Structure of genetic diversity among common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) varieties of mesoamerican and andean origins using new developed microsatellite markers

A common bean genomic library was constructed using the ‘IAC-UNA’ variety enriched for (CT) and (GT) for microsatellite motifs. From 1,209 sequenced clones, 714 showed microsatellites distributed over 471 simple and 243 compound motifs. GA/CT and GT/CA were the most frequent motifs found among these sequences. A total of 123 microsatellites has been characterized. Out of these, 87 were polymorphic (73.7%), 33 monomorphic (26.8%), and 3 (2.4%) did not amplify at all. In a sample of 20 common bean materials selected from the Agronomic Institute Germplasm Bank, the number of alleles per locus varied 2–9, with an average of 2.82. The polymorphic information content (PIC) of each marker varied from 0.05 to 0.83, with a 0.45 average value. Cluster and principal coordinate analysis of the microsatellite data were consistent with the original assignment of the germplasm accessions into the Andean and Mesoamerican gene pools of common bean. Low polymorphism levels detected could be associated with the domestication process. These microsatellites could be a valuable resource for the bean community because of their use as new markers for genetic studies. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

黄颡鱼微卫星标记筛选及特征分析

通过磁珠富集法首次筛选黄颡鱼微卫星标记并对其特征进行分析。用生物素标记的寡核苷酸探针(AC)8、(CT)8、(AT)7、(GATA)8、(GATT)7进行微卫星片断的富集,挑选部分阳性克隆测序获得38个含有微卫星的DNA序列,总计设计并合成了22对微卫星引物。以普通黄颡鱼基因组DNA为模板,进行PCR扩增,结果筛选出18个多态性微卫星标记,每对引物等位基因数2-8个（平均3.78）,遗传杂合度0.2225-0.8101（平均0.5755）,多态信息含量0.3425-0.7900（平均0.5336）。此外18对多态性微卫星引物中有16对在黄颡鱼属其它4种鱼类具有通用性,13对在鲇形目中的3种鱼类具有通用性。因此,可以推断筛选的多态性微卫星标记可用于黄颡鱼属和鲇形目鱼类的遗传分析。     

利用SSR分析小豆种质遗传多样性

摘要：小豆是一类重要的食用豆,本研究利用45对SSR引物对小豆基因组DNA进行了SSR标记的筛选鉴定,共筛选出多态性良好、扩增效果稳定的SSR引物18对。利用筛选出的18个SSR标记,分析了来自我国栽培小豆优异种质53份和日本引进种质27份,旨在阐明其遗传多样性特点,为育种利用提供理论依据。结果表明,在所有参试的80份小豆种质中共鉴定出等位变异92个,平均每个位点为5.1个;其中53份国内小豆和27份日本小豆的等位变异数分别为89个和74个,平均每个位点分别为4.9个和4.1个。所有供试小豆平均每个位点的多态信息含量（PIC）为0.64,变化范围为0.23～0.83,其中国内小豆的平均PIC值为0.63,变化范围为0.23~0.86;日本小豆平均PIC值为0.61,变化范围为0.20~0.81。国内栽培小豆和日本小豆在等位变异数、多态信息含量（PIC）、遗传相似性系数均存在差异,UPGMA聚类分析将参试的80份小豆明显分为五大类,聚类结果与小豆种质地理起源呈现出一定的相关性。试验表明,在小豆遗传育种中,可以通过种质资源相互利用来拓宽育成品种的遗传基础,同时这些SSR标记对于小豆资源DNA指纹图谱构建、遗传作图、基因型鉴定及分子标记辅助育种具有重要意义。     

鲤鱼三、四核苷酸重复微卫星座位的筛选及特征分析*

采用磁珠富集法结合放射性同位素杂交法分离出鲤鱼(Cyprinus carpio)三、四核苷酸重复的微卫星阳性克隆1248个。对其中的384个克隆进行测序分析,共得到微卫星序列325个,其中完美型244个（75.08%）,非完美型59个（18.15%）,混合型22个（6.77%）。依据得到的微卫星序列,用Primer 3软件在线设计并合成引物145对,检测结果显示71.43%（80对/112对）的引物在野生个体间表现出多态性,等位基因数在3~12之间,多态信息含量在0.2109~0.8607之间,80%的位点处于高度多态水平。对群体的遗传结构评估结果显示,四核苷酸重复的微卫星标记在黑龙江鲤野生群体表现出的多态性水平（PIC=0.7740）高于三核苷酸重复（PIC=0.5161）和双核苷酸重复（PIC=0.49）,适用于群体间遗传差异分析和遗传连锁图谱的构建。     

厚朴转录组特征分析及EST-SSR标记的开发

为探明厚朴的基因组信息,开发合适的生物学工具以促进厚朴的遗传育种,本研究利用Illumina-Solexa测序技术对厚朴根、茎皮、叶不同组织的9个样本进行转录组分析,获得大量的序列信息,并以转录组序列为基础进行厚朴SSR分子标记的大规模发掘。结果表明,共获得了109 526条厚朴转录组序列,平均序列长度为1 023 bp,通过与蛋白数据库NR、Swiss-Prot、GO、KOG和KEGG五个数据库比对,分别有6 0361、39 942、65 535、51 351和27 310条厚朴转录组的Unigene被注释;厚朴转录组表达信息进行大通量SSR位点的发掘,发现了含SSR位点的序列25 925条,共27 721个SSR位点,SSR出现的频率为23.67%。厚朴转录组共发现了374种碱基重复模式,且CT/GA和AG/TC是主要的双碱基重复序列,AGA/TCT和AAG/TTC是主要的三碱基重复序列;随机挑选的180对SSR引物中有104对产生清晰可重复的条带,21对引物具有多态性,厚朴SSR引物多态性较高。本研究结果为厚朴及近缘种的遗传多样性分析、遗传图谱构建及比较基因组研究奠定了一定的理论基础。     

Genetic diversity assessment of Bulgarian durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) landraces and modern cultivars using microsatellite markers

The genetic diversity in a Triticum durum Desf. collection, consisting of 102 Bulgarian landraces, nine Bulgarian and 25 introduced cultivars was studied using 14 highly polymorphic microsatellite markers. A total of 100 alleles were identified, with an average of 7.14 alleles per marker. The gene diversity values (He) of the markers for the total samples ranged from 0.23 (WMS357 and WMS631) to 0.77 (WMS46), with an average of 0.52. Within the landraces that were collected from 18 sites in Southern Bulgaria showed 2–11 alleles per locus with an average of 6.07. The microsatellite analysis suggests that the genetic diversity among landraces is lower compared to the diversity levels for durum wheat in countries close to the main centers of wheat domestication. Breeding activities have caused significant reduction of the allelic polymorphism, elimination of rare alleles, and increase in the number of common alleles and the frequency of dominating alleles.     

Molecular diversity of Omani wheat revealed by microsatellites: II. Hexaploid landraces

For millennia, wheat (Triticum spp.) has been grown in traditional aflaj-irrigation systems of remote mountain oases in Oman. However, little is known about the diversity of the ancient landraces used. Given recent reports about the occurrence of novel germplasm in such material, the objective of this study was to evaluate the genetic diversity of hexaploid wheat (Triticum aestivum L.) landraces in relation to their geographic origin using microsatellites. The collection covered most of the cultivation areas in northern Oman where wheat landraces are growing. Total genomic DNA was extracted from six pooled plants representing each accession. A total of 161 wheat accessions were assayed using 35 microsatellite loci in which a total of 305 polymorphic bands were recorded for the 35 microsatellites. The polymorphic information content (PIC) across the 35 microsatellite loci ranged from 0.02 to 0.89 with an average of 0.50. A heterozygosity percentage value of 9.09 was determined and the highest level recorded for accessions from the Batinah district. Rare alleles averaged 1.85 with the highest value being from the Dakhilia district. The results indicated a significant correlation between gene diversity and number of alleles across districts. The correlation coefficient between these two variables over the 35 loci was 0.657, whereby correlation coefficients of 0.718, 0.706, 0.657 and 0.651, respectively, were found for the Batinah, Dhahira, Dakhilia and Sharqia materials. Genetic distances indicated that all landraces were closely related. The cluster analysis discriminated most of the landraces accessions. However, it failed to achieve region-specific groupings of landraces. The present study demonstrated the presence of high diversity in Omani landraces and also indicated the effectiveness of microsatellites to describe it.     

筛选杂交鲤亲子鉴定的微卫星标记(英文)

微卫星的亲子鉴定技术既能减少分池饲养带来的遗传性状的差异又能减少其所占用饲养池的数量和管理强度,在水产养殖和品种选育中得到了广泛的应用。本研究应用21对微卫星标记对鲤(Cyprinus carpio)杂交家系(49-同胞家系:德国镜鲤(C.carpio var.mirror)♀×荷包红鲤(C.carpio var.wuynanensis)♂;24-同胞家系:荷包红鲤♀×德国镜鲤♂)进行遗传多样性及遗传特性的评价,并检测影响微卫星标记鉴定准确性的几个特性。研究结果标明,(1)筛选出的13对标记:在49-同胞家系中,平均有效等位基因数(K)、期望杂合度(He)、观测杂合度(Ho)和多态信息含量(PIC)分别为0.505、0.648、0.791和0.5889;在24-同胞家系中,K、He、Ho和PIC分别为0.548、0.670、0.819和0.6138。(2)筛选出的13对标记:在49-同胞家系和24-同胞家系中的联合排除率(CPE)分别达到0.999997978和0.999999583。(3)在两个杂交家系中,4个核心位点(MFW29,HLJ392,HLJ044,HLJ855)的联合排除率均高于99%。(4)三种情况下(一个亲本已知,NE-1P;另一个亲本已知,NE-2P;双亲都已知,NE-PP;)的联合非父排除率在49全胞家系中分别为1.34E-02、3.91E-04和2.02E-06;在24全胞家系中分别为1.34E-02、3.91E-04和2.02E-06。(5)模拟分析显示(置信度为99%),当使用11个或更多标记时,在49全胞家系中亲本与子代的配对率为100%;当使用9个或更多标记时,在24全胞家系中亲本与子代的配对率大于98%。(6)UPGMA聚类图表明:在混养条件下,筛选出的13对标记可以明显地将子代与无亲缘关系的家系区分开。本研究筛选出13对微卫星标记完全适用于荷包红鲤与德国镜鲤杂交家系的鉴定,为品种选育提供有效的鉴定工具。     

Genetic variation in common and runner bean of the Northern Meseta in Spain

The genetic variation within and between Spanish landraces or varieties of Phaseolus vulgaris L. (common bean) and P. coccineus L. (runner bean) has been estimated by means of isozymes and random amplified polymorphic DNA (RAPD) analyses. Likewise, storage protein and amino acid content in dry seeds have been estimated. Fifteen landraces (60 accessions) of P. vulgaris and six of P. coccineus (six accessions) have been studied. Of the seven isozymatic systems analyzed only three systems and three loci showed variability in each species. Isozyme analyses revealed that genetic variability within and between landraces exist in both species. Even variability within accession was detected in some P. vulgaris landraces. Comparison of isozyme data indicated that Spanish landraces have a lower level of genetic variability than wild American materials and probably also lower than American landraces. RAPD analysis allowed for the uniquely distinguishing of all landraces. Genetic similarity among landraces, estimated by both isozymes and RAPDs, were not related with the seed morphological characters (color, size and shape) which define each variety or landrace. Variation in protein and amino acid content among landraces was also detected. The average protein content in common bean (20.48%) was similar to values previously reported in this species and higher than the average in the runner bean landraces (16.33%). In relation to the amino acid content methionine and cysteine showed the lowest values in all samples, although the content of these two amino acids varied widely among landraces.     

A study on relative abundance,composition and length variation of microsatellites in 18 underutilized crop species

In the present work, genomic DNA libraries for 18 underutilized crop species (including 11 dicot and 7 monocot species) were enriched for several di- and tri- nucleotide microsatellites by using an optimized procedure. About 500–960 clones from each library were sequenced and all the sequences were characterized to have a comparative look on relative abundance, composition and length variations of perfect microsatellites among different crops. Sequence analysis revealed contrasting differences in the abundance of di- and tri-nucleotide microsatellites with the predominance of tri-nucleotide microsatellites in 11 crops and that of di-nucleotides in other 8 crops. Among di-nucleotide microsatellites, AG/GA class was the most abundant in all the crops except for four crops in which AC/CA class was predominant. Among tri-nucleotide repeats, AGC/GCA/CAG class was the most abundant in eight crops followed by AGG/GGA/GAG class in four crops, whereas in the remaining six crops, the most abundant class was highly variable. The longest di- and tri-nucleotide repeats were observed in Chinese cabbage and mungbean with the average lengths of 70 and 57 base pairs, respectively. These observations revealed species-specific in the distributions of microsatellite repeat motifs. The present study provides highly valuable information that can be useful in targeted development of specific microsatellites markers for genetic analysis of these underutilized crops.     

用鲤鱼EST-SSRs分子标记分析长江黑龙江鲤种群结构

从鲤鱼ESTs的数据库（10691个）中进行微卫星序列筛选，共发现微卫星序列127个，占整个ESTs数据库的1.19%，另外从本研究室构建的鲤鱼脑组织cDNA文库中筛选到微卫星序列20个。根据筛选得到的微卫星序列设计引物68对，合成引物40对，其中27对引物能够获得预期的目的片段，20对引物在所检测的群体内及群体间表现出多态性。用此20个多态性标记分析了长江鲤和黑龙江鲤的群体遗传结构，结果表明：长江鲤和黑龙江鲤在多数位点表现出较高的遗传多样性，20个位点的平均有效等位基因数分别为3.9135、3.4820；平均观察杂合度为0.6067、0.5667；平均期望杂合度为0.6737、0.6194；平均多态信息含量为0.6308、0.5714。长江鲤的遗传结构好于黑龙江鲤，两个群体的Hardy-Weinberg偏离指数较小，群体基本保持平衡，群体结构较合理；其中HLJE1、HLJE10位点在长江鲤和黑龙江鲤的扩增结果相差较大，长江鲤的多态性明显高于黑龙江鲤，位点多态信息含量相差一倍以上，可用于群体的鉴别。     

Characterization of Some Italian Common Bean (<Emphasis Type="Italic">Phaseolus Vulgaris</Emphasis> L.) Landraces by RAPD,Semi-random and ISSR Molecular Markers

Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and a semi-random PCR system were used to analyze the genetic diversity of 16 Italian common bean landraces and their relationship to four commercial cultivars. Of the primers tested, 8 ISSR, 6 RAPD and 7 semi-random primers produced polymorphic and reproducible DNA fragments. A higher proportion of polymorphic bands were observed using ISSR (85%) and semi-random (90%) primers than RAPD (69%) method. The combination of any two semi-random markers allowed the identification of all 20 bean genotypes. In contrast ISSR (except for primer (CAC)₃GC) and RAPD markers appeared to be less informative as more than two markers were necessary to achieve the same diagnostic level. Moreover, 7 ISSR, 2 RAPD and 8 semi-random exclusive bands were identified as putative population-specific markers. Semi-random and ISSR derived dendrograms showed similar tendencies in terms of genetic relatedness, whereas clustering of genotypes within groups was not similar when compared with the RAPD technique. Despite the different ability to resolve genetic variation among the investigated landraces, two major clusters with less than 60% (ISSR) and 40% (RAPD and semi-random) genetic similarity were formed with all three marker systems. The two groups were correlated with the phaseolin patterns and seed size of the landraces. The analysis showed that the cultivar ȁ8Lingua di Fuocoȁ9 and most of the landraces (13 out of 16) collected in Italy belong to the Andean gene pool, whereas only the three populations from Pratomagno belong to the Middle American gene pool.     

斑点叉尾鮰趋化因子基因SCYA113内含子1的多态性分析

运用变性聚丙烯酰胺凝胶电泳和PCR产物测序技术检测斑点叉尾鮰CC趋化因子基因SCYA113 内含子1的序列长度和多态性。在3个群体的60个个体中,斑点叉尾鮰CC趋化SCYA113 内含子1存在15种单元型和8种长度类型,长度类型分别为A、B、C、D、E、F、G和H型。对这8种序列进行比对分析发现,斑点叉尾鮰SCYA113 内含子1长度为395~443 bp, 8种长度类型中A、G、T、C的平均百分比为30.01%、15.43%、38.37%、16.19%,而且G+C（31.62%）含量明显小于A+T（68.38%）含量,内含子1与外显子1和2的连接处存在真核基因中mRNA剪接的识别信号GT/AG;引起长度变化的主要原因是短串联重复序列（微卫星序列）TTC和TTA引起的;除了微卫星序列以外,共检测到6个突变位点,其中3个转换位点为118（C→T）,209（G→A）,250（T→C）,3个颠换位点为266（T→A）,289（G→T）,387（G→C）,核苷酸多样性指数(π)为0.004,平均核苷酸差异(K)为1.576;在变异水平上,3个群体表现出一定的遗传差异,84群体中检测到8种长度类型和14种基因型,97群体中检测到8种长度类型和10种基因型,04群体中6种长度类型和10种基因型（缺少A、B等位基因）。从3个群体60个个体拥有22种频率较低(0.017~0.150)的基因型可以看出,斑点叉尾鮰的遗传基因资源较为丰富。     

The influence of European and American wild germplasm in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.) cultivars

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops.     

Cross-amplification and characterization of microsatellite loci for three species of <Emphasis Type="Italic">Theobroma</Emphasis> (Sterculiaceae) from the Brazilian Amazon

This study reports on the cross-species amplification of 23 microsatellite markers previously developed for Theobroma cacao L. (Sterculiaceae), source of chocolate in three economically important Amazonian species of Theobroma (T. grandiflorum, T. subincanum, T. sylvestre). Thirteen of the 23 microsatellite loci tested were polymorphic across the three species at 2–13 alleles per locus. The observed heterozygosity per locus varied from 0.18 to 0.84 and expected heterozygosity ranged from 0.28 to 0.87. The high level of transferability and genetic information content of these microsatellite loci indicate their usefulness for population genetic, mating system and breeding studies of these economically important Amazonian fruit trees.     

花生抗青枯病种质微卫星DNA的分离

提取抗青枯病花生材料基因组DNA,分别经Hae Ⅲ、Rsa Ⅰ酶切后,与生物素标记的6条微卫星探针杂交,富集微卫星DNA。测序分析后,得到非冗余序列180条,其中微卫星序列133条、小卫星序列47条,成功设计出141对引物。其中40对引物用于检测29份花生材料,发现5对为多态性引物,能很好地揭示野生种与栽培种四大类型的...     

The Genetic Diversity and Similarities Among <Emphasis Type="Italic">Kengyilia</Emphasis> Species Based on Random Amplified Microsatellite Polymorphism (RAMP)

The genetic diversity and similarities among 32 Kengyilia accessions, distributed to 14 species and one variety were analyzed by using random amplified microsatellite polymorphism (RAMP) markers. Of the 160 RAMP primer combinations tested, 40 (25%) produced polymorphic and clear bands. A total of 264 bands were produced by 40 primer combinations, among which 231 out of 264 bands (87.5%) were polymorphic. Two to 11 polymorphic bands could be amplified from each primer combination, with an average of 5.8 bands. The data of 264 bands were used for RAMP assay. By NTSYS-pc program, genetic similarity coefficients were generated and dendrogram was constructed using UPGMA. The genetic similarity coefficients ranged from 0.477 to 0.965 with the mean of 0.714. The results showed as follows: (1) distinct genetic differences were present among the different species; (2) the different accessions in a species were clustered together, respectively, which had larger genetic similarities and closer relations; (3) the species with similar morphological characters and the species from the same areas or neighboring geographical regions were clustered together; (4) the lowest genetic similarity was found between K. hirsuta (PI531618) and K. laxiflora (PI531631), while the highest genetic similarity was observed between K. hirsuta (Y2364) and K. hirsuta (Y2368); (5) RAMP results are basically comparable with those obtained from studies on morphology and cytology. It is a useful method for analysis of the genetic diversity and similarities in Kengyilia.     

Genetic diversity and population structure of a common bean (Phaseolus vulgaris L.) collection from Calabria (Italy)

Eighty-seven Phaseolus vulgaris landraces, still cultivated in Calabria (Italy), were investigated in order to study the patterns of common bean genetic diversity in this region, to better understand the evolutionary development of beans in Europe and to properly manage these genetic resources. Four American accessions and five Italian varieties were also included. Different markers, such as 12 microsatellites, seed traits, phaseolins and 100-seed weight were combined with different statistical approaches. For each microsatellite, expected (H _e ) and observed (H _o ) heterozygosities, polymorphism information content (PIC), probability of identity (PI) and homozygosity were calculated. Furthermore, in Calabrian group of bean landraces, total (N _a ) and private (N _pa ) number of alleles, observed (H _o ), expected heterozygosities (H _e ) and allelic richness (AR) were calculated. Genetic distances among landraces were estimated using Nei’s coefficient and a cluster analysis using the UPGMA algorithm was performed. The results clearly indicated that: (1) Calabrian germplasm showed a high level of diversity (H _e  = 0.595); (2) Mesoamerican and Andean gene pools were clearly distinguished in Calabrian germplasm, with the Andean gene pool predominating (83 %); (3) Calabrian landraces were largely hybridized within and between the gene pools. A model-based approach, using the STRUCTURE software, was adopted. Six groups, including 4 of Andean origin and one of Mesoamerican origin were identified. Even more interesting, a small group (8 %) showed a distinct genetic structure, in which interspecific hybridizations with runner bean (Phaseolus coccineus L.) could have occurred. Nevertheless, a relatively high proportion of Calabrian bean landraces (12.6 %) was derived from intra and interspecific hybridizations.     

RAPD markers in the analysis of genetic diversity among common bean germplasm from Central Himalaya

A set of 99 common bean germplasm collected from central Himalaya was investigated for their genetic variability using random amplified polymorphic DNA (RAPD) markers. Ten oligonucleotide primers, selected from 60 initially screened, generated 123 amplicon products. Of these, two amplicons were shared by all the accessions whereas 112 were polymorphic at least in two pair wise comparison. Nine unique bands identified were as low as 0.32 kb M.W. to as high as 3.5 kb and were confined to eight collections. All primers produced polymorphic amplicons though the extent of polymorphism varied with each primer. The primer OPF-17 was found to be most powerful and efficient as it generated a total of 17 bands of which 15 were polymorphic. RAPD markers data were analysed statistically using NTSYSpc.2.02e software and a dendrogram was generated using Jaccard’s similarity coefficient. The similarity coefficient values varied from 0.19 to 0.91. Grouping analysis revealed the categorization of 99 germplasm into 12 major branches with different level of similarity. Three branches namely branch 2, 3 and 5 out of 12 had only one accession. Branch 1 which consisted of three accessions was the most divergent as revealed by Jaccard’s similarity coefficient. Branching pattern of the accessions did not show any correlation with morphological data or altitudinal alignment of the accessions.