Experimental validation of inter-subspecific genetic diversity in rice represented by the differences between the DNA sequences of ‘Nipponbare’ and ‘93-11’

The pedigrees of three sequenced rice cultivars were analyzed to show that a majority of the genetic composition of 'Nipponbare' originates from japonica cultivars while the minority originates from indica cultivars. In contrast, '93-11' is derived mainly from indica cultivars with a smaller contribution from japonica cultivars. All ancestors of 'Guang lu ai 4' appeared to be indica lines. A set of molecular markers (46 InDels and 53 SSRs) polymorphic between 'Nipponbare' and '93-11' were examined in 46 typical indica and 47 typical japonica cultivars selected from 443 accessions according to Cheng's index. All cultivars were divided into indica and japonica groups without overlapping when clustered by Cheng's index, InDels and SSRs. Much higher InDel and SSR diversity between groups than within groups implies that the marker polymorphisms between 'Nipponbare' and '93-11' represent a large proportion of inter-subspecific diversity. About 85% of indica cultivars and more than 90% of japonica cultivars were confirmed to have the same PCR banding patterns as '93-11' and 'Nipponbare', respectively. Some polymorphic loci between 'Nipponbare' and '93-11' cannot be validated in other indica and japonica cultivars, either as subspecies-specific but not predominant alleles, or alleles not specific between the two groups. It was concluded that molecular markers developed from sequence polymorphism between 'Nipponbare' and '93-11' often represent inter-subspecific diversity, although some exceptions were sensitive to either particular marker loci or particular cultivars.     

Genetic structure and diversity of Oryza sativa L. in Guizhou, China

Preserving many kinds of rice resources and rich variations, Guizhou Province is one of the districts with the highest genetic diversity of cultivated rice （Oryza sativa L.） in China. In the current research, genetic diversity and structure of 537 accessions of cultivated rice from Guizhou were studied using 36 microsetellite markers and 39 phenotypic characters. The results showed that the model-based genetic structure was the same as genetic-distance-based one using SSRs but somewhat different from the documented classification （mainly based on phenotype） of two subspecies. The accessions being classified into indica by phenotype but japonica by genetic structure were much more than that being classified into japonica by phenotype but indica by genetic structure. Like Ding Ying＇s taxonomic system of cultivated rice, the subspecific differentiation was the most distinct differentiation within cultivated rice. But the differentiation within indica or japonica population was different： japonica presented clearer differentiation between soil-watery ecotypes than indica, and indica presented clearer differentiation between seasonal ecotypes than japonica. Cultivated rices in Guizhou revealed high genetic diversity at both DNA and phenotypic levels. Possessing the highest genetic diversity and all the necessary conditions as a center of genetic diversity, region Southwestern of Guizhou was suggested as the center of genetic diversity of O. sativa L. from Guizhou.     

Genetic diversity of Chinese summer soybean germplasm revealed by SSR markers

There are abundant soybean germplasm in China. In order to assess genetic diversity of Chinese summer soybean germplasm, 158 Chinese summer soybean accessions from the primary core collection of G max were used to analyze genetic variation at 67 SSR loci. A total of 460 alleles were detected, in which 414 and 419 alleles occurred in the 80 Huanghuai and the 78 Southern summer accessions, respectively. The average number of alleles per locus was 6.9 for all the summer accessions, and 6.2 for both Huanghuai and Southern summer accessions. Marker diversity (D) per locus ranged from 0.414 to 0.905 with an average of 0.735 for all the summer accessions, from 0.387 to 0.886 with an average of 0.708 for the Huanghuai summer accessions, and from 0.189 to 0.884 with an average of 0.687 for the Southern summer accessions. The Huanghuai and Southern summer germplasm were different in the specific alleles, allelic-frequencies and pairwise genetic similarities. UPGMA cluster analysis based on the similarity data clearly separated the Huanghuai from Southern summer soybean accessions, suggesting that they were different gene pools. The results indicate that Chinese Huanghuai and Southern summer soybean germplasm can be used to enlarge genetic basis for developing elite summer soybean cultivars by exchanging their germplasm.     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Study on heterosis of intersubspecies between indica and japonica rice （Oryza sativa L.） using chromosome segment substitution lines

Great heterosis exists in the inter-subspecific crossesbetween indica and japonica rice cultivars[1,2] and the ex-ploitation of this heterosis has long been considered as apromising method to further increase rice yield potential[3].Previous studies indicated that there were large differencesbetween the two subspecies with respect to morphology,isozyme, polymorphism of molecular markers and DNAstructure due to long time genetic differentiation[4,5]. Thosedifferences were identified to be clos…     

Identifying neutral allele Sb at pollen-sterility loci in cultivated rice with Oryza rufipogon origin

Pollen sterility is commonly found in the intra-specific hybrids of indica and japonica rice, which is one of the main constrains for the utilization of heterosis between indica and japonica. Six loci controlling the pollen sterility of F1 between indica and japonica have been identified from previous studies. Neutral alleles at each locus are potential to overcome the F1 pollen sterility associated with the locus. Therefore, exploitation and utilization of neutral alleles are of significant importance. The present research was based on fine mapping of the F1 pollen-sterility gene Sb and the abundant genetic diversity of Oryza rufipogon Griff. indigenous to Gaozhou, Guangdong Province （referred to as Gaozhou wild rice）. Crosses were made using Taichung65 （with the genotype of Sb^jSb^j and referred to as El） and its near-isogenic line of F1 pollen sterility gene Sb（with the genotype of Sb^iSb^i, E2） as female parents, and 12 different accessions of Gaozhou wild rice as male parents. F1 pollen fertility was examined to identify the materials having the neutral alleles at the F1 pollen-sterility locus. Segregation of 4 molecular markers tightly linked with the Sb locus was analyzed in the F2 populations derived from the FlS carrying the neutral gene. The pollen fertility related to the 3 genotypes of the molecular markers was also checked by statistical test to determine whether it was consistent with the hypothesis. The results showed that the pollen fertility of two F1s from one accession of Gaozhou wild rice （GZW099） with E1 and E2 was （89.2±21.07）% and （85.65±1.05）%, respectively. Both of them were fertile and showed no significant difference by t-test. Segregation of the 3 genotypes of the 4 molecular markers followed the expected Mendelian ratio （1：2：1） in the F2 populations. There was no significant difference for the averaged pollen fertility of the plants related to the 3 genotypes, suggesting that no interaction exists between the alleles at the Sb locus in GZW099 and Taichung65 or E2. Evidentially, GZW099 carried the neutral gene （named Sb″Sb″） at the Sb locus, which provides valuable theoretical basis and resources for further studying and overcoming the sterility of indica-japonica hybrids.     

Advances in the understanding of inter-subspecific hybrid sterility and wide-compatibility in rice

Hybrid sterility is a major form of postzygotic reproductive isolation and frequently occurs in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice （Oryza sativa L.）. It has been a major barrier for utilization of the strong heterosis expressed in hybrids between indica and japonica. A large number of loci for rice inter-subspecific hybrid sterility have been identified by genetic analysis. Cytological studies revealed that male and female gamete abortions and reduced affinity between the uniting gametes all occurred in indica-japonica hybrids, suggesting the complexity of the causes for inter-subspecific hybrid sterility. Two genes conditioning embryo-sac and pollen sterility respectively in indica-japonica hybrids have been cloned recently, providing opportunities for molecular characterization of the indica-japonica hybrid sterility and wide-compatibility. Future studies should aim at cloning more genes for indica-japonica hybrid sterility, characterizing the underlying molecular mechanism, and utilization of the findings for the development of inter-subspecific hybrids to increase rice productivity.     

Development, chromosome location and genetic mapping of EST-SSR markers in wheat

A number of 151695 wheat expression sequence tags （ESTs） that originated from GenBank/dbEST from July 14, 2003 to August 24, 2004 were used to search for simple sequence repeats （SSRs） with motif 2-5 bp, and 2038 simple sequence repeats （EST-SSRs）, which accounted for 1.34% of EST database, were identified. Based on these SSR sequences, 249 EST-SSR primer pairs and 166 amplified clear bands in various wheat cultivars were designed. These EST-SSR markers can be used as new molecular markers in wheat and related species. Using Chinese Spring nulli-tetrasomic lines, 93 EST-SSR primer pairs and 193 EST-SSR loci were located on 19 wheat chromosomes except for 4A and 4B. Forty-three loci were mapped on 11 chromosomes of the genetic framework map previously constructed using recombinant inbred lines.     

Genetic linkage map of Brassica campestris L.using AFLP and RAPD markers

A genetic linkage map comprised of 131 loci was constructed with an F2 population derived from an inter-subspecific cross between Brassica campestris L.ssp.chinensis cv.aijiaohang” and ssp.rapifera cv.,”‘isihai”.The genetic map included 93 RAPD loci,36 AFLP loci and 2 morphological loci organized into 10 main linkage groups(LGs) and 2 small groups,covering 1810.9cM with average distance between adjacent markers being approximately 13.8cM.The map is suitable for identification of molecular markers linked to important agronomic traits.QTL analysis,and even for marker-assisted selection in breeding programs of Chinese cabbage and turnip.     

Rapid genome evolution in Pms 1 region of rice revealed by comparative sequence analysis

Pms1, a locus for photoperiod sensitive genic male sterility in rice, was identified and mapped to chromosome 7 in previous studies. Here we report an effort to identify the candidate genes for Pms1 by comparative sequencing of BAC clones from two cultivars Minghui 63 and Nongken 58, the parents for the initial mapping population. Annotation and comparison of the sequences of the two clones resulted in a total of five potential candidates which should be functionally tested. We also conducted com-parative analysis of sequences of these two cultivars with two other cultivars, Nipponbare and 93-11, for which sequence data were available in public databases. The analysis revealed large differences in sequence composition among the four genotypes in the Pms1 region primarily due to retroelement activity leading to rapid recent growth and divergence of the genomes. High levels of polymorphism in the forms of indels and SNPs were found both in intra- and inter-subspecific comparisons. Dating analysis using LTRs of the retroelements in this region showed that the substitution rate of LTRs was much higher than reported in the literature. The results provided strong evidence for rapid genomic evolution of this region as a consequence of natural and artificial selection.     

RAPD Analysis for Genetic Variation within the Endangered Quillwort Isoetes hypsophila （Isoetaceae）

The genetie variation in the critically endangered species Isoetes hypsophila was investigated using Random Amplified Polymorphism DNA (RAPD) markers. Thirteen primers were screened from sixty primers, and a total of 104 DNA fragments were scored, of which, 52 were polymorphic loci. Low-level genetic diversity within populations with PPB values ranging from 7.69% to 25.96% was found. An Analysis of Molecular Variance (AMOVA) indicated that the most of variance (78.30%) occurred between Yunnan and Sichuan. The variances among populations within regions and within populations were only 3.89% and 17.82%, respectively.     

Comparative mapping of rice root traits in seedlings grown in nutrient or non-nutrient solution

The component and amount of nutrient in the growth medium are the major factors affecting root growth.For the systematic dissection of root gene expression,evaluation of nutrient and non-nutrient solutions was conducted for their effect on root traits and quantitative trait loci(QTL)mapping.Three rice root parameters,maximum root length(MRL),root dry weight(RDW),and root/ shoot ratio of dry weight(RSR),were characterized within a double haploid(DH)population from a cross of ZYQ8(indica)and JX17(japonica).The value of the three root traits in two parents all decreased under the nutrient condition compared to those under the nonnutrient condition,of which RSR decreased up to 2.6-fold on average.In the DH population,more than 70 % lines in MRL,94 % lines in RDW,and all the lines in RSR were scored lower.In total,eight QTLs were identified in nutrient system(5 from JX17 alleles and 3 from ZYQ8 alleles)while five QTLs were detected in non-nutrient system(4 from JX17 alleles and 1 from ZYQ8 alleles).Of them,one QTL for RSR was shared by both culturing systems,seven QTLs were specific in nutrient system and the other four QTLs were specific in non-nutrient system.All 13 QTLs were distributed over 7 rice chromosomes-2,3,4,5,6,9 and 10,respectively.     

Genetic analysis and molecular mapping of a presenescing leaf gene psll in rice （Oryza sativa L.）

A rice psl1 （presenescing leaf） mutant was obtained from a japonica variety Zhonghua 11 via radiation of ^60Co-γ in M2 generation. Every leaf of the mutant began to wither after it reached the biggest length, while the leaves of the wild variety could keep green for 25--35 d. In this study, genetic analysis and gene mapping were carried out for the mutant identified. The SSR marker analysis showed that the mutant was controlled by a single recessive gene （psl1） located on chromosome 2. Fine mapping of the psl1 locus was conducted with 34 new STS markers developed around psl1 anchored region based on the sequence diversity between Nipponbare and 93-11. The psl1 was further mapped between two STS markers, STS2-19 and STS2-26, with genetic distances of 0.43 and 0.11 cM, respectively, while cosegregated with STS2-25. A BAC contig was found to span the psl1 locus, the region being delimited to 48 kb. This result was very useful for cloning of the psl1 gene.     

Genome-wide identification of R genes and exploitation of candidate RGA markers in rice

By scanning the whole genomic sequence of japonica rice using 45 known plant disease resistance (R) genes, we identified 2119 resistance gene homologs or analogs (RGAs) and verified that RGAs are not randomly distributed but tend to cluster in the rice genome. The RGAs were classified into 21 families according to their functional domain based on Hidden Markov model (HMM). By comparing the RGAs of japonica rice with the whole genomlc sequence of indica rice, we found 702 RGAs allelic between the two subspecies and revealed that 671 (95.6%) of them have length difference (InDels) in their genomic sequences (including coding and non-coding regions) between the two subspecies, suggesting that RGAs are highly polymorphic between the two subspecies in rice. We also exploited 402 PCR-based and co-dominant candidate RGA markers by designing primer pairs on the regions flanking the lnDels and validating them via e-PCR. The length differences of the candidate RGA markers between the two subspecies are from 1 to 742 hp, with an average of 10.26 hp. All related information of the RGAs is available from our web site(http://ibi.zju.edu.cn/RGAs/index.html).     

Genetic relationships among Chinese and American isolates of Phytophthora sojae assessed by RAPD markers

The genetic diversity of three geographic populations of Phytophthora sojae from China and the United States was determined using random amplified polymorphic DNA （RAPD）. The purpose was to explore genetic relationships among Chinese and American isolates of the organism. 21 random primers were selected among 200 random primers screened. A total of 223 reproducible RAPD fragments were scored among 111 individuals, of which 199 （89.23%） were polymorphic. Analysis of genetic variation showed that there existed higher genetic variation in the United States population in comparison to the Chinese populations. Nei＇s genetic identity and principal component analysis indicated that the populations of Fujian and United States are closer to each other than to Heilongjiang populations. Shannon-Wiener diversity index revealed that the United States populations have a higher genetic di- versity than that of Chinese populations. These data are in support of the hypothesis that P. sojae in the United States might not have been introduced from China.     

Characteristics of the short rachillae of rice from archaeological sites dating to 7000 years ago

The abscission layer formed on a pedicel situated at the basal part of a short rachilla is an important characteristic for discriminating between wild, japonica, and indica rice. The short rachillae of paddy rice grains excavated from the Kuahuqiao, Luojiajiao, and Tianluoshan sites, located in the lower reaches of the Yangtze River and dating to 7000 years old, were observed. The results showed that the short rachillae could be divided into two types: a wild type and japonica type. These results indicated that the rice had been domesticated, but was a primitive cultivated rice that retained some of the characteristics of wild rice. The results also suggested that the rice was changing to resemble japonica type rice. Based on the ratios of wild and japonica types, it was inferred that rice domestication began 10000 years ago.     

Genetic diversity, geographic differentiation and evolutionary relationship among ecotypes of Glycine max and G. soja in China

The knowledge of origin and evolution of cultivated soybeans is one of the basic issues in both biology and agronomy of the crop. In order to investigate the nuclear and cytoplasmic genetic diversity, geographic differentiation and genetic relationship among geographic ecotypes of cultivated （Glycine max） and wild （G. soja） soybeans, the allelic profiles at 60 nuclear simple-sequence repeat （nuSSR） loci and 11 chloroplastic SSR （cpSSR） loci evenly distributed on whole genome of 393 landraces and 196 wild accessions from nation-wide growing areas in China were analyzed. （i） The genetic diversity of the wild soybean was obviously larger than that of the cultivated soybean, with their nuSSR and cpSSR alleles as 1067 vs. 980 and 57 vs 44, respectively. Of the 980 nuclear alleles detected in the cultivated soybean, 377 new ones （38.5%） emerged, while of the 44 chloroplastic alleles in the cultivated soybean, seven new ones （15.9%） emerged after domestication. （ii） Among the cultivated geographic ecotypes, those from southern China, including South-Central China, Southwest China and South China possessed relatively great genetic diversity than those from northern China, while among the wild geographic ecotypes, the Middle and Lower Changjiang Valleys wild ecotype showed the highest genetic diversity. （iii） The analysis of molecular variance, association analysis between geographic grouping and molecular marker clustering and analysis of specific-present alleles of ecotypes demonstrated that the geographic differentiation of both cultivated and wild soybeans associated with their genetic differentiation, or in other words, had their relevant genetic bases. （iv） The cluster analysis of all accessions clearly showed that the wild accessions from Middle and Lower Changjiang Valleys and South-Central ＆ Southwest China had relatively small genetic distances with all cultivated accessions. The UPGMA dendrogram among geographic ecotypes further showed that the genetic distances between all cultivated ecotypes and the Middle and Lower Changjiang Valleys wild ecotype were smaller than those with other wild ones, including their local wild counterparts. Therefore, it is inferred that the wild ancestors in southern China, especially those from Middle and Lower Changjiang Valleys might be the common ancestor of all the cultivated soybeans.     

Genetic structure and eco-geographical differentiation of cultivated Hsien rice (Oryza sativa L. subsp. indica) in China revealed by microsatellites

Indica is not only an important rice subspecies widely planted in Asia and the rest of the world,but it is also the genetic background of the majority of hybrid varieties in China.Studies on genetic structure and genetic diversity in indica germplasm resources are important for the classification and utilization of cultivated rice in China.Using a genetically representative core collection comprising 1482 Chinese indica landraces,we analysed the genetic structure,geographic differentiation and diversity.Model-based structure analysis of varieties within three ecotypes revealed nine eco-geographical types partially accordant with certain ecological zones in China.Differentiation of eco-geographical types was attributed to local ecological adaption and physical isolation.These groups may be useful for developing heterotic groups of indica.To facilitate the identification of different ecotypes and eco-geographical types,we identified characteristic SSR alleles of each ecotype and eco-geographical type and a rapid index of discrimination based on characteristic alleles.The characteristic alleles and rapid discrimination index may guide development of heterotic groups,and selection of hybrid parents.     

Genetic diversity in Penaeus chinensis shrimp as revealed by RAPD technique

The random amplified polymorphic DNA analysis was used to estimate genetic diversity in one successively cultivated stock and three wild stocks of Penaeus chinensis shrimp, two of which were collected from the spawning and wintering grounds in the west coast of Korean Peninsula, and one from the feeding ground in the China coast of the Yellow Sea. A random primer kit was employed to scan the genomic DNA in 20 individuals of each index stock. A total of 110 reproducible RAPD markers were obtained, 68.2 % of which showed a sound eonformability within all the individuals detected, implying that the genetic variability in P. chinensis is relatively low. The proportions of polymorphic loci among these four stocks ranged from 20% to 33.3%, while the degrees of genetic polymorphisms varied from 0.0093 to 0.0307. The genetic variability of inter-stocks was higher than that of intra-stock. The genetic diversity in different stocks differed from each other; that is, a less genetic differentiation in the spawning and wintering stocks from the west coast of Korean Peninsula was revealed and their genetic diversities were higher than that of the spawning stock in the Bohai Sea and the China coast of the Yellow Sea. As detected, the genetic diversity in the successively cultivated stock was the lowest among these four stocks. Through genetic distance analysis between a random pair of individuals, a dendrogram of the above-mentioned four stocks was constructed by unweighted pair group method with arithmetic mean. The results based on cluster analysis well fitted with the geographical distribution of P. chinensis in the Bohai and Yellow Seas.     

Localization of genes for lateral branch and female sex expression and construction of a molecular linkage map in cucumber (Cucumis sativus L. ) with RAPD markers

A cucumber ( Cucumis sativus L. ) molecular linkage map, including 79 random-amplified polymorphic DNAs (RAPD)and two genes , lb for lateral branch and f for female sex expression, is constructed from a cross between a line, S52, with weak lateral growing ability and staminate from Dabieshan Mountains area in China and another line, S06, with strong lateral growing ability and gynoecious from Europe. The map contains nine linkage groups and spans 1110.0 cM with an average distance of 13.7 cM between loci. The lb locus is located in a longer linkage group LG-2 and flanked by two markers, OP-Q5-1 and OP-M-2-2, at 9.3 cM and 15.9 cM, respectively. In the meantime, the RAPD loci, OP-Q5-2 and BC151, in a short linkage group were found to flank f at 13.7 cM and 13.4 cM,respectively. The construction of RAPD map has paved a way for further study of the genes for lateral branch, female sex expression and other agronomic traits in cucumber.