Characterization of the Soybean Genome Using EST-derived Microsatellite Markers

We generated a high-density genetic linkage map of soybean usingexpressed sequence tag (EST)-derived microsatellite markers.A total of 6920 primer pairs (10.9%) were designed to amplifysimple sequence repeats (SSRs) from 63 676 publicly availablenon-redundant soybean ESTs. The polymorphism of two parent plants,the Japanese cultivar ‘Misuzudaizu’ and the Chineseline ‘Moshidou Gong 503’, were examined using 10%polyacrylamide gel electrophoresis. Primer pairs showing polymorphismwere then used for genotyping 94 recombinant inbred lines (RILs)derived from a cross between the parents. In addition to previouslyreported markers, 680 EST-derived microsatellite markers wereselected and subjected to linkage analysis. As a result, 935marker loci were mapped successfully onto 20 linkage groups,which totaled 2700.3 cM in length; 693 loci were detected usingthe 668 EST-derived microsatellite markers developed in thisstudy, the other 242 loci were detected with 105 RFLP markers,136 genome-derived microsatellite markers, and one phenotypicmarker. We examined allelic variation among 23 soybean cultivars/linesand a wild soybean line using 668 mapped EST-derived microsatellitemarkers (corresponding to 686 marker loci), in order to determinethe transferability of the markers among soybean germplasms.A limited degree of macrosynteny was observed at the segmentallevel between the genomes of soybean and the model legume Lotusjaponicus, which suggests that considerable genome shufflingoccurred after separation of the species and during establishmentof the paleopolyploid soybean genome.     

Genetics of actin-related sequences in tomato

Summary The genomic distribution of actin-related sequences in tomato was investigated using a cloned actin gene from soybean. Ten actin loci account for most of the hybridizing fragments observed with Southern analysis. Single loci were found on chromosomes 1, 3 and 10 and two loci on chromosome 4. One locus is linked to an unmapped isozyme marker, Sod-1. The four remaining actin loci are independent of each other and of any of the other markers tested. The number of actin loci in tomato (10) is greater than that estimated for soybean (8). As soybean is apparently a tetraploid and tomato a diploid, these results suggest that the number of actin loci has not been stable during the evolution of dicots. A number of these mapped loci lie in regions of the genome previously devoid of molecular markers and thus may be useful in basic and applied genetic research.     

河北省大豆地方品种遗传基础

以50份河北省大豆地方品种为材料,通过两年对12个农艺性状鉴定和30个SSR位点的分析,结果表明,地方品种农艺性状平均多样性指数PIC为0.83,数量性状平均变异系数为20.15%,30个SSR位点平均等位变异数5.03,平均多样性指数PIC为0.61,参试材料遗传距离平均0.869。利用Bayesian模型将参试材料分为4组。分析结果表明,河北省地方品种的遗传多样性与地理来源或不同熟期类型间没有显著相关。     

河北省大豆地方品种遗传基础

以50份河北省大豆地方品种为材料,通过两年对12个农艺性状鉴定和30个SSR位点的分析,结果表明,地方品种农艺性状平均多样性指数为0．83,数量性状平均变异系数为20．15％,30个SSR位点平均等位变异数5．03,平均多样性指数为0．61,参试材料遗传距离平均0．869。利用Bayesian模型将参试材料分为4组。分析结果表明,河北省地方品种的遗传多样性与地理来源或不同熟期类型间没有显著相关。     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

大豆重要农艺性状的QTL分析

应用栽培大豆科丰1号（♀）和南农1138-2（♂）杂交得到的F9代重组自交系（RILs）群体（201个家系）,构建了含302遗传标记、覆盖2363.8cM、由22个连锁群组成的遗传连锁图谱。采用区间作图法,对该群体的主要农艺性状的调查数据进行QTL分析,表明与开花期、成熟期、株高、主茎节数、每节荚数、倒状性、种子重、产量、蛋白质和含油量等10个重要农艺性状连锁的QTL位点34个,每个数量性状的遗传变异是由多个QTL位点决定的。与产量有关的农艺性状的一些QTL集中在几个连锁群上。     

THE GENETIC BASIS OF DIFFERENCES IN GROWTH AND BEHAVIOR OF SPECIALIST AND GENERALIST HERBIVORE SPECIES: SELECTION ON HYBRIDS OF HELIOTHIS VIRESCENS AND HELIOTHIS SUBFLEXA (LEPIDOPTERA)

Two species of moths and their hybrids were studied to determine the genetic basis of host range. One species, Heliothis virescens (HV), has a broad host range and is an agricultural pest on cotton (Malvaceae), soybean (Fabaceae), and tobacco (Solanaceae). The other species, Heliothis subflexa (HS), has a narrow host range, feeding on plants in the genus Physalis (Solanaceae). Experiments were done to determine whether the abilities to feed on cotton, soybean, and tobacco were each under separate genetic control in HV (genetically uncorrelated) or whether feeding on all three hosts was genetically correlated. By repeatedly backcrossing hybrids to HS while selecting for high larval survival and weight on soybean, loci conferring the ability to feed on soybean were moved from HV into the genetic background of the specialist, HS. After six generations of selection on soybean we tested the selected line on soybean, cotton, Physalis, and tobacco to determine if ability to feed on soybean was correlated with feeding ability on these other hosts. We found no evidence to support the hypothesis that feeding on all three hosts was correlated and controlled by a single locus. Compared to HS, the survival and weight of the selected line were significantly higher on soybean, similar on tobacco and Physalis, and slightly, but not significantly, higher on cotton. We also conducted a behavioral choice test to determine if larval preference for soybean was correlated with preference for other hosts. The selected line larvae had higher preference for soybean and cotton than HS. Preference for tobacco was equal in the selected line and in HS. These results support the trends seen in survival and growth tests where performances on soybean and cotton appeared partially correlated and performances on soybean and tobacco were uncorrelated. In conclusion, the generalist, HV, did not have a single set of loci that governed feeding on soybean, cotton and tobacco. It is likely that some of the loci governing performance and preference for soybean also contributed to performance and preference for cotton and that the soybean loci examined did not interact negatively to lower performance and preference for Physalis, the specialist's host. This approach can be applied more widely to address evolutionary questions about host range and other ecological traits.     

Genetically determined enzyme polymorphism in soy varieties (Glycine max) and in wild soy (Glycine soja)

Analysis of 22 genetic-biochemical systems (42 loci) in 18 varieties of domestic soybean (G. max) and in 3 population of wild soybean (G. soja) was carried out. The part of polymorphous loci (P), intraspecies genetic differentiation (genetic distances--DN) were higher in domestic plants in comparison with wild ones (P = 45%, 17%: DN = 0.038-0.269, 0.059-0129). The preferable polymorphism of loci, coding the enzymes of glycolysis and Kreb's cycle was revealed in wild species. Domestic soybean had more polymorphous enzyme loci, which did not participate in glucose metabolism in comparison with wild species. The presence of the specific part of the gene pool in ancestor species, which was involved in soybean domestication and forming of varieties was discussed.     

基于大豆胞囊线虫病抗性候选基因的SNP位点遗传变异分析

以我国363份栽培和野生大豆资源为材料, 对大豆胞囊线虫抗性候选基因(rhg1和Rhg4)的SNP位点(8个)进行遗传变异分析, 以期阐明野生和栽培大豆间遗传多样性及连锁不平衡水平差异。结果表明, 与野生大豆相比, 代表我国栽培大豆总体资源多样性的微核心种质及其补充材料的连锁不平衡水平较高(R²值为0.216)。在栽培大豆群体内, 基因内和基因间分别有100％和16.6％的SNP位点对连锁不平衡显著, 形成两个基因特异的连锁不平衡区间(Block)。在所有供试材料中共检测到单倍型46个, 野生大豆的单倍型数目(27)少于栽培大豆(31), 但单倍型多样性(0.916)稍高于栽培大豆(0.816)。单倍型大多数(67.4％)为群体所特有(31个), 其中15个为野生大豆特有单倍型。野生大豆的两个主要优势单倍型(Hap_10和Hap_11)在栽培大豆中的发生频率也明显下降, 推测野生大豆向栽培大豆进化过程中, 一方面形成了新的单倍型, 另一方面因为瓶颈效应部分单倍型的频率降低甚至消失。     

Length Polymorphisms of Simple Sequence Repeat DNA in Soybean

The objective of this work was to ascertain the presence and degree of simple sequence repeat (SSR) DNA length polymorphism in the soybean [Glycine max (L.) Merr.]. A search of GenBank revealed no (CA)n or (GT)n SSRs with n greater than 8 in soybean. In contrast, 5 (AT)n and 1 (ATT)n SSRs with n ranging from 14 to 27 were detected. Polymerase chain reaction (PCR) primers to regions flanking the six SSR loci were used in PCR amplification of DNA from 43 homozygous soybean genotypes. At three loci, amplification produced one PCR product per genotype and revealed 6, 7 and 8 product length variants (alleles) at the three loci, respectively. F1 hybrids between parents carrying different alleles produced two PCR products identical to the two parents. Codominant segregation of alleles among F2 progeny was demonstrated at each locus. A soybean DNA library was screened for the presence of (CA/GT)n SSRs. Sequencing of positive clones revealed that the longest such SSR was (CA)9. Thus, (CA)n SSRs with n of 15 or more are apparently much less common in soybean than in the human genome. In contrast to humans, (CA)n SSRs will probably not provide an abundant source of genetic markers in soybean. However, the apparent abundance of long (AT)n sequences should allow this SSR to serve as a source of highly polymorphic genetic markers in soybean.     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

DNA导入和系选大豆品种及其亲本遗传关系的SSR标记分析

利用现有品种的变异进行系选并培育新品种是一种重要的育种方法。利用SSR标记, 对系选大豆(Glycine max)新品种和其亲本的遗传组成进行分析, 明确二者的遗传关系, 为大豆系选育种提供依据。使用48对SSR引物对22个大豆品种及其亲本进行分析, 结果表明, 由外源DNA导入育成的3个品种与其亲本的一致性为35.42%–95.83%, 虽然供体相同, 但由于导入的外源DNA不同, 故育成的品种间差异很大。另外19个系选品种与其亲本的一致性为27.08%–89.58%, 其中有6个品种与亲本间的差异小于30%。聚类分析发现, 所有参试品种分为东北春大豆及黄淮和南方大豆2类, 其中有8个品种不能与其亲本聚在一起, 说明系选品种并不完全是由于亲本通过突变获得。此外, 农艺性状与分子标记分析结果差异较大, 说明利用有限的农艺性状评价品种间的遗传关系存在一定的局限性。通过比较系选品种和亲本之间的保守位点发现, 特定等位变异出现次数≥7的位点有14个, 分布在11个连锁群, 其中有7个位点与产量和抗病性等重要农艺性状有关, 说明DNA导入和系选品种在选择变异性状的同时, 使一些与重要农艺性状有关的等位变异得到了保留。     

Microsatellites from kousa dogwood (Cornus kousa)

Microsatellite loci were identified from Cornus kousa'National'. Primer pairs for 86 loci were developed and of these, eight were optimized and screened using genomic DNA from 22 kousa cultivars. All optimized loci were polymorphic and the number of alleles per locus ranged from three to 17. Observed heterozygosity ranged from 0 to 0.3 and expected heterozygosity ranged from 0.38 to 0.91. These microsatellites will be useful in population studies, and a breeding programme for cultivar development of Cornus species.     

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

Investigating the genetic model for brown stem rot resistance in soybean

Genetic analyses have indicated that brown stem rot (BSR) resistance in soybean is conferred by dominant alleles at three independent loci, the actions of which may be modified by linked or independent loci. A study was conducted to characterize the inheritance of BSR resistance in PI 567609, a soybean plant introduction from China. Segregating progeny from crosses of PI 567609 with BSR-susceptible and -resistant genotypes were evaluated for response to BSR-causal fungus, Phialophora gregata. Genetic analyses indicated that PI 567609 carries a single gene or cluster of linked genes for brown stem rot resistance, and that this gene (or cluster) is allelic to, or tightly linked to previously identified resistance genes, Rbs1, Rbs2, and Rbs3. Because previous allelism tests indicated that Rbs1, Rbs2, and Rbs3 were unlinked, and molecular mapping studies have indicated that Rbs1, Rbs2, and Rbs3 are linked on molecular linkage group J of soybean, a new model is proposed for BSR resistance. In this model, BSR resistance is controlled through the interaction of alleles at four independent loci, at least two of which are necessary to condition a resistance response. Functional redundancy at three of these loci allows any one of the three to interact with a fourth locus to confer resistance to BSR.     

Soybean germplasm pools in Asia revealed by nuclear SSRs

Soybean was domesticated in East Asia, where various kinds of landraces have been established as a result of adaptation to different environments and the diversification of food cultures. Asia is thus an important germplasm pool of soybean. In order to evaluate the genetic structure of the Asian soybean population, we analyzed allelic profiles at 20 simple-sequence repeat (SSR) loci of 131 accessions introduced from 14 Asian countries. The SSR loci produced an average of 11.9 alleles and a mean gene diversity of 0.782 in the accessions tested. Quantification theory III analysis and cluster analysis with the UPGMA method clearly separated the Japanese from the Chinese accessions, suggesting that the Japanese and Chinese populations formed different germplasm pools. The Korean accessions were involved in both germplasm pools, whereas most of the accessions from southeast and south/central Asia were derived from the Chinese pool. Relatively high genetic diversity and the absence of region-specific clusters in the southeast and south/central Asian populations suggest that soybean in these areas has been introduced repeatedly and independently from the diverse Chinese germplasm pool. The present study indicates that the two germplasm pools can be used as exotic genetic resources to enlarge the genetic bases of the respective Asian soybean populations.     

Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soybean

 Simple Sequence Repeat (SSR) allele sizing provides a useful tool for genotype identification, pedigree analysis, and for estimating genetic distance between organisms. Soybean [Glycine max (L.) Merr.] cultivars are identified for Plant Variety Protection (PVP) purposes by standard pigmentation and morphological traits. However, many commercial soybeans arise from a limited number of elite lines and are often indistinguishable based on these traits. A system based on SSR markers would provide unique DNA profiles of cultivars. Fluorescent labeling of alleles combined with automated sizing with internal size standards in each gel lane was used as an alternative to standard [³²P] labeling to assess genetic variability in soybean. Allelic frequencies at 20 SSR loci were determined in 35 soybean genotypes that account for greater than 95% of the alleles in North American soybean cultivars based upon pedigree analysis. An average of 10.1 alleles per locus (range: 5–17), with a mean gene diversity of 0.80 (range: 0.50 to 0.87) were observed at the 20 SSR loci. The 20 loci successfully distinguished modern soybean cultivars that are identical for morphological and pigmentation traits, as well as 7 soybean genotypes reported to be indistinguishable using 17 RFLP probes. Pedigrees of 7 cultivars were studied to estimate stability of SSRs in soybean across generations. Of the 7 pedigrees 6 had one locus in the progeny with an allele(s) that was not present in either parent. These new alleles are most likely the result of mutation. The mutation rate of SSR alleles in soybean was similar to that reported in humans. To avoid difficulty associated with mutation, DNA fingerprint data should be determined from the bulk of 30-50 plants of a cultivar. Received: 24 March 1997 / Accepted: 4 April 1997     

Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi

Twenty-four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty-one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi.     

Identification of quantitative trait loci controlling gene expression during the innate immunity response of soybean

Microbe-associated molecular pattern-triggered immunity (MTI) is an important component of the plant innate immunity response to invading pathogens. However, most of our knowledge of MTI comes from studies of model systems with relatively little work done with crop plants. In this work, we report on variation in both the microbe-associated molecular pattern-triggered oxidative burst and gene expression across four soybean (Glycine max) genotypes. Variation in MTI correlated with the level of pathogen resistance for each genotype. A quantitative trait locus analysis on these traits identified four loci that appeared to regulate gene expression during MTI in soybean. Likewise, we observed that both MTI variation and pathogen resistance were quantitatively inherited. The approach utilized in this study may have utility for identifying key resistance loci useful for developing improved soybean cultivars.     

新考察收集野生大豆与已保存野生大豆的遗传多样性比较

本研究以65份东北地区新收集野生大豆资源以及与其来源相同的已经编目保存的资源为实验材料,利用60对SSR引物进行遗传分析。分析结果表明新收集材料在22个位点的遗传多样性指数都高于以前收集的资源,有62个等位变异是新收集材料所特有。新收集材料与其它资源间的平均遗传相似系数为0．1918。说明新收集的材料使得野生大豆的遗传多样性水平得到提高,对野生大豆进行补充考察和收集是有价值的。