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 Dof (DNA binding with one finger) transcription factor gene-specific primers through data mining as a functional marker and their use for genetic diversity study in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm

The genetic diversity among Hordeum vulgare L. species were assessed based on PCR amplification pattern derived from 75 set of Dof domain and Dof genes specific primers. Multiple bands showing variability in terms of both number and sizes of bands ranging from 0.1 to 3.0 kbp were observed. Out of a total of 2449 bands, 2328 polymorphic and 121 monomorphic bands were obtained and the percentage of polymorphism ranged from 70.27 to 100%. A very high degree of polymorphism was observed with all the primers except HvDof3, HvDof4, HvDof10, HvDof16, HvDof18, HvDof18, HvDof24, Dof4, Dof11, Dof13, Dof15, Dof16, Dof19, Dof20, Dof21, Dof22, Dof23, Dof28, dof38, sbDof23 and sbDof24 primers. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. According to results, the genetic resources and diversity in barley germplasm of H. vulgare were rich. The number of polymorphic fragments per primer detected ranged from 11 to 56 bands with an average of 32.65 bands. Average polymorphic information content (PIC) was 0.81 in overall Dof domain and gene specific primers. HvDof 39 showed the highest PIC (0.99) which can be a good candidate primer to verify genetic diversity in H. vulgare. The unweighted pair-group method of the arithmetic average and principal coordinate analysis showed a clear distinction among the genotypes and the genotypes divided into three clusters in the dendrogram results. A model-based structure analysis revealed the presence of three groups. The study showed that genetic variation and population structure are determined among the species of H. vulgare collected from different geographical origins.     

Comparative evaluation of genetic diversity using RAPD,SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> L. Gaertn.)

Genetic relationships among 52 Eleusine coracana (finger millet) genotypes collected from different districts of Uttarakhand were investigated by using randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P450 gene based markers. A total of 18 RAPD primers, 10 SSR primers, and 10 pairs of cytochrome P450 gene based markers, respectively, revealed 49.4%, 50.2% and 58.7% polymorphism in 52 genotypes of E. coracana. Mean polymorphic information content (PIC) for each of these marker systems (0.351 for RAPD, 0.505 for SSR and 0.406 for cyt P450 gene based markers) suggested that all the marker systems were effective in determining polymorphisms. Pair-wise similarity index values ranged from 0.011 to 0.999 (RAPD), 0.010 to 0.999 (SSR) and 0.001 to 0.998 (cyt P450 gene based markers) and mean similarity index value of 0.505, 0.504 and 0.499, respectively. The dendrogram developed by RAPD, SSR and cytochrome P450 gene based primers analyses revealed that the genotypes are grouped in different clusters according to high calcium (300–450 mg/100 g), medium calcium (200–300 mg/100 g) and low calcium (100–200 mg/100 g). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.95 for all the three marker systems. The dendrograms and principal coordinate analysis (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. Comparison of RAPD, SSR and cytochrome P450 gene based markers, in terms of the quality of data output, indicated that SSRs and cyt P450 gene based markers are particularly promising for the analysis of plant genome diversity. The genotypes of finger millet collected from different districts of Uttarakhand constitute a wide genetic base and clustered according to calcium contents. The identified genotypes could be used in breeding programmes and amajor input into conservation biology of cereal crops.     

Discrimination and genetic diversity of cultivated and wild safflowers (Carthamus spp.) using EST-microsatellites markers

In spite of being one of the major oilseed crops, little is known about genetic diversity and relationships between species of safflower. In this study EST-SSR markers were used to evaluate and characterize 42 genotypes from six species including Carthamus tinctorius, Carthamus palaestinus, Carthamus oxyacanthus, Carthamus lanatus, Carthamus dentatus, and Carthamus boissieri. Thirty three primer pairs produced 123 polymorphic bands with 2–8 alleles per locus. The EST-SSR markers showed different level of gene diversity. The highest Polymorphism Information Content (PIC) values were observed for primers EL510507 and EL390720 (0.49 and 0.45, respectively). The highest genetic diversity and heterozygosity were observed for C. oxyacanthus. Both cluster and principal coordinate analysis (PCoA) clearly separated species into distinct groups. Within each species the accessions were clustered in different subgroups that mainly supported the known origins. The result showed that C. palaestinus had the most genetic similarity with cultivated safflower and C. oxyacanthus was next in this respect. In general, EST-SSR markers effectively revealed the genetic relationships and diversity of Carthamus species. This information is valuable for safflower improvement since C. palaestinus and C. oxyacanthus are both crossable with the cultivated species C. tinctorius.     

Development and characterization of microsatellite markers for genomic analysis of yarrow (Achillea millefolium L.)

Yarrow (Achillea millefoilum L.) is an important medicinal plant with different medicinal and ornamental uses. In this study, SSR markers were developed for the first time from the genome A. millefolium using the slightly modified Hamilton protocol. Three yarrow genomic libraries were constructed, enriched for microsatellite motifs AG, AC and ATG. A total of 30 primer pairs were developed of which 16 were polymorphic in 26 A. millefolium accessions. One accession from A. tenuifolia species was also included to assess the transferability of new developed SSR primers in other species. The average allele number of SSR markers was 8.5 per locus and ranged from 2 to 14. The observed heterozygosity (H _O) varied from 0 to 0.96 with an average of 0.52 and the expected heterozygosity (H _E) ranged from 0.07 to 0.49 with an average of 0.39. Among primers, Am142 showed the highest polymorphic information content (PIC) and the lowest value was obtained from Am59 primer with an average of 0.33. Three loci (AmK59, AmK344 and AmK329) deviated significantly from Hardy-Wrinberg Equlibrium (HWE) and one locus (Am344) might have null alleles. Cluster and PCoA analyses classified A. millefolium accessions according to their geographical distribution and A. tenuifolia species completely separated from A. millefoliulm genotypes.     

Development and use of novel SSR markers for molecular genetic diversity in Italian millet (Setaria italica L.)

Italian millet is a commercially important grain crop. Nineteen polymorphic simple sequence repeat (SSR) markers, developed through construction of an SSR-enriched library from genomic DNA of Italian millet (Setaria italica L., P. Beauv.), were used for assessment of molecular genetic diversity against 40 accessions of S. italica. In total, 85 alleles were detected, with an average of 4.5 alleles per locus. The average gene diversity and polymorphism information content (PIC) values were 0.412 and 0.376, ranging from 0.02 to 0.88 and from 0.02 to 0.87, respectively. Values for observed (H _O) and expected (H _E) heterozygosities ranged from 0 to 0.73 and from 0.03 to 0.89, respectively. Nine loci deviated from Hardy-Weinberg equilibrium. The mean similarity coefficient among accessions was 0.6593. Based on the UPGMA algorithm, six different groups were successfully identified. In this clustering analysis, all Korean accessions grouped in one cluster, indicating that Korean accessions are genetically quite distinct from other introduced accessions. These newly developed microsatellite markers should be very useful tools for several genetic studies, including an assessment of diversity and population structure in Italian millet.     

草鱼EST-SSR标记及5个不同地域群体的遗传结构分析

以草鱼(Ctenopharyngodon idella)脑、肌肉、肝等组织构建cDNA文库,经测序获得unigenes序列45 318个,从中筛选微卫星序列共5 556个,据此设计EST-SSR引物118对,其中19对引物能够扩增出带型清晰且多态性较高的谱带。用这19个EST-SSR标记研究3个长江水系群体(石首、监利和长沙)和2个珠江水系群体(清远和肇庆)草鱼的遗传结构。结果表明,5个群体的平均多态信息含量(PIC)为0.415 4～0.460 4,显示草鱼群体的遗传多样性偏低;平均观测杂合度(Ho)为0.415 8～0.501 3,平均期望杂合度为(He)0.450 6～0.502 8,其中,长沙群体平均期望杂合度最高,为0.502 8,监利群体的平均观察杂合度和平均期望杂合度均最低,分别为0.415 8和0.450 6,即长沙群体的遗传多样性最高,监利群体的遗传多样性最低;对数据进行F-检验,结果表明,群体间的遗传分化程度低。Hardy-Weinberg平衡的卡方检验结果表明,5个群体均一定程度上偏离了平衡;聚类分析显示长沙群体、石首群体与监利群体聚成一支;肇庆群体与清远群体聚成一支,这与草鱼群体的流域分布一致。     

Assessment of genetic diversity of Czech sweet cherry cultivars using microsatellite markers

We analyzed 24 sweet and wild cherry genotypes collected in Czech Republic to determine genetic variation, using previously described 16 SSR primers to adapt a fast, reliable method for preliminary screening and comparison of sweet cherry germplasm collections. All SSRs were polymorphic and they were able all together to distinguish unambiguously the genotypes. These SSR primers generated 70 alleles; the number of alleles per primer ranged from 2 to 7, with a mean of 4.4 putative alleles per primer combination. The primer UDP-98-412 gave the highest number of polymorphic bands (totally 7), while Empa2 and Empa3 gave the lowest number (2). The allele frequency varied from 2.1% to 87.5%. We observed 10% of unique alleles at different loci. The observed heterozygosity value ranged from 0.25 to 0.96 with an average of 0.72 while expected heterozygosity value varied from 0.22 to 0.75 with an average of 0.59. The PIC value ranged from 0.21 to 0.71 with a mean value of 0.523. Cluster analysis separated the investigated cultivars in two groups. High level of genetic diversity obtained in the collection and proved to be sufficiently genetically diverse and therefore these genotypes would be useful to breeders for the development of new cherry cultivars.     

Development of 240 novel EST-SSRs in Eucalyptus L’Hérit

Simple sequence repeat (SSR) markers derived from expressed sequence tag (EST) resources provide great potential for comparative mapping, direct gene tagging of quantitative trait loci and functional diversity studies. Here we report on the development of 240 novel EST-SSRs for the important tree genus Eucalyptus L’Hérit. Of the 240 EST-SSR loci, 218 (90.8 %) were polymorphic among 12 individuals of E. grandis Hill ex Maiden, with the number of alleles per locus (N _a), observed heterozygosity (H _o), expected heterozygosity (H _e) and polymorphic information content (PIC) averaging at 5.0, 0.403, 0.598 and 0.529, respectively. High rates of cross-species/subgenus amplification were observed. The EST-SSRs developed herein would be a valuable addition of functional markers for genetics and breeding applications in a wide range of eucalypt species. The primer sequences for the 240 EST-SSRs have been deposited in the Probe database of GenBank (IDs Pr016588534–773).     

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.     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Assessment of Genetic Diversity among the Elite Maize (Zea mays L) Genotypes Adapted to North-Western Himalayan Region of India using Microsatellite Markers

Maize is an important crop in the North-Western Himalayan states of India for food, feed and nutritional security of human population. Hybrid maize constitutes the major part of the maize area. Twenty four maize lines including the indigenous and exotic inbreds were amplified using 68 SSR primers, spread over the whole genome. The number of alleles across the primers ranged from two to eleven. The genotypes were grouped into different clusters using NTSYSpc2.11 programme. The clusters were well correlated with agronomic traits and resistance against turcicum blight. The PIC value was found to be highest for the primer bnlg1267 (0.84) while the lowest value was for the primer dupssr14 (0.09) with the mean value of 0.60. From this study we concluded that inbred V 359 is expected to give better combinations with CM 128, CM 129, V 340, V 357 and CM 212 for the development of hybrids suitable for the sub-tropical hill regions of India and elsewhere.     

西花蓟马EST-SSR信息分析、标记筛选及其与Genomic-SSR的多态性比较

西花蓟马Frankliniella occidentalis是一种世界性入侵昆虫, 近年来传入我国并不断扩散蔓延。基于简单重复序列（simple sequence repeats, SSRs）的西花蓟马种群遗传结构研究对于揭示其传播途径等具有重要的指导价值。本研究对来源于西花蓟马的13 839条EST序列进行了uni-EST组装、 EST-SSR信息分析以及标记筛选, 并比较了EST-SSR与Genomic-SSR在分析遗传多样性方面的差异。结果表明： 在7 707个singlets中共找到2 623个SSR位点, 分布于1 930个uni-EST中, 平均每2.21 kb就出现一个SSR位点。重复单元中, 以单碱基重复单元为主（83.00%）, 其次是四碱基重复单元（11.17%）, 而二、 三、 五和六碱基重复单元所占比例较低（分别为1.41%, 0.80%, 2.02%和0.91%）。设计出的22对EST-SSR引物中, 4对引物能稳定扩增出清晰的目的条带; 荧光标记毛细管电泳发现3对引物表现出多态性。西花蓟马EST-SSR与Genomic-SSR多态性分析表明, 这3对多态性EST-SSR引物揭示的多态信息含量（PIC）为0.48~0.69, 比5对多态性Genomic-SSR引物揭示的PIC（0.88~0.92）略低。本研究结果可为今后更深入开展西花蓟马的种群遗传结构分析提供帮助。     

Genetic diversity in local and commercial dry bean (Phaseolus vulgaris) accessions based on microsatellite markers

Dry beans are considered to be a crop of great socio-economic importance, because they are an inexpensive source of nutrients and because their cultivation requires considerable manual labor. Studies of genetic diversity have been very important for genetic improvement programs, because they give parameters for the identification of genitors that can provide large heterosis effects and improved segregation in recombinants, increasing the probability of obtaining superior genotypes in the progeny. We evaluated the genetic diversity of 57 dry bean accessions, including 31 local accessions, propagated by small-scale farmers, 20 accessions supplied by the Brazilian Agricultural Research Agency, and six commercial accessions, using 16 microsatellite primers. Among these primers, 13 were found to be polymorphic, giving 29 polymorphic alleles. The largest number of alleles per locus was observed for primer BM141, which had four alleles. The polymorphic information content varied from 0.11 to 0.51, observed for loci BM212 and BM141, respectively. The lowest degree of dissimilarity (0.0) was found between the accession Iapar 81 and the accessions E03, E04, E09, and E13 and between the accession pairs E08 with E16 and Iapar 31 with E06. The highest degree of dissimilarity was found between the accessions Carioca and E22 (1.0). Grouping analysis revealed four groups, according to the place of origin. This tendency was also found in the principal coordinate analysis. The local genotypes were found to have relatively high genetic diversity, while the EMBRAPA and commercial cultivars had a relatively narrow genetic basis.     

Comparative genomics and association mapping approaches for opaque2 modifier genes in finger millet accessions using genic,genomic and candidate gene-based simple sequence repeat markers

Identification of alleles responsible for opaque2 modifiers (Opm) influencing tryptophan content in finger millet is a major aim for further improvement of the quality of the locally adapted finger millet germplasm. Since there is little genome sequence information available, comparative genomics plays a very important role in identification of genes/quantitative trait loci (QTLs) linked to the Opm genes using simple sequence repeat (SSR) markers. In the present study, a total of 74 genic SSRs were developed and then used for genetic diversity and population structure analysis of a global collection of 190 finger millet genotypes. The 74 SSRs yielded 133 scorable alleles and the polymorphism information content values varied from 0.186 to 0.707, with an average of 0.408. The gene diversity was in the range of 0.208–0.752, with an average of 0.501. The SSRs developed from the aspartate kinase2 gene of the lysine pathway showed more polymorphism than the other candidate genes. The 74 genic SSR loci grouped the 190 finger millet genotypes into three major clusters based on their tryptophan content, using both phylogenetic clustering and population structure analysis by STRUCTURE software. Association mapping for Opm was done using 120 (74 genic and 46 genomic) SSR loci for identification of QTLs linked to Opm influencing tryptophan content, and found two QTLs for tryptophan and one QTL for protein content. The QTLs for tryptophan content were associated with the genic marker OM5 at a P value of 0.009 and explained 11 % of phenotypic variance (R ²). The OM5 marker was designed from the 27-kDa γ-zein gene of Opm, which influences the tryptophan content to a large extent, whereas the genomic marker FM8 was linked at a P value of 0.004 and explained 9 % of R ². The QTLs for protein content were associated with the genic SSR marker FMO2EST1, which was designed from the RISBZ1 gene of rice and was linked at a P value of 0.002 and explained 9 % of R ². The 220-bp allele of SSR locus OM5 was found to be present mostly in the high tryptophan-containing genotypes such as exotic genotypes, and among the Indian genotypes it was present in NW Himalayan genotypes. The markers linked to the QTLs for Opm found in the present study can be further used for cloning of the full-length gene, for fine mapping and in the marker-assisted breeding programmes for introgression of alleles into locally well-adapted germplasm.