Analysis of genetic diversity in Indian robusta coffee genepool (<Emphasis Type="Italic">Coffea canephora</Emphasis>) in comparison with a representative core collection using SSRs and AFLPs

Genetic diversity among 40 accessions (Coffea canephora) of robusta coffee genepool available in India was determined in comparison with 14 representative samples from a C. canephora core collection and three accessions of C. congensis, using amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers. Both these molecular approaches were able to generate unique fingerprints for each of the accessions analysed. All the 12 SSR primers used in the present study were found polymorphic, with an average of six alleles per primer pair. Comparative analysis revealed the higher amount of diversity in representatives from a core collection than in the Indian genepool. Moreover, a total of 205 polymorphic AFLP bands were scored in all the 57 accessions analysed. The genetic relationship among 57 accessions was compared on the basis of SSR and AFLP polymorphisms. Genetic similarity dendrograms showed high correlation between the two marker systems. This study clearly established the high amount of diversity present in core samples, which is not represented in Indian genepool. Furthermore, the three accessions of C. congensis did not exhibit any significant diversity from other robusta accessions supporting the school of thought that C. congensis forms a biotype of C. canephora. The potential use of SSRs and AFLP markers in genetic diversity analysis for better ex situ management and also for exploitation of diversity in breeding programmes is discussed.     

Exploration of intra- and inter-population genetic diversity in <Emphasis Type="Italic">Hedysarum coronarium</Emphasis> L. by AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to characterize the genetic diversity within and among natural populations and cultivars of Hedysarum coronarium. Twelve populations within Tunisia were evaluated with three AFLP primer combinations. A total of 207 reproducible bands was detected of which 178 (86%) were polymorphic. The great discriminative power of AFLP markers and their ability to represent genetic relationships among Hedysarum plants was demonstrated. Genetic diversity within and among populations was assessed through Principal Component Analysis (PCA) and cluster analysis by using the Neighbor-joining clustering algorithm. AFLP technology has provided evidence of a high degree of intra- and inter-population genetic diversity in H. coronarium. AFLP banding patterns provided molecular markers correlated with the plants’ geotropism. In addition, AFLP markers can differentiate wild accessions from cultivars. Moreover, geographical origins did not correspond to population clustering.     

Indigenous knowledge and traditional conservation of hulless barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) germplasm resources in the Tibetan communities of Shangri-la,Yunnan, SW China

Hulless barley (Hordeum vulgare L. s. l.) is the staple food crop of Tibetan communities in the Qinghai-Tibet Plateau, covering Tibet, Qinghai, Gansu, Sichuan and Yunnan provinces in China. Participatory Rural Appraisal methodolgies were employed in twenty-seven villages that were randomly selected in Shangri-la region, or Diqing Prefecture of Yunnan Province to document the ethnobotanical and indigenous knowledge related to the production, diversity, use and conservation of hulless barley. For Tibetans in Shangri-la, the crop has a high socio-cultural value. The genetic diversity of landraces managed by the farmers in Shangri-la is an extremely important germplasm resource. We recorded a total of 54 landraces that are primarily described by farmers based on their cooking quality, growth cycle, color and size of the grains. The ethnobotanical and indigenous knowledge recorded in this study contributes to the understanding of the genetic diversity of hulless barley in Shangri-la and even in the Qinghai-Tibet Plateau, and in defining appropriate strategies for its conservation on farm.     

利用SSR和RAPD标记分析中国部分地区晚疫病菌群体遗传多样性

利用SSR和RAPD对来自福建、黑龙江、河北和内蒙古4个地理群体的80个马铃薯晚疫病菌（phytophthora infestans）株进行了遗传多样性分析。13对SSR引物共扩增出76条谱带,多态性条带比率78.9%,相似系数变化范围0.00～0.42之间;筛选出的14条RAPD引物共扩增出189条谱带,多态性条带比率95.2%,相似系数变化范围0.04～0.66之间。遗传多样性分析表明,在4个群体中,福建群体的多样性更为丰富。遗传相似性分析显示,黑龙江和内蒙古两个群体间的遗传相似性最高,而福建和河北两个群体间的遗传相似性最低。聚类分析显示,来自南方福建的菌株与来自北方黑龙江、河北和内蒙古的菌株亲缘关系较远,且福建群体分布于更多的聚类组,显示出更高的遗传变异度。     

Microsatellites and RAPD Markers to Study Genetic Relationships Among Cowpea Breeding Lines and Local Varieties in Senegal

Genetic diversity in local cowpea varieties and breeding lines from Senegal were studied using random amplified polymorphic DNA (RAPD) and microsatellite (SSR) techniques. Among the 61 RAPD primers used, twelve show polymorphism. Fifteen of the 30 microsatellite primer pairs were polymorphic, detecting one to nine alleles per locus. The RAPD and SSR data were analyzed both separately and in combination to assess relationships among genetic lines. Although RAPD provided information on levels of genetic diversity, microsatellite markers are most effective in determining the relationship among cowpea accessions and varieties. The SSR results support the genetic diversification of cowpea in Senegal and underscore their potential in elucidating patterns of germplasm diversity of cowpea in Senegal. An erratum to this article is available at .     

江西野生毛花猕猴桃群体SSR遗传多样性研究

为分析江西野生毛花猕猴桃群体遗传多样性,以江西省境内的5个野生毛花猕猴桃雄性群体(72个个体)为试验材料,采用SSR分子标记技术,选取15对多态性引物,利用聚丙烯酰胺电泳对PCR产物进行检测。结果表明,15对SSR引物共检测到86个位点,多态性位点百分率为100.0%;观察到的平均等位基因数为5.733,有效等位基因数为3.002,Shannon信息指数为1.046。表观杂合度介于0.111~0.819之间,预期杂合度介于0.041~0.876之间,遗传分化系数为2.01,野生毛花猕猴桃雄性群体间存在较大的遗传分化。5个毛花猕猴桃雄性群体遗传距离范围为0.102~0.409,遗传相似度范围为0.665~0.903,群体间遗传距离与地理距离无相关性。群体的遗传多样性丰富度依次为庐山>井冈山>南源山>武功山>麻姑山,江西地区供试的野生毛花猕猴桃雄性群体在分子水平上具有丰富的多态性。本研究结果为毛花猕猴桃雄性品种选育、种质创新与利用提供了一定的理论基础。     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

浙江省南部地方梨品种的SSR和AFLP遗传多样性研究

为了解浙江省南部地方梨品种的遗传多样性,采用10对苹果和3对梨上的SSR引物以及6对AFLP引物对来自温岭和丽水的18个地方梨品种或类型进行了鉴定分析。结果表明10对苹果引物在梨上扩增出62条条带。13对SSR引物的观察杂和度(He)为0.056～0.944,平均值为0.57,而香农指数(I)的范围0.340～2.013,平均值为1.22。6对AFLP引物组合共扩增出364条带,平均每对引物扩增60.67条,平均多态性比例为73.35%。采用UPGMA聚类分析法构建的SSR和AFLP系统树中,云和梨、云和粗花雪梨与云和细花雪梨在2个系统树中聚在一起,虽然相互间有较高的遗传相似度,但属于不同的品种;3个同名为老雪梨的品种(老雪梨1、老雪梨2和老雪梨3)分散在不同的组中,可能为异物同名。SSR和AFLP聚类结果基本吻合,但AFLP的结果更符合品种的地域分布情况。     

甘蓝SSR标记在近缘种青花菜的通用性及其应用

本研究对50条甘蓝SSR引物在其近缘种青花菜中的通用性进行了分析,结果表明,38对引物在青花菜上可有效扩增,扩增产物分子量在100～1500bp,有效扩增比率为76%,其中18%具有较好的多态性,揭示了两种作物基因组间存在一定的相似性。同时利用获得的多态性较好的9对引物对青花菜进行基因型鉴定和遗传多样性分析,20份青花菜基因型中共检测到51个基因位点,平均每个引物组合可扩增出5.67个条带,多态性为66.7%。多引物组合可鉴别出所有青花菜基因型。聚类分析结果表明同一来源的基因型间具有相近的遗传基础,且聚类与熟性具有一定的相关性。本研究为今后青花菜种质资源的收集、利用及分子标记辅助育种提供了新的SSR标记和参考。     

Assessment of genetic diversity in Egyptian barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) genotypes using SSR and SNP markers

A barley core collection can be studied extensively and the derived information can be used to identify loci/genes for the genetic improvement of quantitative and qualitative traits. To assess genetic diversity, allelic variation and population structure of Egyptian barley, 134 barley genotypes collected from a different region along with 19 cultivated genotypes obtained from of the Egyptian Agricultural Research Center. All genotypes were analyzed with 261 polymorphic SSR and SNP alleles. The mean number of alleles per locus was 4 and PIC was 0.49, while the level of genetic diversity was 0.55 ranging from 0.03 to 0.82. The genotypes were assigned to three subpopulations that were consistent with their origins. The genetic variation within population was higher (51%) than among population at the molecular levels (F_ST =?0.491 when P?<?0.10). The level of polymorphic variation was highest in subpopulations-II, due to collected from different regions with different ear-types thus, expected to contain more diversity than local genotypes in subpopulations-I and subpopulations-III. The structured study found that the 153 barley genotypes are in harmony with clustering approaches using the SSR and SNP genotypic data in a neighbor-joining tree and principal components analysis, which identified three subpopulations. These results demonstrated genetic diversity among the Egyptian barley genotypes can be applied to suggest approaches, such as association analysis, classical mapping population development, and parental line selection in breeding programs. Therefore, it is necessary to use the exotic genotypes as the genetic resources for developing new barley cultivars in Egypt.     

我国部分冬小麦新品种（系）SSR标记遗传差异的研究

本研究利用53对SSR引物对全田1999－2000年北方冬麦区及黄淮冬麦区观察圃中选出的48个新品种（系）进行遗传差异研究，共检测出58个SSR位点上的367个等位变异，平均每个位点有6.33个等位变异，其中B组每个位点的等位变异最多，这表明B基因组化更快，分化更大。48个品种（系）在全基因组及A、B、D基因组聚类结果表明这些品种的相似系数聚类的范围较小，为0.75－0.98。全基因组聚类结果与品种的系谱来源及育成地区相吻合。研究结果表明我国冬小麦品种的种质基础相对较狭窄。加强不同来源种质的利用和特异亲本类型的培育对我国冬小麦遗传改良非常重要，利用5个多态性高的SSR标记就可以将这48个小麦新品种（系）区分开，每个品种（系）都有各自独特的指纹图谱。     

Comparison of genetic variation among accessions of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> using AFLP and SSR markers

Genetic diversity of 54 accessions of Aegilops tauschii from five countries was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphisms (AFLPs). In the case of AFLP analysis, a total of 256 amplification products obtained, 234 of them were polymorphic across all the 54 accessions. A total of 224 fragments were obtained from the 24 SSR primers and 219 of fragments were polymorphic across all the genotypes screened. Based on both AFLP and SSR markers, the highest percentage of polymorphisms were obtained in Iranian and accessions of unknown origin. The highest polymorphic information content (PIC) value was observed for SSRs (0.82) while the highest marker index (MI) value was for AFLPs (8.5) reflecting the hyper-variability of the first and the distinctive nature of the second system. Principal co-ordinate analysis (PCO) revealed congruent patterns of genetic relationships for both data sets, but did not group accessions strictly according to their geographical origins. Poor correlation was found between AFLP and SSR marker loci. This low association may be due to low number of AFLP and SSR markers. These results show that molecular markers can help to organize the genetic variability and expose useful diversity for breeding purposes.     

The domestication and dispersal of the cultivated ramie (Boehmeria nivea (L.) Gaud. in Freyc.) determined by nuclear SSR marker analysis

Ramie (Boehmeria nivea) was domesticated in China. However, the geographic region of domestication is not exactly known. Genetic diversity and population structure of ramie and their wild relatives were assessed using microsatellite markers. The 8 microsatellite primers revealed 96 alleles in 50 ramie populations, with an average of 10.25 alleles per locus. Cultivated ramie gene pool harbors approximately 82.9 % of the SSR diversity presented in wild B. nivea var. nivea. It is suggested that ramie has experienced a relatively moderate domestication bottleneck. The distribution of genetic diversity shows that genetic diversity is relatively high in populations along the Yangtze River compared to the peripheral ones. The scatter plots of principal coordinates analysis indicated that there are three well-supported varieties in B. nivea. The NJ tree and the distribution of genetic diversity showed that ramie has been domesticated in the middle and lower regions of the Yangtze valley, and populations in Sichuan province have been introduced from this region, forming naturalized populations.     

基于SSR标记的梨资源遗传多样性分析

本研究利用SSR标记技术对56份梨资源进行了遗传多样性分析。利用筛选出的6对SSR引物共扩增40条谱带,其中多态性位点38个,多态性位点比例为95%,每对引物产生有效等位基因6.3个。各位点期望杂合度H值在0.0354～0.491,平均为0.1964;有效等位基因Ne值在1.0367～1.9648,平均值为1.2958;香农指数I值平均值为0.3256,说明了供试梨材料的遗传多样性较低。利用SSR标记可将44个栽培品种区分开,但无法区分芽变和原种。根据SSR标记揭示的多态性,采用NTSYS-pc软件,以UPGMA法进行聚类分析,结果显示所检测的56份梨材料在相似系数0.71处可分为4组,其中中国的白梨、秋子梨和砂梨相互交错在一起,没有独自各自成组。     

甘蔗抗褐锈病基因定位亲本间多态性SSR标记筛选

为获得更多用于构建甘蔗遗传连锁图谱可用的多态性SSR标记,本研究以6个高抗甘蔗褐锈病品种和6个高感甘蔗褐锈病品种为亲本,根据感病母本×抗病父本选配12个杂交组合,筛选在亲本间条带清晰、多态性明显、重复性较好的引物。结果表明,组合柳城03-1137×德蔗93-88、Mex105×粤糖00-236亲本间的多态性引物数最多,分别占总数的52.38%和47.62%;其中,10对引物(18-mSSCIR34、22-mSSCIR38、25-mSSCIR48、32-mSSCIR67、51-mSSCIR50、57-MSSCIR21、71-SMC1490CL*、73-SMC278CS*、75-SMC31CUQ*、77-SMC336BS*)在组合柳城03-1137×德蔗93-88中多态性最好,7对引物(22-mSSCIR38、25-mSSCIR48、45-mSSESTC04、51-mSSCIR50、67-mSSCIR9*、76-SMC334BS*、80-SMC486CG*)在Mex105×粤糖00-236中多态性最好,利用筛选得到的引物,更有利于构建分子遗传图谱。本研究结果为抗褐锈病新基因定位和开发与其紧密连锁的分子标记研究提供了一定的理论依据。     

玫瑰黄链霉菌Men-myco-93-63与其突变株间的DNA多态性分析和序列特征性扩增区域(SCAR)标记

玫瑰黄链霉菌(Streptomyces roseoflavus )Men-myco-93-63是分离自马铃薯疮痂病自然衰退土壤中的一株拮抗菌。前期研究表明,该菌株及其发酵液对棉花黄萎病菌（Verticillium dahliae）等多种重要的植物病原真菌具有较强的抑制作用。利用AFLP对Men-myco-93-63及其8株抗生素生物合成阻断突变株进行了基因组DNA多态性分析。结果表明,38对AFLP引物共产生3 142条谱带,其中2 362条为多态性谱带,占总带数的75.2 %。从96对引物中筛选得到了3对引物：E-CA/M-GA,E-GA/M-AC和E-GA/M-AG,分别扩增出254、312和209 bp 3条仅在Men-myco-93-63中存在,命名为EM254、EM312和EM209。将这3个片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对突变株的分子检测。     

Genetic diversity and differentiation in Ethiopian populations of Phytolacca dodecandra as revealed by AFLP and RAPD analyses

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analyses were performed on six populations (a total of 89 individuals) of Phytolacca dodecandra (endod) collected in Ethiopia. The populations were selected based on our previous investigation to represent two altitude groups: lowland/central-highland (1600–2500 m) and highland (2501–3000 m). A total of 197 AFLP and 68 RAPD markers were scored from 5 primer pairs and 12 random primers, respectively. The overall patterns obtained for AFLPs and RAPDs from diversity, cluster and principal component analyses were very comparable. However, the moderate correlation (r = 0.56) between AFLP and RAPD similarity matrices as well as the discrepancies in diversity estimates between the two techniques in some populations and in the lowland/central-highland plants could be due to differences in sensitivity of reaction conditions, bias in scoring of bands, number of markers used for analyses, and/or parts of the genome surveyed. For both AFLP and RAPD, the lowland/central-highland populations showed higher polymorphism and Shannon information measure (H) than the highlands. Cluster and principal component analyses performed for both marker types have also clearly demonstrated the differentiation of all the lowland/central highland plants from those of the highlands, in agreement with our previous conclusion. Markers scored from any of the five AFLP primer pairs were sufficient to clearly distinguish the two altitude groups; with RAPD, selection of about 8 informative markers produced by seven random primers was needed for the same purpose.     

基于石蒜属转录组序列的SSR分子标记开发应用

为开发石蒜属简单重复序列(SSR)分子标记,并研究SSR引物在石蒜属内的通用性,本研究对石蒜属石蒜、忽地笑、中国石蒜、长筒石蒜、换锦花、香石蒜6个种质转录组测序,检测SSR位点并设计引物,通过PCR扩增和毛细管电泳判断引物的有效性和多态性,绘制石蒜属17个种质资源的指纹图谱并对杂交后代的真实性进行早期检测。结果表明,共获得404 481条Unigenes,利用数据库进行同源比对和功能注释,并对Unigene进行SSR位点挖掘和分析,共检测到59 612个SSR位点。其中,单核苷酸重复>二核苷酸重复>三核苷酸重复,分别占SSR总数的62.88%、20.06%和14.66%,四核苷酸及以上重复单元相对较少。选取并合成8对荧光引物进行PCR扩增,通过毛细管电泳检测发现,8对荧光引物共检测到60个多态位点,多态位点数平均为7.50,多态性信息含量(PIC)值变化范围为0.148 0~0.940 8,平均值为0.593 0。利用引物扩增带型组合法构建了石蒜属17个种资源的指纹图谱,其中引物QZ209可区分所有供试材料,并可用于杂交后代鉴定。本研究开发的SSR标记具有丰富的多态性,在石蒜属植物的资源多样性分析、杂交种鉴定及遗传图谱的构建应用中具有重要意义。     

大蒜种质遗传多样性的SSR分析

为了探索中国大蒜种质个体的SSR位点的分布情况,为品种鉴定、保存及遗传改良提供分子生物学依据,利用6对SSR引物对40个大蒜(Allium sativumL.)品种进行聚类分析、主成分分析及遗传多样性评价。共检测到21个多态性位点,平均每对引物可扩增出约3.5条多态性片段,多态性百分率为56.76%;SSR引物组合平均有效等位基因数、Nei基因多样度和Shannon信息指数分别为1.5551、0.3414和0.5188。聚类分析显示,6对SSR引物可把40份大蒜种质资源从0.59相似系数水平上3个类群。第一类群包含28份种质,在相似系数为0.73的水平上进一步又被分成了3个亚类;第二亚类仅包含2份种质;第三亚类包含10份种质,在0.68的相似系数水平上分成了2个亚类。主成分分析和UPGMA的结果基本一致。不同地理来源的大蒜种质的Shannon-Weaver多样性指数的变幅为0.0576~0.4179,说明大蒜种质遗传多样性丰富。本研究利用SSR分子标记技术较准确地解析大蒜不同材料间的亲缘关系及遗传多样性,为中国大蒜SSR分子标记提供基础资料。     

Development of EST-SSR in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

The development of EST-SSR in foxtail millet (Setaria italica) for polymorphism and transferability study was reported here. From 1213 EST sequences, 30 SSRs were obtained and primers were designed for 26 SSRs. Among them, four pairs of SSR primers amplified polymorphic products in 12 foxtail millet cultivars and one accession of Setaria viridis, a wild relative of foxtail millet, with 10 alleles detected for the four loci and 2.5 alleles per locus. In addition, ten SSR markers could be transferred to other nine Gramineae species. The putative functions of 11 ESTs containing polymorphic and transferable SSRs were also identified.