中国灌木辣椒种质遗传多样性的SRAP和SSR分析

应用SRAP和SSR分子标记对8份辣椒种质进行了遗传多样性分析,结果表明,15对SRAP引物组合共扩增出321条带,平均每对引物扩增出21.40条,多态性位点比率为72.90%;18对SSR引物共扩增出109条带,平均每对引物扩增出6.06条,多态性位点比率为98.17%。与SRAP比较,SSR检测到的Shannon多样性指数(I)、观测等位基因数(Na)和有效等位基因数(Ne)等遗传多样性参数都较大,说明SSR有更高的多态性检测效率。基于SRAP的聚类与基于SSR的聚类之间存在极显著正相关,且都能将中国灌木辣椒种质与美洲灌木辣椒种质及一年生辣椒种质有效区分。     

基于SSR标记分析小豆及其近缘植物的遗传关系

本研究利用87对SSR引物分析了80份栽培小豆(Vigna angularis)、22份野生小豆(V.angularis var.nipponensis)以及10份豇豆属(共7个种)近缘植物,旨在比较豇豆属不同种的遗传多样性,并分析种间的遗传关系.结果显示87对SSR引物在112份小豆及其近缘植物资源中共检测到667个等位变异.其中有75个、71个和82个SSR位点分别在栽培小豆、野生小豆和近缘植物中表现为多态.随机抽样分析发现,平均每SSR位点检测到的等位变异数目为近缘植物>野生小豆>栽培小豆,与多态信息含量(PIC)值一致,说明近缘植物及野生小豆中蕴含着丰富的遗传变异,是栽培小豆育种的重要基因来源.聚类分析显示,栽培小豆、野生小豆和近缘植物间的遗传分化比较明显,分别聚为三大类,其中栽培小豆的遗传背景与其生态环境相对应;近缘植物又可以分为三个亚类,亚类间的遗传距离与其亲缘关系相对应.本研究结果也说明利用SSR标记辅助豇豆属的种间分类是可行的.     

基于棉花参比种质的SSR多态性核心引物筛选

棉花品种间遗传多样性的评价和遗传图谱构建都需要大量多态性的SSR标记。本研究通过构建棉花参比种质(panel), 高效快速地筛选出棉花多态性核心引物。本实验选用12份表型性状和遗传差异较大的棉花种质作为参比种质, 对来自Cotton Microsatellite Database (CMD)网站上 (http://www.cottonssr.org) 的5,914对棉花SSR引物进行了PCR扩增。结果表明: 在不同棉种间多态性的引物有4,800对, 约占有效扩增引物(5,300对)的90.6%; 能区分不同品种间差异的引物大约有500对, 占有效扩增引物的9.4%。我们推荐其中319对扩增效果好、条带清晰的引物作为棉花种质资源鉴定和分子指纹分析的核心引物, 其中277对能把陆地棉品种间的差异鉴别出来。此外, 我们还找到了在陆地棉、海岛棉及亚洲棉三大栽培棉中都有差异的13对共同SSR引物, 建议作为分子指纹分析和种质鉴定的首选引物。本研究公开了319对核心SSR引物及其多态性信息, 有利于规范引物筛选程序、提高引物筛选效率, 对棉花种质资源的遗传多样性分析、指纹图谱构建, 以及种子真伪性和纯度的鉴定等都具有现实意义。     

用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较

由Ralstonia solanacearum E.F.Smith引起的青枯病是若干亚洲和非洲国家花生生产的重要限制因子,利用抗病品种是防治这一病害最好的措施。虽然一大批抗青枯病花生种质资源材料已被鉴定出来,但对其遗传多样性没有足够的研究,限制了在育种中的有效利用。本研究以31份对青枯病具有不同抗性的栽培种花生种质为材料,通过简单序列重复(SSR)和扩增片段长度多态性(AFLP)技术分析了它们的遗传多样性。通过78对SSR引物和126对AFLP引物的鉴定,筛选出能显示抗青枯病种质多态性的SSR引物29对和AFLP引物32对。所选用的29对多态性SSR引物共扩增91条多态性带,平均每对引物扩增3.14条多态性带;32对多态性AFLP引物共扩增72条多态性带,平均扩增2.25条多态性带。在所筛选引物中,4对SSR引物(14H06,7G02,3A8,16C6)和1对AFLP引物(P1M62)检测花生多态性的效果优于其他引物。SSR分析获得的31个花生种质的遗传距离为0.12-0.94,平均为0.53,而AFLP分析获得的遗传距离为0.06～0.57,平均为0.25,基于SSR分析的遗传距离大于基于AFLP分析的遗传距离,疏枝亚种组的遗传分化相对大于密枝亚种组。基于两种分析方法所获得的聚类结果基本一致,但SSR数据聚类结果与栽培种花生的形态分类系统更为吻合。根据分析结果,对构建青枯病抗性遗传图谱群体的核心亲本和抗性育种策略提出了建议。     

苦荞地方种质资源的遗传多样性分析

利用SSR分子标记技术对中国苦荞主产区陕西、云南、四川、西藏等地的82份苦荞地方种质的遗传多样性进行了分析,以揭示中国特有的作物种质--苦荞地方种质资源的遗传多样性,促进苦荞优良品种的选育.结果显示:(1)所用25个SSR引物中有13个引物在苦荞地方品种中具有多态性,且扩增条带的稳定性较好,共扩增出208条条带,其中多态性条带200条,占总数的96.2%;(2)聚类分析结果显示,82份苦荞材料的遗传相似系数(GS)分布于0.52～0.85之间,平均值为0.69,在GS值为0.722的水平上,82份材料被聚为10大类群.研究表明82份苦荞品种间遗传多样性明显,具有丰富的遗传基础.     

中国引种桉树种质资源的遗传多样性分析

该研究从110对SSR引物中,选用47个扩增稳定、条带特异的SSR多态位点,对42种159份桉树种质材料进行PCR扩增,通过统计条带构建二维数据库的方法,进行遗传多样性分析和聚类分析。结果显示:(1)共检测到137个等位基因,平均每个位点等位基因数为2.915个,各位点等位基因变异范围为2~7个。(2)遗传多样性分析结果表明,平均Shannon’s信息指数为0.181,平均观察杂合度Ho为0.068,平均多态信息含量PIC为0.182。综合各指标分析发现,位点eSSR-GR018、eSSR-GR083和eSSR-GR109的多态性程度最高,反映的遗传信息量更大,能够在桉树种质资源的遗传多样性分析和种质鉴定等方面发挥更大的作用。(3)非加权类平均法(UPGMA法)和主坐标分析法(PCoA法)聚类分析均表明,159份桉树种质材料分为两大类群,且昆士兰桉和少花桉间具有较高的遗传相似度,很可能产生杂交种;聚类结果与基于形态学的HillJohnson分类系统基本一致。研究表明,中国引种的桉树种质资源具有较高水平的遗传多样性,该研究所选用的47对SSR引物可有效地应用于桉树种质资源的鉴定分析。     

利用RAPD标记评价小豆种质遗传多样性

本研究利用10个RAPD引物对180份小豆种质的基因组DNA进行扩增,共扩增出44条带,其中35条具有多态性,比例为79.5%,平均每个引物扩增出3.5条多态性带;平均遗传距离为0.274,变异幅度为0.05～0.60,平均遗传多样性指数为0.692;基于RAPD标记,把180份小豆种质聚类划分为4个组群,该组群的划分与小豆的生态地域性似乎不存在明显的相关性.     

广西普通油茶种质资源遗传多样性的SSR分析

普通油茶( Camellia oleifera)是我国分布最广、产量最多的山茶属中一个重要油料树种。广西是普通油茶的重要分布区,种质资源十分丰富。为深入了解广西普通油茶种质资源的遗传变异,服务于种质保存和品种选育,该研究首先对已开发的SSR分子标记进行多态性筛选和评价,在此基础上利用多态性较高的引物,对97份广西有代表性的普通油茶种质资源进行遗传多样性分析。结果表明：(1)在已开发的10对油茶SSR分子标记中,7对能稳定扩增且表现为共显性,2对扩增不稳定,另外1对无法扩增出产物。(2)7对共显性SSR标记总共检测到33个等位基因,每对标记检测到等位基因数目的变化范围为3~6个,平均每个位点等位基因数为4.7143个,有效等位基因数目的变化范围为2.0842~4.3148,平均有效等位基因数为2.8288;基因多样性变化范围为0.5202~0.7682,平均每个位点基因多样性为0.6281。(3)参试群体中绝大多数位点未处于Hardy-Weinberg平衡,存在遗传结构;观测杂合度和期望杂合度的变化范围分别为0.4130~0.6701和0.5233~0.7724,其平均值分别为0.5698和0.6316。(4)种质资源间遗传距离变化范围为0.05~0.7917,平均遗传距离为0.3545;UPGMA聚类显示相同来源的种质资源无法聚成一类,在同一聚类分支上混有不同来源的种质资源。这表明已开发的油茶SSR分子标记适用于广西普通油茶,广西普通油茶种质资源拥有较丰富的遗传多样性。该研究结果为广西普通油茶资源的深度开发和高效利用提供了科学依据。     

绿豆种质资源的ISSR遗传多样性分析

利用ISSR标记对33份绿豆种质进行了遗传差异和亲缘关系分析。 结果表明,从46条ISSR引物中筛选出10条扩增条带清晰、多态性高的引物,利用这10条引物从33份绿豆种质中共扩增出118条条带,其中多态性条带115条,多态性位点比例98.18%。遗传相似性与聚类分析结果表明,供试绿豆种质间的遗传相似系数范围为0.50～0.98之间,平均0.68。当遗传相似性系数为0.682时,供试33份绿豆种质资源分为4个ISSR类群( ISSR Groups,IGs),第Ⅰ类群包括产地为黑龙江、吉林共9份和河北的1份绿豆种质资源,第二类群包括河南、山西和陕西的所有和河北的4份绿豆种质资源,第三类群为来自泰国的5份绿豆抗虫资源,第四类群包括山东和内蒙各2份绿豆资源;当遗传相似系数为0.98时,供试的33份绿豆种质资源可被全部区分开。 ISSR分析结果表明, ISSR类群划分与绿豆的地理来源存在一定相关性;而同一类群中,地理来源相同的绿豆种质间也存在一定的遗传差异。     

黔南60份茶树种质资源遗传多样性的SSR分析

为探索黔南野生茶树种质资源的遗传多样性,利用SSR分子标记技术,对黔南60份茶树资源进行了DNA遗传多样性分析。结果表明:15对引物均显示多态性,基因多态性百分率为98.64%。15对SSR引物共扩增出147个观测等位基因和73.778 6个有效等位基因,平均每个引物扩增9.8个观测等位基因,4.918 6个有效等位基因。15个通用位点共产生280种基因型,平均每个位点18.7种基因型。遗传多态信息量变异范围为0.123 9~0.926 8,平均0.572 5,平均观测杂合度、平均期望杂合度和平均Shannons信息指数分别为0.470 0、0.602 3、1.464 4。经聚类分析后,60份材料间遗传相似系数在0.205 1~0.863 6之间,以平均遗传相似系数0.477 5为阈值,可将60份种质资源聚为8个类群,其在分子遗传水平上的分类结果与其材料来源分类的结果并不完全一致,而且材料来源地间遗传距离与地理距离不存在显著的相关性,有少部分同一来源的材料分散在各个类群中。研究认为,黔南茶树资源间的遗传差异较大,遗传基础较宽,具有丰富的遗传多样性。     

Molecular Diversity in some Ghanaian Cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. (Walp)] Accessions

Cowpea [Vigna unguiculata L. (Walp)] is grown mainly for its protein-rich grains and is consumed in various forms in sub-Saharan Africa. Average grain yield in farmers’ fields is generally low due to a number of biotic and abiotic stresses. One hundred and six cowpea accessions from Ghana, which had previously been evaluated for seedling drought tolerance, were used for this study. This paper attempts to use three multi-locus PCR-based molecular markers; simple sequence repeats (SSR), inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphisms (REMAP), to analyse genetic diversity in the cowpea accessions. Analysis of the polymorphic bands data indicated that 101 alleles were amplified among 121 cowpea genotypes (83.4%) from 16 SSR primer pairs out of a total of 30 SSR primer pairs. Likewisely, a total of 66 (54.5%) polymorphic bands were obtained from IRAP and a total of 114 (94.2%) highly polymorphic bands obtained from REMAP analysis. The outcome indicated the highly polymorphic nature of the DNA markers, as small groups of these molecular markers were found to be able to identify each of the accessions used. Microsatellite markers (SSRs) and retrotransposon-based markers, like IRAP and REMAP, were found to be highly polymorphic and informative, suggesting that genomic fingerprinting has a major role in characterizing populations.     

Rapid Development of Microsatellite Markers for Callosobruchus chinensis Using Illumina Paired-End Sequencing

Background

The adzuki bean weevil, Callosobruchus chinensis L., is one of the most destructive pests of stored legume seeds such as mungbean, cowpea, and adzuki bean, which usually cause considerable loss in the quantity and quality of stored seeds during transportation and storage. However, a lack of genetic information of this pest results in a series of genetic questions remain largely unknown, including population genetic structure, kinship, biotype abundance, and so on. Co-dominant microsatellite markers offer a great resolving power to determine these events. Here, we report rapid microsatellite isolation from C. chinensis via high-throughput sequencing.

Principal Findings

In this study, 94,560,852 quality-filtered and trimmed reads were obtained for the assembly of genome using Illumina paired-end sequencing technology. In total, the genome with total length of 497,124,785 bp, comprising 403,113 high quality contigs was generated with de novo assembly. More than 6800 SSR loci were detected and a suit of 6303 primer pair sequences were designed and 500 of them were randomly selected for validation. Of these, 196 pair of primers, i.e. 39.2%, produced reproducible amplicons that were polymorphic among 8 C. chinensis genotypes collected from different geographical regions. Twenty out of 196 polymorphic SSR markers were used to analyze the genetic diversity of 18 C. chinensis populations. The results showed the twenty SSR loci were highly polymorphic among these populations.

Conclusions

This study presents a first report of genome sequencing and de novo assembly for C. chinensis and demonstrates the feasibility of generating a large scale of sequence information and SSR loci isolation by Illumina paired-end sequencing. Our results provide a valuable resource for C. chinensis research. These novel markers are valuable for future genetic mapping, trait association, genetic structure and kinship among C. chinensis.     

Genetic relationship of cowpea (Vigna unguiculata) varieties from Senegal based on SSR markers

Genetic diversity and phylogenetic relationships among 22 local cowpea (Vigna unguiculata) varieties and inbred lines collected throughout Senegal were evaluated using simple sequence repeat molecular markers. A set of 49 primer combinations were developed from cowpea genomic/expressed sequence tags and evaluated for their ability to detect polymorphisms among the various cowpea genotypes. Forty-four primer combinations detected polymorphisms, with the remaining five primer sets failing to yield PCR amplification products. From one to 16 alleles were found among the informative primer combinations; their frequencies ranged from 0.60 to 0.95 (mean = 0.79). The genetic diversity of the sample varied from 0.08 to 0.42 (mean = 0.28). The polymorphic information content ranged from 0.08 to 0.33 (mean = 0.23). The local varieties clustered in the same group, except 53-3, 58-53, and 58-57; while Ndoute yellow pods, Ndoute violet pods and Baye Ngagne were in the second group. The photosensitive varieties (Ndoute yellow pods and Ndoute violet pods) were closely clustered in the second group and so were inbred line Mouride and local cultivar 58-57, which is also one of the parents for inbred line Mouride. These molecular markers could be used for selection and identification of elite varieties for cowpea improvement and germplasm management in Senegal.     

Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek)

Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard’s similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.     

Assessment of genetic diversity in Ethiopian cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.] germplasm using simple sequence repeat markers

The genetic diversity of cowpea (Vigna unguiculata L. Walp.) in Ethiopia was analyzed using 19 uniform accessions, 62 variable accessions (yielding 185 sub-types), and two mungbean (Vigna radiata) accessions (four subtypes) as outgroup. A set of 23 polymorphic simple sequence repeat (SSR) markers was identified, and polymorphism in the various accessions was scored by determining amplicon variability. Allele frequency, genetic diversity, and polymorphism information content (PIC) were determined for each SSR marker, and a neighbor joining dendrogram was generated to show the genetic relationship among the individual accessions. A total of 75 allelic variants was defined, with the average number of alleles per locus calculated to be three. The average genetic diversity (D) was 0.47, and PIC was 0.4. Three main clusters were identified by phylogenetic analysis, and the clusters and sub-grouping were supported by STRUCTURE and principal component analysis. This grouping had a moderate fixation index value of 0.075 and gene flow (Nm) of 3.176, indicating that the accessions possess wide diversity within individuals and populations. The accessions showed no clustering by geographical origins. Three well-characterized molecular markers (SSR1, C42-2B, and 61RM2) for race specific resistance to Striga gesnerioides in the cowpea cultivar B301 were used to evaluate the accessions for their potential for use in genetic improvement against this pest. Based on this analysis, only two accessions, 222890–2 from Gambela and 286–2 from the Southern Nations, Nationalities, and Peoples (SNNP) region, were found to cluster with B301 and contain the SSR1 resistance allele. These findings will assist in germplasm conservation efforts by the Institute of Biodiversity and Conservation of Ethiopia, and contribute to future studies aimed at the genetic improvement of local germplasm for improved overall agronomic performance as well as Striga resistance in particular.     

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats - SSRs and amplified fragment length polymorphisms - AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger's modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98% and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm.     

Development and polymorphism of Vigna unguiculata ssp. unguiculata microsatellite markers used for phylogenetic analysis in asparagus bean (Vigna unguiculata ssp. sesquipedialis (L.) Verdc.)

Asparagus bean (V. unguiculata ssp. sesquipedialis), a specific form of cowpea (V. unguiculata L. Walp.), is cultivated as a vegetable crop throughout eastern and southern Asia for its tender long pods. Little is known about the genetic relationship between asparagus bean and the broader species, particularly the dominant ssp. unguiculata. We report here the development and transferability of simple sequence repeat (SSR) markers, over 40% of which are EST-derived, from ssp. unguiculata to asparagus bean and the use of a subset of the polymorphic markers to assess the genetic diversity of asparagus bean cultivars from diverse geographic origins across China. A total of 410 EST derived SSR (eSSR) markers and 600 SSR markers derived from cowpea genespace sequences (GSS) were developed, with a cross-subspecies transferability of 100% and 98.5%, respectively. In a recombinant inbred line population of asparagus bean, a 1:1 segregation was observed for most loci. Principal coordinate analysis (PCA) and phylogenetic clustering based on 62 alleles detected by 14 polymorphic SSR markers distinguished ssp. unguiculata and sesquipedialis into separate groups. Improved asparagus bean cultivars in China generally have a narrow genetic basis compared with landraces varieties. This suggests that asparagus bean breeding programs need to consider utilizing landrace germplasm to enhance genetic variability and ensure long-term gains from selection and reduce genetic vulnerability to pathogen/pest epidemics. Because of their transferability across subspecies, the SSR markers described in this study could be effectively employed in cross-subspecies trait introgression breeding from ssp. unguiculata to sesquipedialis.     

Characterization of new microsatellite markers in mung bean,Vigna radiata (L.)

The present work reports the isolation and characterization of new polymorphic microsatellites in mung bean (Vigna radiata L.). Of 93 designed primer pairs, seven were found to amplify polymorphic microsatellite loci, which were then characterized using 34 mung bean accessions. The number of alleles ranged from two to five alleles per locus with an average of three alleles. Observed and expected heterozygosity values ranged from 0 to 0.088 and from 0.275 to 0.683, respectively. All seven loci showed significant deviations from Hardy–Weinberg equilibrium, whereas only one pairwise combination (GBssr‐MB77 and GBssr‐MB91) exhibited significant departure from linkage disequilibrium. These newly developed markers are currently being utilized for diversity assessment within the mung bean germplasm collection of the Korean Gene Bank.