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.     

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.     

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 (∼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.     

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.     

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.     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

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 and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

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     

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.     

Characterization of microsatellite loci in the brown treecreeper (Climacteris picumnus) and cross‐species amplification in the white‐throated treecreeper (Cormobates leucophaeus)

Eight polymorphic microsatellite markers were developed for the brown treecreeper, Climacteris picumnus. The number of alleles ranged from three to 25 per locus with observed heterozygosities between 0.05 and 0.76. Seven of the eight primer pairs also amplified polymorphic microsatellite loci in the white‐throated treecreeper (Cormobates leucophaeus). These markers are likely to be useful for population genetic and parentage studies in any of the Australasian treecreepers (Climacteridae) and are the first genetic markers developed for any member of this passerine family.     

Development and characterization of microsatellite markers in black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned and sequenced microsatellite loci from black poplar (Populus nigra L.) and developed primers for sequence-tagged microsatellite (STMS) analysis. Twelve primer pairs for dinucleotide repeats produced fragments of sufficient quality which were polymorphic in P. nigra. Some of them also showed amplification in other Populus species (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, and/or P. lasiocarpa). The best nine and (GT) (GA) microsatellite markers were tested on a set of 23 P. nigra genotypes from all over Europe. The microsatellites were highly polymorphic, with 10–19 different alleles per microsatellite locus among these 23 genotypes. WPMS08 sometimes amplified three fragments. Using the other eight marker loci, the level of heterozygosity among the plants was on average 0.71 (range 0.25–1.00). The microsatellite markers developed will be useful for screening the genetic diversity in natural populations and in gene bank collections. Received: 21 October 1999 / Accepted: 24 November 1999     

Polymorphism of microsatellite loci of European chickpea cultivars

The polymorphism of 13 microsatellite loci in 61 chickpea varieties from the National Center of Genetic Plant Resources of Ukraine was studied. Forty-seven alleles were detected. Three loci were monomorphic; the rest showed polymorphism. The genetic diversity of chickpea varieties in all loci was 0.41 according to the Nei polymorphism index (D). It was concluded that chickpea varieties from Europe had insignificant microsatellite loci diversity. The most polymorphic group included chickpea varieties from Russia (D = 0.38); the least polymorphic group, Spanish varieties (D = 0.25). A consensus tree representing the most credible divergence of Europe chickpea varieties was constructed. The possible reasons for clustering chickpea varieties in a common cluster are discussed.     

Development and characterization of simple sequence repeat DNA markers for Zelkova serrata

We developed 14 microsatellite loci from an enriched genomic DNA library of a broad‐leaved deciduous tree, Zelkova serrata. Of 198 clones from the library, 112 contained microsatellite repeat regions. The M13‐tailed primer method was used for economy. Sequence‐specific primer pairs were designed for 58 of 76 candidate clones. Fourteen of these primer pairs successfully amplified polymorphic single loci among 34 individuals collected from the Kanto breeding region in Japan. The expected heterozygosity for the 14 microsatellite markers ranged from 0.378 to 0.876, suggesting that these will prove valuable for breeding and ecological studies on Z. serrata.     

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.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

Wild gibbons' parentage tested by non-invasive DNA sampling and PCR-amplified polymorphic microsatellites

Elucidating the genetic relationships among members of a social group is indispensable in studying any social system of primates.Hylobates spp. are believed to be monogamous, although some long-term monitoring studies have provided conflicting evidence. We applied a parentage testing technique to a group ofHylobates muelleri in the wild. Forty-five microsatellite loci were screened in the 12 unrelated gibbons' DNA, and 16 of the 45 loci were found to be polymorphic. Hair and fecal samples from 15 gibbons in the field were collected non-invasively. In each sample, the 16 polymorphic loci were amplified by PCR using appropriate primer pairs and separated by electrophoresis. We estimated three pairs of parents-offspring, a pair each of both father-offspring, and mother-offspring genetic relationships. Further, in two of the five cases, we revealed the family a subadult lived with was not a natal one of the subadult. The non-invasive sampling methods and polymorphic primer pairs used in this study would greatly enhance the understanding of gibbon's society in the wild.     

Eleven polymorphic microsatellite loci in Lindera benzoin,Lauraceae

Microsatellite markers were developed for studies of the genetic diversity and population substructure of Lindera benzoin, Lauraceae (spicebush). Nuclear microsatellite sequences were obtained from DNA libraries that were enriched for (CA), (GA), (AAG) and (ATG) repeat motifs. From 69 microsatellite sequences, 20 primer sets were developed. Of these, 11 primer pairs resulted in amplified polymorphic loci. In 29 samples of eastern Pennsylvania spicebush plants, the number of microsatellite alleles ranged from two to 16 per locus with observed heterozygosity values ranging from 0.10 to 0.82.     

Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)

The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73.