Molecular analysis of kabuli and desi type of Indian chickpea (Cicer arietinum L.) cultivars using STMS markers

Fifty one Sequence tagged microsatellite sites (STMS) primer pairs were employed to assess the genetic diversity and relationships with morphological characters among the sixty-eight chickpea (Cicer arietinum L.) cultivars of India. A total of 32 out of 51 STMS primers were found polymorphic and a total of 121 alleles were generated out of which 102 (83 %) were detected for the 32 polymorphic STMS markers with an average of 2.22 alleles per locus. The PIC values of all the polymorphic loci ranged from 0.15 (TS82) to 0.69 (TS29) with the mean value of 0.27. Three primers showed PIC value of more than 0.60. The highest PIC value was observed for the primer TS29 (0.69), succeeded by the primer GA 11 (0.61) and TS71 (0.60). Gene diversity (He) was observed in the range of 0.16 (TS82) to 0.74 (TS29) with an average value of 0.33. The heterozygosity (Ho) was observed to be 0.39 (average) with a range of 0.04 (TA18) to 1.00 (TA76, STMS 5, TA72 and TA122). Based on the above STMS marker analysis by considering the parameters of PIC value (≥0.55), gene diversity (≥0.62), and polymorphic alleles (≥4), six highly polymorphic STMS loci GA11, TA76S, TA89, TS29, TS43 and TS71 were observed which can effectively be used in further molecular studies. Dendrogram generated by the UPGMA analysis and POWER MARKER v3.0 showed similar results and there was no clear demarcation of Kabuli and Desi genotypes. The present study resulted in identification of highly distinct genotypes JG 130 and C 235 (57 %) followed by two pairs of genotypes B 108 and JG 11 (57.8 %) and, JG 315 and RSG 2 (59 %) which can be used effectively in a breeding programs in order to develop transgressive segregants with wider genetic base and better promising genotypes. Effective use of these three pairs of chickpea genotypes is expected to give better products for the development of higher yielding Kabuli and Desi genotypes with tolerance/resistance to biotic and abiotic stresses and quality traits.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome

A size-selected genomic library comprising 280,000 colonies and representing ≈18% of the chickpea genome, was screened for (GA)_n, (GAA)_n and (TAA)_n microsatellite-containing clones, of which 389 were sequenced. The majority (～75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%–9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613?cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P?≥?0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.     

Use of morphological traits and microsatellite markers to characterize the Tunisian cultivated and wild figs (Ficus carica L.)

We used 8 morphological traits and 17 simple sequence repeats loci to characterize 71 cultivated and wild Tunisian fig trees (Ficus carica L.). Significant morphological differences were inferred from leaf traits. The statistical analysis showed two major fig groups that indicated a common morphological basis. A total of 74 SSR alleles was revealed, defining 63 unique multilocus genotypes indicating a substantial genetic diversity. Based on multilocus SSR genotypes an identification key was established using MFC30, MFC3, MFC11 and MFC19 loci to identify figs. Analysis of variance components of linkage disequilibrium shown that among 136 pairs of loci, 32 present a significant gametic disequilibrium. The parameter D′²_IS (0.1284) was greater than D′²_ST (0.0079), a pointer of close to zero variance in total simple, and consequently the more pronounced independence of the 17 SSR loci. The majority of Ohta's variance components of linkage disequilibrium followed a pattern caused by genetic drift or a non-systematic disequilibrium profiles and natural selection occurs only for LMFC24-MFC8 pair loci in cultivated figs. Our results suggest that the morphological and SSR markers are suitable to characterize figs and should be recommended in conservation management strategy.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

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.     

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 dissection of drought tolerance in chickpea (Cicer arietinum L.)

Key message

Analysis of phenotypic data for 20 drought tolerance traits in 1–7 seasons at 1–5 locations together with genetic mapping data for two mapping populations provided 9 QTL clusters of which one present on CaLG04 has a high potential to enhance drought tolerance in chickpea improvement.

Abstract

Chickpea (Cicer arietinum L.) is the second most important grain legume cultivated by resource poor farmers in the arid and semi-arid regions of the world. Drought is one of the major constraints leading up to 50 % production losses in chickpea. In order to dissect the complex nature of drought tolerance and to use genomics tools for enhancing yield of chickpea under drought conditions, two mapping populations—ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261) segregating for drought tolerance-related root traits were phenotyped for a total of 20 drought component traits in 1–7 seasons at 1–5 locations in India. Individual genetic maps comprising 241 loci and 168 loci for ICCRIL03 and ICCRIL04, respectively, and a consensus genetic map comprising 352 loci were constructed (http://cmap.icrisat.ac.in/cmap/sm/cp/varshney/). Analysis of extensive genotypic and precise phenotypic data revealed 45 robust main-effect QTLs (M-QTLs) explaining up to 58.20 % phenotypic variation and 973 epistatic QTLs (E-QTLs) explaining up to 92.19 % phenotypic variation for several target traits. Nine QTL clusters containing QTLs for several drought tolerance traits have been identified that can be targeted for molecular breeding. Among these clusters, one cluster harboring 48 % robust M-QTLs for 12 traits and explaining about 58.20 % phenotypic variation present on CaLG04 has been referred as “QTL-hotspot”. This genomic region contains seven SSR markers (ICCM0249, NCPGR127, TAA170, NCPGR21, TR11, GA24 and STMS11). Introgression of this region into elite cultivars is expected to enhance drought tolerance in chickpea.     

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.     

Application of EST-derived microsatellite markers for analysis of genetic variation in tall fescue and its comparison with morphological markers

Analysis of genetic diversity in germplasm collections is an important component of crop improvement programs. This study was conducted to analyze genetic variation and to classify tall fescue genotypes based on phenotypic evaluation and EST-SSR molecular markers. Twenty-five genotypes were assessed based on phenotypic and 42 EST-SSR molecular markers according to a completely randomized block design with three replications during eight years (2007–2014). Results indicated that the effect of year, genotype and their interaction were significant for all of the measured traits. Both morphological and molecular assessments showed considerable genetic variation among genotypes. The estimates of broad-sense heritability (h²_b) were moderate to high (h²_b = 42.1–78.4) for the traits studied. Based on EST-SRR analysis, a total number of 229 alleles were detected with an average of 4.58 alleles per marker. Average PIC value was 0.49 with a range of 0.014 for NFA140 to 0.95 for NFA047. Phenotypic evaluations and EST-SSR molecular marker classified genotypes into 3 and 7 clusters, respectively which mainly supported geographical origins. The general correspondence was observed between morphological and molecular classification. Therefore, combining the molecular markers with morphological responses could be more beneficial to describe genetic variation and distinguish superior genotypes for future breeding programs.     

Inheritance studies of SSR and ISSR molecular markers and phylogenetic relationship of rice genotypes resistant to tungro virus

Multivariate analyses were performed using 13 morphological traits and 13 molecular markers (10 SSRs and three ISSRs) to assess the phylogenetic relationship among tungro resistant genotypes. For morphological traits, the genotypes were grouped into six clusters, according to D² statistic and Canonical vector analysis. Plant height, days to flowering, days to maturity, panicle length, number of spikelet per panicle, number of unfilled grain per panicle and yield were important contributors to genetic divergence in 14 rice genotypes. Based on Nei's genetic distance for molecular studies, seven clusters were formed among the tungro resistant and susceptible genotypes. Mantel's test revealed a significant correlation (r = 0.834*) between the morphological and molecular data. To develop high yielding tungro resistant varieties based on both morphological and molecular analyses, crosses could be made with susceptible (BR10 and BR11) genotypes with low yielding but highly resistant genotypes, Sonahidemota, Kumragoir, Nakuchimota, Khaiyamota, Khairymota and Kachamota. The chi-square analysis for seven alleles (RM11, RM17, RM20, RM23, RM80, RM108 and RM531) of SSR and five loci (RY1, MR1, MR2, MR4 and GF5) of three ISSR markers in F₂ population of cross, BR11 × Sonahidemota, showed a good fit to the expected segregation ratio (1:2:1) for a single gene model.     

Patterns of subspecies genetic diversity among oilseed <Emphasis Type="Italic">Brassica rapa</Emphasis> as revealed by agro-morphological traits and SSR markers

Brassica rapa (2n = 20, AA genome) is an important oil yielding species of the family Brassicaceae and characterized by wide range of genetic and morphological subtypes suitable for cultivation under diverse agro-climatic regions of India. In this study, genetic diversity among three subspecies of B. rapa including yellow sarson, toria and outlier brown sarson was estimated using various agro-morphological traits and simple sequence repeat (SSR) markers. Maximum variability was recorded for siliqua angle (Coefficient of variation = 30.9%), followed by seeds/siliqua (CV = 18.8%), leaf length (CV = 10%) and plant height (CV = 16.8%). Principal component analysis explained more than 50% of the total observed morphological variability for first two components. Of the 107 SSR markers tested, 80 generated reproducible, clear and distinct amplicons of which, 65 (81.25%) were found polymorphic. The number of alleles at each locus ranged from 2 to 7, with an average of 3.03 alleles per marker. A total of 197 alleles were detected at 65 SSR loci with average PIC value of 0.457 and a mean resolving power of 3.04. Neighbor-Joining cluster analysis based on morphological traits and SSR markers separately classified all the 28 genotypes into five major groups. The population structure analysis resulted into three sub-populations with certain extent of admixture among the earlier established taxonomic sub-groups. Twenty-three unique alleles were detected in thirteen B. rapa varieties. The clustering analysis and principal coordinate analysis outlined the genetic relationships among different varieties belonging to the three subspecies of B. rapa. Genetically diverse genotypes as illustrated by score plots and from the clustering patterns brought out the wide range of diversity present among B. rapa genotypes and the underlying options available for selecting parental genotypes for hybridization and developing high yielding cultivars suitable for Indian conditions.     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

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.     

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea ( Cicer arietinum L.) genome

A size-selected genomic library comprising 280,000 colonies and representing ≈18% of the chickpea genome, was screened for (GA)_n, (GAA)_n and (TAA)_n microsatellite-containing clones, of which 389 were sequenced. The majority (∼75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%–9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613 cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P ≥ 0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome. Received: 14 January 1999 / Accepted: 29 April 1999     

Molecular Analysis of Chickpea (Cicer arietinum L) Cultivars Using AFLP and STMS Markers

Fifteen AFLP and eighteen STMS primer pairs were employed to reveal genetic diversity and relationship in twenty-one cultivars of chickpea (Cicer arietinum L). Fifteen AFLP primer pairs generated 1804 amplicons, out of which 1732 amplicons (96%) were polymorphic and 600 amplicons (&#x223C;33%) were genotype specific. Eighteen polymorphic STMS primer pairs generated 64 amplicons with an average of 3.55 amplicons per primer pair. Polymorphic information content (PIC) varied from 0.52 to 1.0 for STMS markers. The genetic similarity between cultivars varied from 0.30 to 0.85 for AFLP and 0.22 to 0.83 for STMS markers. Dendrogram constructed after combining both AFLP and STMS markers data with Bootstrap analysis, grouped all the cultivars into four clusters. Association of varietal type and flower colour was observed as cultivars E 100Ymu and Nabin (Both Desi type and pink flower) clustered together in the dendrogram.     

A linkage map of chickpea (Cicer arietinum L.) based on populations from Kabuli × Desi crosses: location of genes for resistance to fusarium wilt race 0

Two recombinant inbred line (RIL) populations derived from intraspecific crosses with a common parental line (JG62) were employed to develop a chickpea genetic map. Molecular markers, flower colour, double podding, seed coat thickness and resistance to fusarium wilt race 0 (FOC-0) were included in the study. Joint segregation analysis involved a total of 160 markers and 159 RILs. Ten linkage groups (LGs) were obtained that included morphological markers and 134 molecular markers (3 ISSRs, 13 STMSs and 118 RAPDs). Flower colour (B/b) and seed coat thickness (Tt/tt) appeared to be linked to STMS (GAA47). The single-/double-podding locus was located on LG9 jointly with two RAPD markers and STMS TA80. LG3 included a gene for resistance to FOC-0 (Foc0₁/foc0₁) flanked by RAPD marker OPJ20₆₀₀ and STMS marker TR59. The association of this LG with FOC-0 resistance was confirmed by QTL analysis in the CA2139 × JG62 RIL population where two genes were involved in the resistance reaction. The STMS markers enabled comparison of LGs with preceding maps.     

Transferability of sequence tagged microsatellite site (STMS) primers across four major pulses

The transferability of genome-specific sequence tagged microsatellite site (STMS) primers from field pea (P. sativum) and chickpea (C. arietinum) to other major pulses was examined. Overall, field pea STMS primers amplified products in most of the accessions in comparison to that of the chickpea STMS primers, which amplified products in relatively few accessions. The highest level of successful amplifications with a single primer was 89% for field pea and 33% for chickpea primers respectively. The potential transferability of the STMS primers among species, expressed as the total mean percentage of positive amplifications, was 53% for the field pea STMS primers and 9% for the chickpea STMS primers. The individual mean percentage of successful transferability of field pea STMS primers across lentil, vetch and chickpea/Cicer sp. accessions was 60%, 39% and 62%, respectively. Whereas, for the chickpea STMS primers successful transferability was 5%, 3% and 18% for lentil, vetch and field pea, respectively. The trnasferability of these STMS primers indicates a high level of sequence conservation in these regions across species. Together with their locus-specificity, co-dominant nature and potential to amplify multiple alleles, their transferability makes STMS markers a powerful tool for genetic mapping, diversity analysis and genotyping.