Analysis of Diversity in Chinese Cultivated Barley with Simple Sequence Repeats: Differences Between Eco-Geographic Populations

The genetic diversity of 116 barley accessions, representing five Chinese eco-geographic populations, was studied using simple sequence repeat (SSR) markers. The 21 SSR loci revealed 128 alleles with an average of 6.1 alleles per locus. The highest values of proportion of polymorphic loci (P) and gene diversity index (He) were obtained in the Northern (P = 1.00; He = 0.60) and the Yangtze River reaches and Southern populations (P = 1.00; He = 0.59). The lowest values were in the populations of the Yellow River reaches (P = 0.86; He = 0.44). The highest average number of alleles per locus (4.52) and number of unique alleles (7) were found in the Qinghai–Tibet plateau population. Cluster analysis revealed that together with the row type, strong eco-geographic variables influenced the classification. Associations of SSR and eco-geographic values were established for 11 SSR loci. Four to six markers were found to discriminate among geographic groups, which may serve as tools for diagnosis of the eco-geographic populations and provide evidence for the adaptive nature of SSR markers.     

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.     

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.     

Generation and analysis of expressed sequence tags for microsatellite marker development in Calamus simplicifolius C. F. Wei

Rattans serve as an important source of raw non-wood materials for furniture and handicraft industries worldwide. However, their genomic sequence information in public databases is very limited. In this study, a set of 2,528 good-quality expressed sequence tags (ESTs) were generated from a full-length cDNA library constructed previously with root, stem and male inflorescence tissues of Calamus simplicifolius C. F. Wei, a rattan species native to Hainan Island, China. The ESTs were assembled into 1,588 unigenes, including 1,221 singletons and 367 contigs. BlastX searches against the GenBank non-redundant protein database revealed that 1,248 (78.6 %) unigenes had at least one significant match (E ≤ 10^?5). The gene ontology functional classification assigned 991, 669 and 977 of the unigenes to the cellular component, molecular function and biological process categories, respectively. A total of 71 simple sequence repeat (SSR) loci were developed among these ESTs, including 65 polymorphic across 19 rattan species representing three genera. High levels of cross-species/genus transferability were observed for the EST-SSRs. For the polymorphic EST-SSR markers, the number of alleles per locus and polymorphic information content ranged from 2 to 25 (mean 11.1) and from 0.135 to 0.949 (mean 0.695), respectively. The EST sequences and the EST-SSR primers have been deposited in GenBank databases of EST (IDs JK838364–40891) and Probe (IDs Pr16718978–9048, to be assigned).     

Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2–5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.     

Assessment of molecular diversity and evolutionary relationship of kenaf (Hibiscus cannabinus L.), roselle (H. sabdariffa L.) and their wild relatives

Kenaf (Hibiscus cannabinus L.) and roselle (H. sabdariffa L.) are valuable fibre crop species with diverse end use. Phylogenetic relationship of 73 accessions of kenaf, roselle and their wild relatives from 15 countries was assessed using 44 inter-simple sequence repeat (ISSR) and jute (Corchorus olitorius L.) specific simple sequence repeats (SSR) markers. A total of 113 alleles were identified of which 61.95 % were polymorphic. Jute specific SSR markers exhibited high polymorphism and resolving power in kenaf, although ISSR markers exhibited higher resolving power than SSR markers. Number of polymorphic alleles varied from 1 to 5 for ISSR and 1 to 6 for SSR markers. Cultivated species exhibited higher allele polymorphism (57 %) than the wild species (35 %), but the improved cultivars exhibited lower genetic diversity compared to germplasm accessions. Accessions with common genetic lineage and geographical distribution clustered together. Indian kenaf varieties were distinct from cultivars bred in other countries and shared more genetic homology with African accessions. High genetic diversity was observed in the Indian (J = 0.35–0.74) and exotic kenaf germplasm collections (J = 0.38–0.79), suggesting kenaf might have been introduced in India from Africa through Central Asia during early domestication. Genetic similarity-based cluster analysis was in close accordance with taxonomic classification of Hibiscus.     

Population structure and genetic diversity analysis of Indian and exotic rice (Oryza sativa L.) accessions using SSR markers

In order to understand the population structure and genetic diversity among a set of 82 rice genotypes collected from different parts of the Asian countries including India were characterized using 39 microsatellite loci. The Population structure analysis suggested that the optimum number of subpopulations was four (K = 4) among the rice genotypes, whereas phylogenetic analysis grouped them into three populations. The results obtained from phylogenetic and STRUCTURE analysis proved to be very powerful for the differentiation of rice genotypes based on their place of origin. The genetic diversity analysis using 39 SSR loci yielded 183 scorable alleles, out of which 182 alleles were observed to be polymorphic with an average of 4.8 alleles per locus. The Polymorphism Information Content (PIC) values for all the polymorphic primers across 82 rice genotypes varied from 0.02 to 0.77, with an average of 0.50. Gene diversity (He) was found to be in the range of 0.02 (RM484) to 0.80 (OSR13) with an average value of 0.55, while heterozygosity (Ho) was observed with an average of 0.07, ranging from 0.01 (RM334) to 0.31 (RM316). The present study resulted in identification of seven highly polymorphic SSR loci viz., OSR13, RM152, RM144, RM536, RM489, RM259 and RM271 based on the parameters like PIC value (≥0.70), gene diversity (≥0.71), and polymorphic alleles (≥6). These seven polymorphic primers can effectively be used in further molecular breeding programs and QTL mapping studies of rice since they exhibited very high polymorphism over other loci. SSR analysis resulted in a more definitive separation of clustering of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions.     

Evaluation of the genetic diversity and population structure of sesame (Sesamum indicum L.) using microsatellite markers

Sixteen polymorphic microsatellite (SSR) markers, developed from an SSR-enriched genomic DNA library of sesame (Sesamum indicum L.), were used to assess genetic diversity, phylogenetic relationships, and population structure among 150 sesame accessions collected from 22 countries. A total of 121 alleles were detected among the sesame accessions. The number of detected alleles varied from 2 to 18, with an average of 7.6 alleles per locus. Polymorphism information content values ranged from 0.03 to 0.79, with an average of 0.42. These values indicated an excess of heterozygous individuals at 16 loci and an excess of homozygous individuals at three loci. Of these, 32 genotype-specific alleles were identified at 11 of 16 polymorphic SSR markers. Cluster analyses were performed by accession and population, revealing a complex accession distribution pattern with mean genetic similarity coefficient of 0.45 by accession and 0.52 by population. The wide variation in genetic similarity among the accessions revealed by SSRs reflected a high level of polymorphism at the DNA level. Model-based structure analysis revealed the presence of three groups that were basically consistent with the clustering results based on genetic distance. These findings may be used to augment the sesame germplasm and to increase the effectiveness of sesame breeding.     

Determination of the population structure of common bean (Phaseolus vulgaris L.) accessions using lipoxygenase and resistance gene analog markers

The common bean (Phaseolus vulgaris L.) is an important food legume throughout the world. Because of the conservation across different plant species, it is possible to evaluate the degree of genetic diversity in the common bean using gene-based marker techniques. The lipoxygenase (LOX) and resistance gene analog (RGA) genes play an important role in the response to biotic and abiotic stresses. Eighty-six common bean accessions were genotyped using gene-based LOX and RGA markers. The total number of polymorphic bands ranged from 193 for LOX to 17 for RGA markers. We detected considerable diversity with a mean of 8.7 alleles per primer for the LOX analysis. For the RGA markers, the number of alleles per polymorphic locus varied from 1 to 4 with an average allele number of 2.8. The genetic similarity between the accessions based on the LOX and RGA markers ranged from 0.12 to 0.55. Using STRUCTURE, 3 groups were revealed among the accessions. The results of this study should provide valuable information for future studies on the genetic diversity of common bean accessions and for association mapping studies examining the relationships between the genotypic and phenotypic traits related to the stress response.     

Identification,characterization, validation and cross-species amplification of genic-SSRs in Indian Mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

Brassica juncea is an economically important oilseed crop worldwide. It has limited genomic resources at present. We generated 47,962,057 expressed sequence reads which were assembled into 45,280 unigenes. A total of 4108 SSR loci (≥10 bp) were identified in these unigenes. Trinucleotide was the most frequent repeat unit (59.91 %) followed by di- (38.66 %), tetra - (0.71 %), hexa - (0.49 %) and pentanucleotide repeats (0.24 %). Primers were designed for 2863 SSR loci among which 460 were selected for primer synthesis. A total of 339 loci amplified successfully of which 134 (39.5 %) exhibited polymorphism among six B. juncea genotypes with PIC values ranging from 0.18 to 0.81. Further, 25 polymorphic SSRs were used for analysis of genetic variability in 25 genotypes of Brassicas and their wild relatives. Two to five alleles with PIC values 0.22–0.66 were detected at these loci. The dendrogram grouped the genotypes according to their known pedigree/systematic position.     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

Diversification of indigenous gene- pool by using exotic germplasm in lentil (Lens culinaris Medikus subsp. culinaris)

Genetic diversity was studied among 21 accessions of lentil using SSR markers and morphological traits in order to assess the diversification of Indian gene-pool of lentil through introgression of exotic genes and introduction of germplasm. Among these , 16 genotypes either had ‘Precoz’ gene, an Argentine line in their pedigree or genes from introduced lines from ICARDA. Sixty five SSR markers and eight phenotypic traits were used to analyse the level of genetic diversity in these genotypes. Forty three SSR markers (66 %) were polymorphic and generated a total of 177 alleles with an average of 4.1 alleles per SSR marker. Alleles per marker ranged from 2 to 6. The polymorphic information content ranged 0.33 to 0.80 with an average of 0.57, suggesting that SSR markers are highly polymorphic among the studied genotypes. Genetic dissimilarity based a dendrogram grouped these accessions into two main clusters (cluster I and cluster II) and it ranged 33 % to 71 %, suggesting high level of genetic diversity among the genotypes. First three components of PCA based morphological traits explained higher variance (95.6 %) compared to PCA components based on SSR markers (42.7 %) of total genetic variance. Thus, more diversity was observed for morphological traits and genotypes in each cluster and sub-cluster showed a range of variability for seed size, earliness, pods/plant and plant height. Molecular and phenotypic diversity analysis thus suggested that use of germplasm of exotic lines have diversified the genetic base of lentil germplasm in India. This diversified gene-pool will be very useful in the development of improved varieties of lentil in order to address the effect of climate change, to adapt in new cropping systems niches such as mixed cropping, relay cropping, etc. and to meet consumers’ preference.     

Development and characterization of 15 microsatellite loci for Cariniana estrellensis and transferability to Cariniana legalis, two endangered tropical tree species

From a genomic library enriched for GA/CA repeats, 15 highly polymorphic microsatellite markers were developed for Cariniana estrellensis, a tropical forest tree. The microsatellite loci were screened in 49 mature trees found between Pardo river and Mogi-Guaçu river basins, in the state of São Paulo, Brazil. A total of 140 alleles were detected with an average of 9.33 alleles per locus. The expected heterozygosity ranged from 0.37 to 0.88. These loci showed a high probability of paternity exclusion. Additionally, 12 loci were effectively transferred to Cariniana legalis. High levels of polymorphism make the present SSR markers useful for population genetic studies.     

Microsatellite marker-based diversity and population genetic analysis of selected lowland and mid-altitude maize landrace accessions of India

Maize (Zea mays L.) harbours significant genetic diversity not only in its centre of origin (Mexico) but also in several countries worldwide, including India, in the form of landraces. In this study, DNA fingerprinting of 48 landrace accessions from diverse regions of India was undertaken using 42 fluorescent dye-labeled Simple Sequence Repeat (SSR) markers, followed by allele resolution using DNA sequencer and analysis of molecular diversity within and among these landraces. The study revealed a large number of alleles (550), with high mean number of alleles per locus (13.1), and Polymorphism Information Content (PIC) of 0.60, reflecting the level of diversity in the landrace accessions. Besides identification of 174 unique alleles in 44 accessions, six highly frequent SSR alleles were detected at six loci (phi014, phi090, phi112, umc1367, phi062 and umc1266) with individual frequencies greater than 0.75, indicating that chromosomal regions harboring these SSR alleles are not selectively neutral. F statistics revealed very high genetic differentiation, population subdivision and varying levels of inbreeding in the landraces. Analysis of Molecular Variance showed that 63 % of the total variation in the accessions could be attributed to within-population diversity, and 37 % represented between population diversity. Cluster analysis of SSR data using Nei’s genetic distance and UPGMA revealed considerable genetic diversity in these populations, although no clear separation of accessions was observed based on their geographic origin.     

Development of genomic simple sequence repeat markers in faba bean by next-generation sequencing

Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. Development of genetic markers for faba bean is important to study diversity and for molecular breeding. In this study, we used Next Generation Sequencing (NGS) technology for the development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5 % and 20.9 % of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96 %) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5 % polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions.     

Identification and characterization of 43 novel polymorphic EST-SSR markers for arum lily,Zantedeschia aethiopica (Araceae)

• Premise of the study: A new set of microsatellite or simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) was developed for arum lily (Zantedeschia aethiopica), which is one of the most iconic and widely recognized ornamental plants in the world. • Methods and Results: Using 2175 unigenes derived from 4283 random ESTs in arum lily, 166 primer pairs were designed and tested for amplification in 24 accessions from Asia, Europe, and Africa. A total of 43 loci were polymorphic, with the number of alleles per locus ranging from two to 10. The observed heterozygosity, expected heterozygosity, and polymorphism information content ranged from 0.2313 to 0.8480, 0.3034 to 0.8648, and 0.1015 to 0.7364, respectively. • Conclusions: These novel polymorphic EST-SSR markers will facilitate future studies of genetic variation and molecular-assisted breeding systems in arum lily.     

Comparison of SSR and cytochrome P-450 markers for estimating genetic diversity in Picrorhiza kurrooa L.

Picrorhiza kurrooa L., a high altitude medicinal plant, is known for its drug content called Kutkin. In the present study, DNA-based molecular marker techniques, viz. simple sequence repeats (SSR) and cytochrome P-450 markers were used to estimate genetic diversity in Picrorhiza kurrooa. Twenty five accessions of Picrorhiza kurrooa, collected from ten different eco-geographical locations were subjected to 22 SSR and eight cytochrome P-450 primer pairs, out of which 13 SSR markers detected mean 5.037 alleles with a mean polymorphic information content (PIC) of 0.7718, whereas eight cytochrome P-450 markers detected mean 5.0 alleles with a mean PIC of 0.7596. Genetic relationship among the accessions was estimated by constructing the dendrograms using SSR and cytochrome P-450 data. There was a clear consistency between SSR and cytochrome P-450 trees in terms of positioning of most Picrorhiza accessions. SSR markers could cluster various Picrorhiza kurrooa accessions based on their geographical locations whereas cytochrome P-450 markers could cluster few accessions as per their geographical locations. The Mantel test between SSR and cytochrome P-450 markers revealed a good fit correlation (r = 0.6405). The dendrogram constructed using the combined data of SSR and cytochrome P-450s depicted two clusters of accessions based on its eco-geographical locations whereas two clusters contained the accessions from mixed eco-geographical locations. Overall, the results of the present study point towards quiet high degree of genetic variation among the accessions of each eco-geographic region.