52) was achieved. The allotetraploid hybrid has steadily passed on the merit of having no gland in seeds but having it in plant body for 8 generations, which makes it possible to breed a cultivated vatiety, with the comprehensive utilization of cotton fibre, protein and oil and resistance to insects. The allotetraploid has reproduced for 8 generations. Crossing G. hirsutum and G. barbadase with the allotetraploid produced the triple hybrid.

The whole plant in the tri-species hybrid looked roughly like upland cotton. While the character of pubescence was more like G. bickii, denser than G.arboreum and G.hirsutum; The gossypol contents of the triple hybrids ranged from 0.00125 percent to 0.0481 percent, much lower than the glanded upland cotton varieties and close to the standard (0.02%-0.04%) set by WHO and FAO.The analysis of (A₂×G₁ ) ×(AD)₁ chromosome karyotype is following as:

The karyotype formula of (A₂×G₁): 2n=4x=52=50m(6SAT)+2Sm;

The karyotype formula of (AD)₁: 2n=4x=52=50m+2Sm+(2SAT);

The karyotype formula of(A₂×G₁)×(AD)₁: 2n=4x=52=2M+44m(SAT)+6Sm(SAT).

Total of 137 RAPD amplified bands of the triple hybrid (A₂×G₁)×(AD)₁ F₁ were acquired, among those 73.7% of the DNA bands are similar to the parents. 65.6% of 131 (A₂×G₁)×(AD)₂ F₁ bands amplified and 61.4% of 145 (A₂G₁)×(AD)₂ F₁ bands amplified are similar to their parents. The amplification results using primer OPAV-19 shows that the triple hybrid not only has the bands of amphiploid G.arboreum × G.bickii, but also has its own specific one.

Breeding durable resistance to pathogens is a major task for modern plant breeders. Pyramiding different resistant genes into a genotype is one way to breed wheat cultivars with durable and broad-spectrum powdery mildew resistance. With the large number of pathogen isolates, the traditional method of Pm gene identification based on resistance to individual isolate is time-consuming. Molecular marker that can be used to identify the specific resistant gene quickly and precisely will provide a tool in pyramiding multiple genes and speeding up the gene pyramiding process in breeding.

Biochemical marker, STS-PCR marker and SCARS-PCR marker, which are tightly linked with Pm4b, Pm12, Pm13 and Pm21b, were used to identify DH2 population that potentially contain the multiple Pm resistant genes. The results showed that four plants confer with Pm4b+ Pm12+ Pm21b, two plants with Pm4b+ Pm13+ Pm21b, one plant with Pm13+ Pm21b, four plants with Pm4b+Pm21b and four plants with Pm12+Pm21b, respectively. We will validate the plants with identified genes using gene-to-gene method and genetic analysis. These results will be useful for molecular marker-assisted breeding in wheat.

Key words: common wheat powdery mildew; gene-pyramid; DH population; molecular marker

The genus Populus comprises about 30 woody perennial species of great economical and environmental value and is represented by such familiar trees as poplars, cottonwoods, and aspens. Populus has also been used as a model system for addressing biological questions related to recalcitrant forest trees with long generation intervals and outcrossing mating systems. The biological attributes of Populus as a model system in forest-tree biological includes rapid growth, rapid seed formation, small genome size (2C=1.2 pg), identical chromosome number of all species (2n=38), and efficient transformation system. It is especially the ease of vegetative propagation of individual Populus genotypes that greatly facilitates the estimates of quantitative and molecular genetic parameters. This plays an important role in bringing traditionally separate disciplines into an era of integrative biology.

Marssonina leaf spot of poplar is an important forest disease in China. Marssonina brunnea has been considered the most important of these pathogens because it occurs in most regions of China and has been associated with significant economic damage poplar plantation. The fungus causes very small, dark brown spots on leaves and petioles and sometimes on young green twigs and capsules. Poplar trees attacked by Marssonina display premature leaf fall and reduced photo -synthetic capacity and growth..

Supported by National 973 program (G1998010206).

Starch properties in common wheat are important factors influencing the noodle quality. Waxy proteins of more than 80 wheat varieties were screened through an improved 1-D SDS-PAGE method, 14 varieties were absent in Wx-B1 protein, one variety Kanto107 was absent in Wx-A1 and Wx-B1 proteins, and Baihuomai was absent in Wx-D1 protein. Fifty-eight waxy wheat lines were used to analyze the paste properties through the Rapid Viscosity Analyser (RVA).

The result showed that the averages of peak viscosity, breakdown and peak time are 208.8,115.7 and 3.8, respectively, while those of Kanto107, one of the parents, are 314.6,126.1 and 6.3, respectively. Similar to the results of other reports, major paste properties of waxy wheat lines are lower than their parents. Furthermore, a STS-marker and a SSR-marker were used to tag the waxy gene. A 440 bp band was obtained with the STS-marker if the Wx-B1 gene is normal, while a 265 bp band and a 204 bp band were amplified with the SSR-marker if the Wx-A1 and Wx-D1 genes are normal, respectively. The results show that the two markers are very useful to the molecular marker assisted noodle wheat breeding. Further researches are under a way in our lab. These studies will enable us to determine the composition of waxy proteins of the current major wheat varieties in China and have great significance in the assisted selection of wheat breeding.

Rice seedling characteristics largely determines its grain yield. A better genetic understanding of the seedling characteristics in rice could be helpful to improve variety. In this study, a population of 240 recombinant inbred lines derived from a cross between Zhenshan 97 and Minghui 63 was used to identify QTLs for seedling characteristics. All materials were grown by hydroponic culture. Data for the following characters were collected at 25 and 35 days after germination: plant height (PH), tillers per plant (TP), shoot dry matter weight (SDW), root length (RL), number of root (NR), root dry weight (RDW) and the seedling character index (SCI, the ratio of SDW weight to PH). Analysis using composite interval mapping detected 15 QTLs for the seven traits of 25 days old seedlings, Zhenshan 97 alleles at fourteen of the QTLs increased trait values. The QTL in the vicinity of R3166 on chromosome 5 simultaneously influenced SDW, RL, NR, RDW and SCI with the same direction. Twenty four QTLs were detected for the seven traits of the 35 days old seedlings. However, Minghui 63 alleles at 13 of the QTLs increased trait values. The QTL linked to R3166 was also identified affecting the SDW, RL, NR and RDW. Only 5 QTLs were commonly detected at two stages. The QTL analysis also clearly indicated that Zhenshan 97 alleles offered positive effects in the first 25 days of seedling growth, while after that the positive effects of Minghui 63 alleles on seedling growth gradually became pronounced.

Germination rate and seedling growth were measured to quantify seedling vigor. Total amylase activity, a-amylase activity, reducing sugar content, root activity, and seed weight were determined. Correlations were observed between the seedling vigor and physiological traits. A total of 31 QTLs were detected for the five seedling vigor traits, of which eight were for germination rate, eight for total dry weight, seven for shoot dry weight, four for root dry weight, and four for maximum root length, accounting for 54.7%, 58.0%, 52.7%, 47.5%, and 44.6% of phenotypic variations, respectively. Three QTLs were detected for total amylase activity, two for a-amylase activity, six for reducing sugar content, three for root activity, and nine for seed weight, explaining 20.5%, 14.4%, 51.9%, 27.2%, 64.9% of phenotypic variation, respectively.

Generally, QTL analysis reveals that the intervals of RG393-C1087-RZ403 on chromosome 3, C246- RM26-C1447 and R830- R3166-RG360-C734b on chromosome 5, and the interval of Waxy on chromosome 6 are the four main chromosomal regions controlling seedling vigor. Several QTLs for amylase activities, reducing sugar content, and root activity were localized in the similar regions as the QTLs for seedling vigor. The results suggest that these traits were under the control of pleiotropic and/or closely linked QTLs. In summary, the study demonstrated that QTL mapping for biochemical or metabolic characteristics might provide a strategy for analyzing the physiological basis of morphological trait variation. The co-localization of QTLs for seedling vigor and physiological characters may be considered as the explanation for the genetic basis of the correlation between the two sets of traits in rice.

Stay-green refers to a process in which yellowing was delayed for a longer time compared with a standard reference genotype. It often conferred plant some extra beneficial characteristics such as resistance to lodging, diseases, drought stress, etc. Stay-green of leaves can keep plants vigorous in photosynthesis during the later growth period, and therefore may lead to increase of yield. This study was intended to investigate QTLs for leaf stay-green traits in order to use them in rice to prolong the photosynthetic time. The materials included a population made up of 190 DH lines derived from the cross between an indica rice cultivar Zhenshan 97 and a japonica rice cultivar Wuyujing 2, the former yellowed rapidly and the latter yellowed slowly which was regarded a stay-green genotype. A total of 174 polymorphic SSR markers were used for constructing the genetic marker linkege map. The following observations were used as measurements for evaluating stay-green of the leaves: visual rate of green area retention (rgaf, rgas), retention of the green degree of the first and second leaf measured by SPAD-502 (rdgf, rdgs), retention of chlorophyll content (including rchlaf, rchlas, rchlbf, rchlbs, rchlf, rchls) for the first and second leaf at 30 d after heading, the date of flag (dfl) and second leaf (dsl) completely yellowed, and some other traits derived from the above measurements. A total of 52 QTLs, located on 2, 3, 4, 6, 7, 8, 9, 10 chromosomes, were detected for these traits. Many QTLs were overlapping with each other indicating the possibility that common pathways governed the different respects of senesce. The QTLs with relatively big effects included rchlbs1 located at RM324-RM301 on chromosome 2, rgaf1 located at RM82-RM125 on chromosome 7, dfy1, dsl1 located at RM125-RM2 on chromosome 7, dfy2, dsl2 at RM118-RM248 on chromosome 7, rrdgs1 (which related to the retention rate of the degree of the second leaf green derived from rdg/dg, where dg referred to the degree of leaf green measured by SPAD-502 at head date.) located at RM30-RM340 on chromosome 6. They explained 35.04%, 28.04%, 27.38%, 34.1%, 20.1%, 23.26% and 23.34% the phenotypic variations, respectively. The QTL located at RM125-RM2 also controlled heading date, plant height, the maturity date. These results were based on the data in 2001 and repeated experiment in 2002 was underway. The QTLs consistently detected in two years would be very meaningful for the improvement of stay-green traits.

Wheat sharp eyespot caused by Rhizoctonia cerealis is one of the serious diseases in China, which has resulted in great loss of wheat grain yield. Immune variety has not been found in both cultivated wheat and related wild species. However, considerable variation exists among different wheat germplasm for quantitatively resistance to this fungus. To map and localize resistance QTLs associated with disease using quantitative trait loci (QTLs) analysis can contribute to providing theoretical guide to breeding for disease resistance and the basis on which molecular marker-assisted selection can be implemented.

In this study, two mapping populations were used to analyze QTLs associated with the resistance to R. Cerealis. The population of (Opata85×W7984) recombinant inbred lines was kindly provided by ITMI. Another population is (Wenmai6×Shanhongmai) recombinant inbred lines which was cultured in this study. The marker genotype of this population was assessed with 112 wheat microsatellites and 97 AFLP markers. For the construction of the genetic map, linkage analysis was performed with the program MAPMAKER (Lander et al. 1987), and the map comprised 152 marker loci (3148 cM) with the average marker density of 20.7cM.

Through mixed-model composite interval mapping, six putative QTLs associated with seedling resistance to Rhizoctonia cerealis were detected in the population of (Opata85×W7984), totally accounted for 49.9% of phenotypic variation. Three putative QTLs associated with adult resistance to Rhizoctonia cerealis were detected in the population of (Opata85×W7984) and (Wenmai6×Shanhongmai), respectively. In the prior population, the adult resistance QTL has the same interval with one of seedling resistance QTL located on 7B chromosome accounted for 19.11% of phenotypic variation. In the population of (Wenmai6×Shanhongmai), one QTL under field environments was localized on 2B chromosome, accounted for 13.11% of phenotypic variation, derived from susceptible parent-Wenmai6, another QTL under greenhouse environments was localized on 6B chromosome, accounted for 14.34% of phenotypic variation, derived from resistant parent-Shanhongmai. Several significant digenic interactions associated with seedling and adult resistance were detected in two populations. It is shown that the epistatic effect is one of the main genetic factors contributed to the resistance to Rhizoctonia cerealis.

Key words: Wheat Sharp eyespot; QTL

Aili Li, Jizeng Jia

The phenomenon of heterosis or hybrid vigor is poorly understood despite its perceived importance in evolution and its practical significance in breeding programs that aim to increase yield of crop plants. Previous studies have showed that gene expression in hybrid F₁ alters obviously as compared to their parental inbreds, but for further elucidation of the molecular mechanism of heterosis, it should be to clone and identify the genes that differentially expressed between hybrid F₁ and its parental inbreds.

The above results indicated that heterosis is a complex biology phenomenon, and many kinds of genes may participate in its developing. Therefore, the further study will be to fish the full-length cDNAs of gene expression alteration and analyze their detailed function.

Key words: wheat, heterosis; DDRT-PCR; molecular mechanism.

Badila which was virus free by way of tissue culture and Badila infected SCMV were studied by DD (Differential display), a set of flouresent DD operating method was founded.9 differentially expressed cDNA fragments were obtained by this set of operating method and selected 23 primer combinations. The size of 9 fragments was between 250 bp and 350 bp. These fragments were grouped into two types: 4 differentially expressed cDNA fragments which exist or not in the two compared experimental materials; 5 differentially expressed cDNA fragments which had the intensity difference between the two compared experimental materials. 5 fragments were obtained from Badila which was virus free, and 4 fragments were obtained from Badila infected SCMV.The 9 fragments were amplified by agarose gel again and had the same size as the fragments in the fluoresent DD Gel. Some influence factors of DD were discussed.

Grain hardness is one of the most important characters in wheat quality improvement. Based on the homologues of Pina and Pinb gene, which are major genes controlling the grain hardness in cereal crops, two pairs of degenerate primers were designed. The specific fragments of about 450bp in size were obtained, respectively, after PCR amplification using the genomic DNA of wheat variety Jing411 as template. Then they were cloned into vector pGEM-3Z f(+), and sequenced after the identification of the recombinants and endonuclease analysis. The results indicated that, compared with the Pina gene from wheat variety Heron, the Pina gene in Jing411 shared 98.9% and 98% homology in nucleotide acid sequence and amino acid sequence, respectively. The whole length of Pina gene was 447bp, encoding 149 amino acid residues, having the signal peptide of 19aa and the tryptophan-rich domain (WWKWWK) which are specific to Pina gene in cereal crops. Similarly, the Pinb gene shared 99.8% and 99.3% homologies in nucleotide acid sequence and amino acid sequence respectively compared with the cloned Pinb gene in soft wheat variety Heron. The whole length of Pinb gene was 447bp,encoding 149 amino acid residues, having the signal peptide of 19aa and the tryptophan-rich domain (KWWK) which were specific to Pinb gene in cereal crops. The isolation of Pina and Pinb genes laid a fundamental basis for further improvement of the grain hardness of wheat variety through genetic engineering. At present, these two genes have been constructed into the bacterial expression vector and expressed in the E coli (BL21). Construction of the high efficient monocot plants expression vector harboring these two genes, its antisense genes and the fusions of these two genes and wheat transformation via pollen tube pathway and biolastic mediated transfer of these genes are under way.

Key words: Wheat; Grain hardness; Gene cloning; Tryptophan-rich domain

Shen Wenbiao, Wang Ren, Wang Yihua, Wan Jianlin, Zhai Huqu and Wan Jianmin*

loxE1 transcript levels in various tissues, such as embryo, stem and leaves, were investigated by Northern blot analysis of total RNA samples. The results showed loxE1 conferred embryo-specific expression pattern. On the other side, southern blot analysis was performed using above loxE1 as probe. Several bands of strong intensity were observed with three different restriction enzymes, indicating that several other Lox family members occur in rice.

The majority of R genes are the type of nucleotide binding site-leucine rich repeat (NBS-LRR). The conserved motifs in NBS domain had been used to isolate resistance gene analogs (RGAs) from soybeans and other plants. In the present study, The RGAs were used to clone R gene candidates from Kefeng 1, a soybean cultivar resistant to soybean mosaic virus (SMV) and soybean cyst nematode (SCN).

The oligonucleotide primers were designed according to the tobacco R gene N and Arabidopsis R gene RPS2. The PCR products from Kefeng 1 were cloned and 358 positive clones were isolated. Four open-reading NBS analogs were finally characterized and designated as KNBS1, KNBS2, KNBS3 and KNBS4.

In order to clone R genes from soybean, a high titer of soybean cDNA library (4.2´ 10⁷Pfu/mL) was constructed. The library was screened using the mixed probes of the resistance analogs mentioned above. Four positive clones were obtained, which were designated as KR1 , KR2, KR3 and KR4.

The length of KR1 was 3672bp with a complete open reading frame encoding a protein of 1124 amino acids which consisted of a Toll/Interluekin receptor (TIR) domain, a nucleotide binding site (NBS) domain, a leucine-rich repeats (LRR) domain and two C-terminal transmembrane segments. The KR1-related sequences were present as 2-4 copies within the soybean genome and two of them were mapped onto L linkage group. The transcripts of KR1 gene were detected in roots, hypocotyls, cotyledons, epicotyls and leaves. The gene appeared to be induced by salicylic acid treatment and soybean mosaic virus infection. Like N and L6, the KR1 gene gave rise to two mRNAs via alternative splicing. The larger one (KR1, 609bp) was unique transcript in resistance parent (Kefeng1); the smaller one (NR1^tr, 588bp) that aroused via alternative splicing was predominant in susceptible cultivar Nannnong1138-2 under normal condition. When the plants were inoculated with SMV, the transcripts were all accumulated positively in both parents but the ratio of NR1/NR1^tr transcripts was reversed in susceptible parent . These results demonstrated that KR1 is possiblely a functional resistance gene in infection of soybean mosaic virus and mediated by salicylic acid.

The KR2 , KR3 and KR4 were incomlete ORFs. Using 5’ RACE, the whole sequences of KR3 and KR4 were acquired. KR3 (AF502079) was 2,353 bp in length including a 41 bp poly-A tail, and encoded a protein of 636 amino acids. Structural analysis revealed that it had conserved motifs of R genes such as TIR and NBS, but no LRR domain. KR3 shared 28.1% amino acid sequence identity with N, suggesting that they might arise from close ancestral genes before speciation. Southern blot analysis showed that KR3 was probably in single- or low-copy numbers in soybean genome. Under normal condition, KR3 was expressed in a relatively low level in soybean leaf. However, application of 2.0 mmol/L SA to the plants resulted in accumulating of KR3 mRNA. The mRNA level reached a peak at 36 h after initiation of the SA treatment and then began to decline.

KR4 (AF502079) was 3,818 bp in length encoding a protein of 1211 amino acids. It had only conserved motifs of NBS and shared 16.0% amino acid sequence identity with Xa1 of rice.

Key words: Momordica charantia; chitinase; gene cloning

Brassinosteroids (BRs) are natural growth-promoting products found at low levels in pollen, seeds, and young vegatative tissues throughout the plant kingdom. Recently, the notion that BRs are essential for plant growth and development has been widely accepted by the discovery of BR dwarf mutants of Arabidopsis, pea and tomato. The observation that only BRs could rescue the mutant phenotypes to the wild type provided convincing evidence that BRs are indeed essential plant hormones for normal plant growth and development. Cell elongation is developmental process that is regulated by light and phytohormones and is of critical importance for plant growth. The dwarfed phenotype results from a failure in normal cell elongation. The cells of BR dwarf mutants are far shorter than the corresponding wild-type cells. Furthermore, exogenous application of BRs could elongate the cells and rescue the dwarfed phontype. These confirmed the role of BRs in cell elongation. However, the mechanisms of BRs acting on cell elongation, especially in cotton (Gossypium hirsutum) fiber development are largely unknown. The fibers of cotton are single-cell trichomes that result from elongation of epidermal cells of the ovule and undergo rapid and synchronous elongation. Because the longation occurs at a fast rate over a relatively long period, uninterrupted by cell division, The fibers of cotton are a good experimental system for studying cell elongation. In addition, changes in the cell wall structure of elongating cotton fibers have been well characterized.

Funded by the National Natural Science Foundation of China (Grant No. 30170588 )

The fiber cells of cotton (Gossypium Hirsuturm L.) represent a unique cell type that undergoes a period of very rapid elongation. Fiber cells are also single-celled trichomes, which arise in near synchrony from the epidermis of the ovule and may elongate at peak rates in excess of 2mm d^-1 during the rapid polar-expansion phase of development. So the cotton fibers are a good experimental system for studying cell elongation.

Funded by the National Natural Science Foundation of China (Grant No. 30170588 )

Funded by the National Natural Science Foundation of China (Grant No. 39870532 )

The isolation of flanking sequences is a crucial step in the study of gene cloning and expression. On the basis of Y-shaped adaptor (Prashar and Weissman, 1996), we designed a new method which could be used to amplify the flanking region from both genomic DNA and cDNA. This method was designated as Y-shaped adaptor dependent extesion (YADE). The schematic of this method is showed in Fig.1.

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