Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

Genomic diversity among sorghum genotypes with resistance to sorghum shoot fly, Atherigona soccata

Host plant resistance is one of the important components for management of sorghum shoot fly, Atherigona soccata. The levels of resistance in cultivated germplasm are low to moderate, and therefore, it is important to identify sorghum genotypes with diverse mechanisms of resistance based on physico-chemical and or molecular markers. We assessed the genetic diversity of 15 sorghum genotypes with different levels of resistance/susceptibility to shoot fly, A. soccata using 93 sorghum simple sequence repeat (SSR) primer pairs and simultaneously characterized for 15 morpho-biochemical traits associated with shoot fly resistance. Of these 93 SSR primer pairs, amplification products from 79, thought to correspond to single-copy loci distributed across all ten sorghum chromosome pairs, showed good polymorphism across the 15 sorghum genotypes. The polymorphic information content (PIC) values of these 79 SSR markers ranged from 0.06 to 0.86. The Principal Coordinate Analyses (PCoA) and cluster analyses based on dissimilarity matrices derived from SSR based allelic variation (Neighbor-Joining distance) and variation in 15 morpho-biochemical traits (based on Gower??s distance), revealed grouping of most susceptible genotypes in single cluster. The improved breeding lines grouped with resistant or susceptible genotypes, based on shared pedigree. Based on these results, three resistant accessions viz., IS 1054, IS 1057 and IS 4664 were found diverse to IS 18551, which is widely used as shoot fly resistance donor. These diverse sources, after further characterization for resistance mechanisms, can be used in breeding programs for improving shoot fly resistance.     

Molecular characterization and genetic diversity analysis of soybean (Glycine max (L.) Merr.) germplasm accessions in India

Molecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard’s similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0266-y) contains supplementary material, which is available to authorized users.     

Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus×domestica borkh. core subset collection

 A collection of 66 Malus×domestica Borkh. accessions from the USDA-ARS Plant Genetic Resources Unit’s core collection was screened with a set of eight SSR (simple sequence repeat) primers developed at the PGRU in order to determine genetic identities, estimate genetic diversity, and to identify genetic relationships among these accessions. All eight primer pairs generated multiple fragments when used in amplification reactions with DNA from these accessions. High levels of variation were detected with a mean of 12.1 alleles per locus and a mean heterozygosity across all eight loci of 0.693. The eight primer pairs utilized in this study unambiguously differentiated all but seven pairs of accessions in this collection of 66 M.×domestica Borkh. genotypes. The probability of matching any two genotypes at all eight loci in this study was approximately 1 in 1 billion. The markers detected two misnamed accessions in the collection. Genetic-identity data produced a genetic-relatedness phenogram which was concordant with geographic origins and/or known pedigree information. These SSR markers show great promise as tools for managing Malus ex situ germplasm collections as well as for collection and preservation strategies concerning wild Malus populations in situ. Received: 28 March 1998 / Accepted: 29 April 1998     

Molecular characterization and similarity relationships among apricot ( Prunus armeniaca L.) genotypes using simple sequence repeats

A collection of 48 apricot genotypes, originated from diverse geographic areas, have been screened with 37 SSR primer pairs developed in different species of Prunus in order to identify and characterize the genotypes and establish their genetic relations. Thirty one of those primer pairs resulted in correct amplifications and 20 produced polymorphic repeatable amplification patterns with the 48 genotypes studied. A total of 82 alleles were detected for the 20 loci. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used. The results obtained evidence for the cross-species transportability of microsatellite sequences, allowing the discrimination among different genotypes of a given fruit-tree species with sequences developed in other species. UPGMA cluster analysis of the similarity data grouped the genotypes studied according to their geographic origin and/or their pedigree information. Received: 5 April 2001 / Accepted: 4 May 2001     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.)

We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L.). Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci. Five primer pairs amplified polymorphic products of the expected size range. SSR polymorphism was explored in a set of 46 olive cultivars. A total of 26 alleles were detected for the five loci. Heterozygosity ranged from 0.46 to 0.71. Ninety one per cent of the cultivars had unique multilocus genotypes. Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’. Received: 3 February 2000 / Accepted: 21 March 2000     

Development, characterization and inheritance of new microsatellites in olive (Olea europaea L.) and evaluation of their usefulness in cultivar identification and genetic relationship studies

Twelve new microsatellites have been developed in olive. For that purpose, a genomic library of the olive cultivar ‘Arbequina’ was enriched for GA, GT and ACT repeats. Two methods of screening yielded 27 sequences containing microsatellites out of the 119 clones sequenced. The GA repeat seems to be the most abundant motif. Among sequences containing microsatellites, 4 (14.8%) were redundant, 1 (3.7%) was previously described in the literature and 12 (44.4%) could not be used for primers design because the repeat motifs were incomplete. Suitable primer pairs were obtained for the remaining 10 (37.0%) sequences plus an additional 14 recovered from a formerly developed library. For the 24 primer pairs designed, 4 failed to amplify, 8 produced a complex bands pattern and 12 succeeded in giving amplification products. Considering these 12 primer pairs, 10 showed single locus amplification, whereas the other 2 revealed two loci each. This was demonstrated by studying allele segregation in two olive progenies. Sixty-eight alleles were detected for the 12 microsatellites when 51 olive cultivars were analysed. The number of alleles per locus ranged from 1 to 13. The expected heterozygosity varied between 0 and 0.83. All pairs of cultivars could be distinguished using only three microsatellites due to their great discrimination power value. The data coming from genotyping the 51 olive cultivars for 7 out of the 12 new microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice similarity coefficient. Cultivar association according to their geographical origin was observed.     

SSR标记揭示的云南地方稻品种遗传多样性及其保育意义

为了探索水稻(Oryza sativa L.)地方品种的遗传多样性及其有效保育方法,对采自云南省17个村寨的82个水稻地方品种和3个国际常用的典型籼稻和粳稻品种进行了微卫星(SSR)分子标记的分析.利用19对SSR引物在85个水稻品种中共扩增出了83个基因型,其分子量变异在100～500 bp之间.基于各品种SSR基因型遗传相似系数聚类分析而获得的UPGMA树状图表明各水稻品种之间存在较大的遗传多样性,其相似系数变异在0.15～0.90之间.但这些地方品种的遗传多样性并非呈均等的地理分布.这85个水稻品种在相似系数为0.52之处分为二组,其中一组包括几乎所有的籼稻品种,而另一组包括全部的粳稻品种,表明SSR标记能很好揭示水稻籼-粳分化.同时,有些来自不同采集地的同名品种表现出一定的遗传差异,说明同名异物的现象存在.云南水稻地方品种具有丰富的遗传多样性,对其有效保育十分重要和迫切,但只有根据遗传多样性的水平和分布特点,采用正确的保育对策和取样方法才能确保对云南水稻地方品种的有效保育.结果进一步表明,选用适当的微卫星引物,可以为准确鉴定籼稻和粳稻品种及研究其进化规律提供有效的分子标记方法,并有利于有目标的水稻遗传资源保育和育种创新.     

SSR标记揭示的云南地方稻品种遗传多样性及其保育意义

为了探索水稻(Oryza　sativa　L.)地方品种的遗传多样性及其有效保育方法,对采自云南省17个村寨的82个水稻地方品种和3个国际常用的典型籼稻和粳稻品种进行了微卫星(SSR)分子标记的分析。利用19对SSR引物在85个水稻品种中共扩增出了83个基因型,其分子量变异在100～500　bp之间。基于各品种SSR基因型遗传相似系数聚类分析而获得的UPGMA树状图表明各水稻品种之间存在较大的遗传多样性,其相似系数变异在0.15～0.90之间。但这些地方品种的遗传多样性并非呈均等的地理分布。这85个水稻品种在相似系数为0.52之处分为二组,其中一组包括几乎所有的籼稻品种,而另一组包括全部的粳稻品种,表明SSR标记能很好揭示水稻籼-粳分化。同时,有些来自不同采集地的同名品种表现出一定的遗传差异,说明同名异物的现象存在。云南水稻地方品种具有丰富的遗传多样性,对其有效保育十分重要和迫切,　但只有根据遗传多样性的水平和分布特点,采用正确的保育对策和取样方法才能确保对云南水稻地方品种的有效保育。结果进一步表明,选用适当的微卫星引物,可以为准确鉴定籼稻和粳稻品种及研究其进化规律提供有效的分子标记方法,并有利于有目标的水稻遗传资源保育和育种创新。     

Development, characterization, and cross-species/genera transferability of SSR markers for rubber tree (Hevea brasiliensis)

Genomic simple sequence repeat (SSR) markers are particularly valuable in studies of genetic diversity, evolution, genetic linkage map construction, quantitative trait loci tagging, and marker-assisted selection because of their multi-allelic nature, reproducibility, co-dominant inheritance, high abundance, and extensive genome coverage. The traditional methods of SSR marker development, such as genomic-SSR hybrid screening and microsatellite enrichment, have the disadvantages of high cost and complex operation. The selectively amplified microsatellite method is less costly and highly efficient as well as being simple and convenient. In this study, 252 sequences with SSRs were cloned from the rubber tree (Hevea brasiliensis) genome from which 258 SSR loci were obtained. The average repeat number was six. There were only 10 (3.9%) mononucleotide, trinucleotide, and pentanucleotide repeats, whereas the remaining 248 (96.1%) were dinucleotide repeats, including 128 (49.6%) GT/CA repeats, 118 (45.7%) GA/CT repeats, and 2 (0.8%) AT/TA repeats. A total of 126 primer pairs (see ESM) were successfully designed of which 36 primer pairs generated polymorphic products from 12 accessions of the cultivated species, 4 related species, and 3 species of the family Euphorbiaceae. In addition, investigations based on four genomic SSRs (GAR4, ACR22, CTR25, and GTR28) by cloning and sequencing provided evidence for cross-species/genera applicability, and homologous sequences were obtained from the rubber tree and Euphorbiaceae. Further analysis about the variation of the flanking regions of the four markers was carried out.     

Genetic Diversity and Population Structure Among Oat Cultivars and Landraces

In this study, genetic diversity among 177 oat (Avena sativa L.) accessions including both white and red oat landraces and 36 commercial cultivars was studied for simple sequence repeat (SSR) loci. Thirty-one genomic and expressed sequence tags (EST)-derived primer pairs were selected according to high polymorphism from an initial 66 SSR batch. Markers revealed a high level of polymorphism, detecting a total of 454 alleles. The average gene diversity for the whole sample was 0.29. Genetic similarity, calculated using the Dice coefficient, was used for cluster analysis, and principal component analysis was also applied. In addition, population structure using a Bayesian clustering approach identified discrete subpopulation based on allele frequency and showed similar clustering of oat genotypes in four groups. Accessions could be classified into four main clusters that clearly separated the commercial cultivars, the red oat landraces and two clusters of white oat landraces. Cultivars showed less diversity than the landraces indicating a reduction of genetic diversity during breeding, whereas white oat landraces showed higher diversity than red ones. The average polymorphic information content of 0.80 for the SSR loci indicated the usefulness of many of the SSR for genotype identification. In particular, two markers, MAMA5 and AM04, with a total of 50 alleles and a high discrimination power (>0.90), were sufficient to discriminate among all commercial cultivars studied highlighting their potential use for variety identification.     

Substantial genetic diversity in cultivated Moroccan olive despite a single major cultivar: a paradoxical situation evidenced by the use of SSR loci

To assess the genetic diversity in Moroccan cultivated olive, Olea europaea L. subsp. europaea, we performed molecular analysis of olive trees sampled in four geographic zones representing all areas of traditional olive culture. The analysis of 215 trees using 15 simple sequence repeat (SSR) loci revealed 105 alleles distributed among 60 SSR profiles. The analysis of chloroplast deoxyribonucleic acid polymorphism for these 60 olive genotypes allowed to identify four chlorotypes: 42 CE1, one CE2, nine COM1 and eight CCK. Among the 60 SSR profiles, 52 corresponded to cultivated olive trees for which neither denomination nor characterisation is available. These local olive genotypes displayed a spatial genetic structuring over the four Moroccan geographic zones (northwest, north centre, Atlas and southwest), as pairwise Fst values ranged from 0.0394 to 0.1383 and varied according to geographic distance. As single alleles detected in local olive were also observed in Moroccan oleaster populations, results suggest that plant material was mainly selected from indigenous populations. The assumption that Picholine marocaine cultivar is a multi-clonal cultivar was not supported by our data because we found a single genotype for 112 olive trees representing 31 to 93% of the olives sampled locally in the 14 different areas. Picholine marocaine and the few other named cultivars do not seem to belong to the same gene pools as the unnamed genotypes cultivated only locally. The situation is paradoxical: a substantial genetic diversity in Moroccan olive germplasm, probably resulting from much local domestication, but a single cultivar is predominant.     

Identification of unique alleles and assessment of genetic diversity of rabi sorghum accessions using simple sequence repeat markers

Genetic diversity among 42 sorghum accessions representing landraces (19), advanced breeding lines (16), local cultivars (2) and release varieties (5) with 30 simple sequence repeat (SSR) markers revealed 7.6 mean number of alleles per locus showing 93.3% polymorphism and an average polymorphism information content of 0.78 which range from 0.22 (Xtxp12) and 0.91(Xtxp321). The average heterozygosity and effective number of alleles per locus were 0.8 and 6.65 respectively. Cluster analysis based on microsatellite allelic diversity clearly demarcated the accessions into ten clusters. A total of 24 unique alleles were obtained from seven SSR loci in 23 accessions in a size range of 110–380 bp; these unique alleles may serve as diagnostic tools for particular region of the genome of respective genotypes. Selected SSR markers from different linkage groups provided an accurate way of determining genetic diversity at the molecular level.     

野生花生种质的SSR遗传多样性

以花生属(Arachis)6个区组24种(包括栽培种)84份种质为材料,用SSR技术对其亲缘关系和遗传多样性进行了分析.从206对SSR引物中筛选到59对能扩增出稳定的多态性条带的引物,这些引物能在花生属基因组DNA中扩增出1～6个DNA片段.结果表明,84份种质的遗传距离为0.04～0.93,平均为0.64,其中匍匐区组的A.appressipila的2份种质(G4与G5)的遗传距离最小(0.04),匍匐区组的A.rigonii(G14)与根茎区组的A.glabrata(G28)的遗传距离最大(0.93).聚类分析结果与花生属的区组分类基本一致,栽培种花生被聚在花生区组中,而且7份栽培种被聚在同一亚亚组中,相同植物学类犁(相当于变种)的材料均被分别聚在一起.异形花区组与直立区组的亲缘关系最近,与花生区组的亲缘关系较近的是匍匐区组.花牛区组的二倍体野生种A.villosa、A.duranensis和A.benensis与栽培种化生关系较近,可以作为桥梁物种来转移其他野生花生的优良基因.     

Evaluation of SSR Markers for the Assessment of Genetic Diversity and Fingerprinting of Gossypium hirsutum Accessions

A total 177 simple sequence repeat (SSR) markers were screened using a set of 47 Upland cotton genotypes comprising 14 commercial varieties, 14 germplasm accessions and 19 advanced breeding lines to identify informative markers for genetic diversity assessment and fingerprinting in G. hirsutum. Only 21% (381177) of SSR markers tested showed polymorphism with a mean of 2.18 alleles per locus and with average polymorphism information content (PIC) of 0.32. The SSR markers revealed a Jaccard’ similarity coefficient ranging between 0.43 and 0.89, with an average of 0.67 among accessions. Cluster analysis using unweighted pair group method with arithmetic averages (UPGMA) and principal component analysis (PCA) indicated that majority of the genotypes were very closely related. All the 47 genotypes showed heterorygosity for at least one of the SSR loci. We discovered 19 rare and 6 unique alleles among the tested genotypes of cotton. Fingerprint based on all the 38 loci revealed a probability of identical match by chance of 3.98x10. A set of ten SSR markers was identified which could distinguish all the 47 genotypes with a moderate probability of identical match by chance (X?_D ⁿ = 0.01).     

Genetic diversity of loquat germplasm (Eriobotrya japonica (Thunb) Lindl) assessed by SSR markers.

Genetic relationships among 40 loquat (Eriobotrya japonica (Thunb) Lindl) accessions that originated from different countries and that are part of the germplasm collection of the Instituto Valenciano de Investigaciones Agrarias (IVIA) (Valencia, Spain) were evaluated using microsatellites. Thirty primer pairs flanking microsatellites previously identified in Malus x domestica (Borkh.) were assayed. Thirteen of them amplified polymorphic products and unambiguously distinguished 34 genotypes from the 40 accessions analyzed. Six accessions showing identical marker patterns were Spanish local varieties thought to have been derived from 'Algerie' by a mutational process very common in loquat species. A total of 39 alleles were detected in the population studied, with a mean value of 2.4 alleles per locus. The expected and observed heterozygosities were 0.46 and 51% on average, respectively, leading to a negative value of the Wright's fixation index (-0.20). The values of these parameters indicate a smaller degree of genetic diversity in the set of loquat accessions analyzed than in other members of the Rosaceae family. Unweighted pair-group method (UPGMA) cluster analysis, based on Nei's genetic distance, generally grouped genotypes according to their geographic origins and pedigrees. The high number of alleles and the high expected heterozygosity detected with SSR markers developed in Malus x domestica (Borkh.) make them a suitable tool for loquat cultivar identification, confirming microsatellite marker transportability among genera in the Rosaceae family.