Exploring the genetic diversity of Ethiopian grass pea (Lathyrus sativus L.) using EST-SSR markers

Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequence-based markers for less-studied species. In this study, EST-simple sequence repeat (SSR) markers developed from Lathyrus sativus L. EST sequences and cross-transferable EST-SSRs derived from Medicago truncatula L. were utilized to investigate the genetic diversity among grass pea populations from Ethiopia. A total of 45 alleles were detected using eleven EST-SSRs with an average of four alleles per locus. The average polymorphism information content for all primers was 0.416. The average gene diversity was 0.477, ranging from 0.205 for marker Ls942 to 0.804 for MtBA32F05. F(ST) values estimated by analysis of molecular variance were 0.01, 0.15, and 0.84 for among regions, among accessions and within accessions respectively, indicating that most of the variation (84%) resides within accessions. Model-based cluster analysis grouped the accessions into three clusters, grouping accessions irrespective of their collection regions. Among the regions, high levels of diversity were observed in Gojam, Gonder, Shewa and Welo regions, with Gonder region showing a higher number of different alleles. From breeding and conservation aspects, conducting a close study on a specific population would be advisable for genetic improvement in the crop, and it would be appropriate if future collection and conservation plans give due attention to under-represented regions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9662-y) contains supplementary material, which is available to authorized users.     

Analysis of durum wheat germplasm adapted to different climatic conditions

A study of the extent and patterns of microsatellite diversity in 234 genotypes from Ethiopian durum wheat (Triticum turgidum) landraces was conducted to identify areas of diversity that could be used as a source of new germplasm for developing high yielding and stable varieties. Landraces belonging to nine populations, from three Ethiopian regions [Tigray (T), Gonder (G) and Shewa (S)] with different climates, were analysed by using 28 simple sequence repeat (SSR) markers. The level of polymorphism was high and quite consistent among populations underlining the great diversity existing. The highest level of diversity was found within populations, about 75.9%, while about 5.3% was attributed to differences between regions. The level of expected heterozygosity was on an average, rather high, ranging from 39% to 56%, whereas the observed heterozygosity was, on an average, limited to 14%. An average of about five alleles per locus was detected in each population. Nevertheless, alleles were not equally present in populations as confirmed by the high level of expected heterozygosity. The polymorphism information content (PIC) for the markers assessed showed a wide range of values from 0.14 to 0.92. The likelihood relationships among the nine Ethiopian populations indicated that the material collected in the Gonder region (a wet climate) was genetically more diverse than the materials from Shewa and Tigray (dryer climates). The high number of loci in linkage disequilibrium (LD), up to 23, has demonstrated that the loci were associated irrespective of their physical location. This holds true even if the loci are located on different chromosome arms. Genetic diversity values between populations was very different and was used to produce a dendrogram showing population relationships.     

Use of RAPD and ISSR Markers in Detection of Genetic Variation and Population Structure among Fusarium oxysporum f. sp. ciceris Isolates on Chickpea in Turkey

Genetic variation among the isolates of Fusarium oxysporum f. sp. ciceris, the causal agent of chickpea wilt worldwide, was analysed using pathogenicity tests and molecular markers – random amplified polymorphic DNA (RAPD) and inter‐simple sequence repeat (ISSR) polymorphism. Hundred and eight isolates were obtained from diseased chickpea plants in 13 different provinces of Turkey, out of which 74 isolates were assessed using 30 arbitrary decamer primers and 20 ISSR primers. Unweighted pair‐grouped method by arithmetic average cluster analysis of RAPD, ISSR and RAPD + ISSR datasets provided a substantially similar discrimination among Turkish isolates and divided into three major groups. Group 1, 2 and 3 consisted of 41, 18 and 15 isolates, respectively. These methods revealed a considerable genetic variation among Turkish isolates, but no correlation with regard to the clustering of isolates from different geographic regions. Analysis of molecular variance confirmed that most genetic variability resulted from the differences among isolates within regions. Our results also indicated that the low‐genetic differentiation (F_ST) and high gene flow (Nm) among populations had a significant effect on the emergence and evolutionary development of F. oxysporum f. sp. ciceris. This is the first report on genetic diversity and population structure of F. oxysporum isolates on chickpea in Turkey.     

Genome-wide insertion–deletion (InDel) marker discovery and genotyping for genomics-assisted breeding applications in chickpea

We developed 21,499 genome-wide insertion–deletion (InDel) markers (2- to 54-bp in silico fragment length polymorphism) by comparing the genomic sequences of four (desi, kabuli and wild C. reticulatum) chickpea [Cicer arietinum (L.)] accessions. InDel markers showing 2- to 6-bp fragment length polymorphism among accessions were abundant (76.8%) in the chickpea genome. The physically mapped 7,643 and 13,856 markers on eight chromosomes and unanchored scaffolds, respectively, were structurally and functionally annotated. The 4,506 coding (23% large-effect frameshift mutations) and regulatory InDel markers were identified from 3,228 genes (representing 11.7% of total 27,571 desi genes), suggesting their functional relevance for trait association/genetic mapping. High amplification (97%) and intra-specific polymorphic (60–83%) potential and wider genetic diversity (15–89%) were detected by genome-wide 6,254 InDel markers among desi, kabuli and wild accessions using even a simpler cost-effective agarose gel-based assay. This signifies added advantages of this user-friendly genetic marker system for manifold large-scale genotyping applications in laboratories with limited infrastructure and resources. Utilizing 6,254 InDel markers-based high-density (inter-marker distance: 0.212 cM) inter-specific genetic linkage map (ICC 4958 × ICC 17160) of chickpea as a reference, three major genomic regions harboring six flowering and maturity time robust QTLs (16.4–27.5% phenotypic variation explained, 8.1–11.5 logarithm of odds) were identified. Integration of genetic and physical maps at these target QTL intervals mapped on three chromosomes delineated five InDel markers-containing candidate genes tightly linked to the QTLs governing flowering and maturity time in chickpea. Taken together, our study demonstrated the practical utility of developing and high-throughput genotyping of such beneficial InDel markers at a genome-wide scale to expedite genomics-assisted breeding applications in chickpea.     

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R² at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea.     

SPAR methods revealed high genetic diversity within populations and high gene flow of Vanda coerulea Griff ex Lindl (Blue Vanda), an endangered orchid species

Molecular genetic fingerprints of seven populations of Vanda coerulea comprising of thirty-two genotypes from Northeast India were developed using PCR based markers. Genetic variability in the wild genotypes of V. coerulea was analyzed using two different single primer amplification reactions (SPAR) methods, viz., random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR). A total of 32 genotypes were used to investigate the existing natural genetic diversity at intra-specific level. Two hundred and twenty six (226) amplification products were scored by RAPD and ISSR, both of which collectively showed 58.88% polymorphism with a mean intra-population genetic diversity (Hpop) of 0.119. However, their level of diversity at inter- and intra-population levels was significant, with the percentage of polymorphic loci (Pp) ranging from 17.70% to 45.13%, Shannon's information index (I) from 0.105 to 0.268 and Nei's gene diversity (h) from 0.072 to 0.185 with mean Nei's gene diversity 0.174 and the overall estimate of gene flow being (Nm) 1.165. Analysis of molecular variance (AMOVA) showed 96.07% of variation at intra-population level, whereas 3.93% variation was recorded at inter-population level. Only one major cluster was detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Present investigation suggests the efficiency of SPAR methods to estimate the genetic diversity of V. coerulea and can be seen as a starting point for future research on the population and evolutionary genetics of this species.     

SSR analysis of genetic diversity and structure of the germplasm of faba bean (Vicia faba L.)

Assessing the diversity and genetic structure of faba bean (Vicia faba L.) germplasm is essential to improve the quality and yield of this economically important crop. In this study, simple sequence repeats (SSRs) were utilized to evaluate the diversity and structure of 35 faba bean genotypes originating from three different geographical regions (Northern Africa, Eastern Africa, and Near East). All 15 SSR loci generated a total of 100 alleles. The allele number per locus varied from 4 to 11, with a mean of 6.67. The expected heterozygosity (H_e) of SSR loci ranged between 0.51 and 0.81, with a mean of 0.63. The PIC value also varied from 0.44 to 0.78, with an average of 0.58. The expected heterozygosity of 22 faba bean genotypes was higher than the observed one. Interestingly, AMOVA analysis showed that much of variability resided within accessions (79.2%). A highly significant difference among regions was also evidenced, and represented 5.3% of the total variation. Moreover, cluster analysis divided the 35 faba bean genotypes into two main clusters. The first main cluster comprised all faba bean genotypes originating from the Near East region, whereas the second main cluster comprised all the genotypes originating from the Northern and Eastern Africa regions, indicating that the Northern and Eastern African faba bean genotypes were more closely related to each other than to the Near East genotypes. Structure analysis also revealed that the 35 faba bean genotypes might be assigned to two populations, in complete accordance with cluster analysis data. In conclusion, this study showed high levels of diversity in the analysed genotypes of faba bean, and could be utilized in future breeding programmes to develop new cultivars of high yield.     

Potential of DNA markers in detecting divergence and in analysing heterosis in Indian elite chickpea cultivars

 Molecular markers such as RAPDs and microsatellites were used to study genetic diversity in 29 elite Indian chickpea genotypes. In general, microsatellites were more efficient than the RAPD markers in detecting polymorphism in these genotypes. Among the various microsatellites, (AAC)₅, (ACT)_5, (AAG)₅ and (GATA)₄ were able to differentiate all 29 chickpea cultivars. The mean value of probability of identical match by chance was 2.32×10^-25 using DraI-(ACT)₅, TaqI-(AAC)₅, TaqI-(AAG)₅ and TaqI-(GATA)₄ enzyme-probe combinations. The dendrogram, constructed on the basis of similarity index values, grouped the chickpea genotypes into five main clusters with 8 cultivars genetically distant and outgrouped from the main clusters. To investigate if DNA markers are useful in predicting F₁ performance and heterosis in chickpea, we crossed 8 genotypes having important agronomic characters in a diallel set. The F₁s and their parents in the diallel set were analysed for agronomic traits for better parent and midparent heterosis. Heterosis was found to be much higher for yield than for yield components that fit a multiplicative model. The analysis of genetic divergence using D² statistics clustered the 8 cultivars into two groups. Although molecular marker-based genetic distance did not linearly correlate to heterosis, two heterotic groups could be identified on the basis of the general marker heterozygosity. Received: 1 August 1998 / Accepted: 29 September 1998     

Population structure and genetic analysis of different utility types of mango (Mangifera indica L.) germplasm of Andhra Pradesh state of India using microsatellite markers

Genetic analysis of 90 mango genotypes including juicy, table, dual and pickle types from different parts of Andhra Pradesh of India was carried out employing 143 mango-specific microsatellite markers. Of the 143, 34 were new mango-specific microsatellite loci isolated in the course of the present investigation by constructing an (CA) _n and (TG) _n -enriched genomic library. Characterization of the 90 genotypes resulted in the detection of 301 alleles from 106 polymorphic loci with an average of 2.87 alleles per locus and polymorphism information content of 0.67. UPGMA cluster analysis grouped all the genotypes into two major groups with a genetic similarity range of 47–88 %. Grouping of the genotypes based on the utility type was observed only at sub-cluster level. Study of population structure by a model-based STRUCTURE analysis revealed the germplasm to exist in four gene pools. Overall F _st of 0.11 indicated genetic differentiation between the populations to be low. Analysis of molecular variance revealed that major proportion of the variation was within the individuals (62.25 %). The molecular marker-based study of genetic diversity suggests that the germplasm studied representing the kind of variability would be a valuable genetic resource for future breeding and association mapping in search for new and novel alleles.     

Genetic Diversity of Iranian Accessions,Improved Lines of Chickpea (<Emphasis Type="Italic">Cicer arietinum L.</Emphasis>) and Their Wild Relatives by Using Simple Sequence Repeats

Biodiversity information in available germplasm is very useful for the success of any breeding program. To establish genetic diversity among 44 genotypes of chickpea comprising cultigen, landraces, internationally developed improved lines and wild relatives, genetic distances were evaluated using 19 simple sequence repeat markers with 100 marker loci. Estimation of the number of alleles per locus (n _a), the effective allele number (n _e), and Wright fixation index F were 6.25, 3.67, and 0.44, respectively. Polymorphism information content values ranged from 0.84 (locus NCPGR6 and TA135) to 0.44 (locus NCPGR7) with an average of 0.68. Dice’s coefficient similarity matrix for studied chickpea genotypes varied from 0.07 to 1.0 indicating a broader genetic base among genotypes studied. The highest similarity, 1.0, was observed between genotypes Sel 96TH11484 and Sel 96TH11485; while, the lowest, 0.07, was observed between genotypes Sel 95TH1716 and Azad. Based on the UPGMA clustering method, all genotypes were clustered in eight groups, which indicated the probable origin and region similarity of landraces and local Iran landraces over the other cultivars and wild species. It also represents a wide diversity among available germplasm. Analysis of molecular variance revealed that 41% of the total variance was due to differences among groups while 59% was due to differences within groups. The results of principal coordinate analysis approximately corresponded to those obtained through cluster analysis. Genetic variation detected in this study can be useful for selective breeding for specific traits and in enhancing the genetic base of breeding programs.     

iPBS-Retrotransposons-based genetic diversity and relationship among wild annual Cicer species

Lack of requisite genetic variation in cultivated species has necessitated systematic collection, documentation and evaluation of wild Cicer species for use in chickpea variety improvement programs. Cicer arietinum has very narrow genetic variation, and the use of a wild relative in chickpea breeding could provide a good opportunity for increasing the available genetic variation of cultivated chickpea. Genetic diversity and the relationship of 71 accessions, from the core area of chickpea origin and domestication (Southeastern Turkey), belonging to five wild annual species and one cultivated species (Cicer arietinum) were analysed using iPBS-retrotransposon and ISSR markers. A total of 136 scorable bands were detected using 10 ISSR primers among 71 accessions belonging to 6 species, out of which 135 were polymorphic (99.3 %), with an average of 13.5 polymorphic fragments per primer, whereas iPBS detected 130 bands with 100 % polymorphism with an average of 13.0 bands per primer. C. echinospermum and C. pinnatifidum were the most diverse among species, whereas C. arietinum exhibited lower polymorphism. The average polymorphism information contents (PIC) value for both marker systems was 0.91. The clustering of the accessions and species within groups was almost similar, when iPBS and ISSR NeighborNet (NNet) planar graphs were compared. Further detailed studies are indispensable in order to collect Cicer germplasm, especially C. reticulatum, from southeastern Turkey particularly, from Karacada? Mountain for preservation, management of this species, and to study their genetic diversity at molecular level. This study also demonstrates the utility and role of iPBS-retrotransposons, a dominant and ubiquitous part of eukaryotic genomes, for diversity studies in wild chickpea and in cultivated chickpea.     

Efficiency of RAPD, SSR and Cytochrome P450 gene based markers in accessing genetic variability amongst finger millet (Eleusine coracana) accessions

Finger millet (Eleusine coracana L.) is an important crop used for food, forage, and industrial products. Three DNA marker techniques, random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P₄₅₀ gene based markers were used for the detection of genetic polymorphism in 83 accessions of finger millet collected from various geographical regions of India and Africa. A total of 18 RAPD, 10 SSR and 10 pairs of cytochrome P₄₅₀ gene based markers were generated 56.17, 70.19 and 54.29% polymorphism, respectively. Mean polymorphism information content (PIC) for each of these marker systems (0.280 for RAPD, 0.89 for SSR and 0.327 for cytochrome P₄₅₀ gene based markers) suggested that SSR marker were highly effective in determining polymorphism. The phenograms based on the three markers data indicate that genotypes from different geographical regions are clearly distinguishable as separate clusters. Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.90 for all the three marker systems. The dendrograms and PCA plots derived from the binary data matrices of the three marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. Based on the results of present study, SSR and cytochrome P₄₅₀ gene based markers appear to be particularly useful for the estimation of genetic diversity. This study reveals the potential of RAPD, SSR and gene based markers for characterizing germplasm of Eleusine coracana and narrow down the vast germplasm into distinct core groups.     

Molecular Variability and Genetic Structure of Chrysodeixis includens (Lepidoptera: Noctuidae), an Important Soybean Defoliator in Brazil

This study provides the first genetic characterization of the soybean looper, Chrysodeixis includens (Walker, 1857), an important defoliating pest species of soybean crops in Brazil. Population genetic variability and the genetic structure of C. includens populations were evaluated by using ISSR markers with samples from the major soybean producing regions in Brazil in the growing seasons 2011/2012. Seven different primers were applied for population characterization of the molecular variability and genetic structure of 8 soybean looper populations from 8 states of Brazil. The seven ISSR loci generated 247 bands in 246 individuals of C. includens sampled. The expected heterozygosity (H _E) in the populations varied between 0.093 and 0.106, while the overall H _E was 0.099, indicating low genetic diversity. The analysis of molecular variance indicated that 98% of the variability was expressed among individuals within populations (F _ST = 0.021, p = 0.001). The low level of polymorphism over all populations, the high levels of gene flow, and the low genetic structure are indicatives of the exchange of genetic information between the different sampled regions. Population structuring suggests the presence of two major groups which do not correlate with their geographic sampling location in Brazil. These results may indicate recent recolonization of C. includens in Brazil or migration patterns following source-sink dynamics. Furthermore, the presence of two groups within C. includens suggests that a study on development of resistance or any other genetic-based trait needs to be evaluated on both groups, and pest management in soybean fields should be aware that differences may come to the control strategies they use.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.