Sequence-tagged Microsatellite Site Research Articles

Investigation of resistance using STMS markers against Ascochyta blight in the chickpea varieties

Chickpea (Cicer arietinum L.), a prominent legume plant, is an important agricultural plant that is widely grown both in Türkiye and around the world. Ascochyta blight, caused by the fungal phytopathogen Ascochyta rabiei, is one of the major causative agents responsible for yield reductions across the spectrum of chickpea diseases. The impact of diseases varies depending on crops, countries, seasons and cropping systems, and yield loss data collected under well-defined conditions is limited. It is noteworthy that this pathogen shows significant genetic diversity in Türkiye's agricultural environment. In light of this, this study aimed to conduct a research to determine the resistant/tolerant and susceptible genotypes of 34 certificated chickpea varieties grown in different regions of Türkiye by using Sequence Tagged Microsatellite Site (STMS) markers that are related to the genes that provide resistance against Ascochyta blight. The results obtained in this study showed that the primers Ta2, Ta146 and Ts54 used as STMS markers have distinctive features in providing highly effective results in the detection of resistant/tolerant and susceptible varieties of Ascochyta blight.

STMS markers related to Ascochyta blight resistance in chickpea

Chickpea (Cicer arietinum L.) is one of the important legume crops and is cultivated in large-scale throughout Türkiye as well as the world. Ascochyta blight, caused by the fungal phytopathogen Ascochyta rabiei, is the leading reason for the highest yield losses among the diseases known for chickpea. The pathogen exhibits high genetic diversity in Türkiye. Therefore, resistancy using Sequence Tagged Microsatellite Site (STMS) markers related with the genes that provide resistant against Ascochyta blight was investigated for the 205 chickpea breeding lines grown in different parts of Türkiye. The analysis for Ascochyta blight resistance was performed using Ta2, Ta146 and Ts54. It was demonstrated that Ta2, Ts54 and Ta146 were the STMS markers having distinguishable features for the detection of Ascochyta blight resistance and were shown to be used in credible fashion for the selection of resistant chickpea breeding lines.

Development of low-phytate maize inbred lines through marker-assisted introgression of lpa1

Context Phytic acid is the major storage form of phosphorus in cereals and is considered an anti-nutritional factor because it chelates major mineral micronutrient cations, resulting in micronutrient malnutrition in humans. For monogastric animals fed maize (Zea mays L.) grains, the stored phosphorus does not release into the digestive tract, leading to phosphorus deficiency and environmental pollution. Aims The aim of the study was to develop maize lines with a lower level of phytic acid that might substantially enhance the nutritional value of maize. Methods The lpa1 mutant allele conferring low phytic acid was transferred into the parental lines of popular maize hybrid DMH 121 (i.e. BML 6 and BML 45) through marker-assisted backcross breeding. Foreground selection was performed using a co-dominant single nucleotide polymorphism marker through a high-resolution melting approach, and background selection was undertaken using 50–55 polymorphic sequence-tagged microsatellite site markers. Key results Near-isogeneic lines were produced with >90% recurrent parental genome and reduction of phytic acid content by up to 44–56% compared with the original lines. Conclusions The near-isogeneic lines carrying lpa1 can be used to reconstitute DHM 121 with low phytate content. Implications The low-phytate maize hybrids produced can be useful in reducing micronutrient malnutrition in humans, as well as environmental pollution.

Unclasping potential chickpea resources for the antioxidant enzyme superoxide dismutase

AbstractBackgroundThe investigation was conducted to assess the diverseness and identify novel resources to be utilized in chickpea abiotic‐biotic stress resistance breeding for the antioxidant enzyme Superoxide Dismutase. The plants were grown in a randomized block design in triplicates under all suitable agronomic practices using PUSA 256 as a check. Fresh leaves were collected for the estimation of enzyme superoxide dismutase (SOD) and DNA extraction. Number of pods were recorded on 20 individual plants from the middle of the row for each of the 12 genotypes. Employing 32 Sequence Tagged Microsatellite Site (STMS) markers together with morpho‐biochemical data, Jaccard's similarity coefficients along with dendrograms were generated to compare and assess the diversity among the genotypes.ResultsAmong the 12 chickpea genotypes, poorest versus best performers were identified as BGD‐70 versus ICRISAT‐3668 for SOD activity. Out of 32 STMS primers, 80 alleles with 2.5 an average per loci were found. The marker TA‐80 with the highest polymorphic information content (PIC) could be identified to be used in molecular characterization. The meticulous perusal revealed that the genotypes ICRISAT‐3668 and SBD 377, distantly located on different molecular clusters, expressed the higher SOD activity with the observed distances indicating genetic governance, probably by a limited number of polygenes/OTLs and might be utilized as a potential source for the SOD activity for chickpea abiotic‐biotic stress resistance breeding programs.ConclusionThe identified novel genetic resources may be used to widen the germplasm base, prepare a maintainable catalog and identify systematic blueprints for future chickpea abiotic‐biotic stresses breeding strategies.

The core set of sequence-tagged microsatellite sites markers between halophytic wild rice Oryza coarctata and Oryza sativa complex

For breeding of salinity tolerant rice, halophytic species Oryza coarctata is considered as a valuable resource. Non-availability of molecular markers in the species is a major limitation and identification of markers applicable with wider gene pool will be resource-saving. Even after the recent advancement in high throughput genotyping techniques, sequence-tagged microsatellite sites (STMS) markers are considered as valuable resource for pre-breeding, especially when usable for a large number of species. Using a novel bioinformatics pipeline for cross transferable marker identification, whole genome sequences of nine Oryza species were surveyed with 23,499 STMS markers of rice. With this highly reproducible strategy, band sizes or polymorphism prediction among different species is possible before in-vitro validation. Only 359 STMS markers were cross-transferable to O. coarctata and 77 of those were common with O. sativa complex. These core markers were distributed over 11 chromosomes and nearly 76% were located within various genes of Oryza. The markers also showed unique genome specific polymorphism pattern with high levels of inter- and intraspecific variations in “AA” genome and complete absence of intra- or inter-population variations in O. coarctata (KKLL). The numbers of microsatellite motifs and the repeat numbers in different motifs were much lesser in O. coarctata leading to the exceptionally high level of polymorphism with all “AA” genome species. Two markers are also useful for species identification. The hyper-variable markers are effective resource for pre-breeding of O. sativa, O. glaberrima and new rice for Africa involving O. coarctata or any other species of O. sativa complex.

SSR Marker Based Molecular Genetic Diversity Analysis Among Saccharum spontaneum (L.) Collected from North Western Region of India

Saccharum spontaneum L. is the most diverse species of the genus Saccharum with the widest distribution from Africa-Mediterranean region to Far East including South Pacific islands. Its contribution in the development of present day sugarcane cultivars is well recognized. In the present study, 30 S. spontaneum L. accessions collected from North Western India including the states of Gujarat and Rajasthan were characterized with respect to their ecological distribution using 20 STMS (Sequence Tagged Microsatellite Site) primers. The study revealed the ability of STMS markers in discriminating the accessions with specific markers with 95.4% polymorphism. The pair wise genetic distance among 30 S. spontaneum L. accessions ranged from 0.17 to 0.48 with an average of 0.32. The maximum genetic distance was observed between the pairs IND 07-1462 and IND 08-1501 and IND 07-1464 and IND 08-1501. All the 30 S. spontaneum L. accessions were grouped into five major clusters. Among the five clusters, Cluster II had maximum of nine accessions viz., IND 07-1461, IND 07-1462, IND 07-1463, IND 07-1465, IND 07-1466, IND 07-1467, IND 07-1468, IND 07-1470 and IND 07-1474. All the accessions except IND 07-1474 were from Kachchh district of North West arid zone with low rainfall and high salinity. Majority of the accessions clustered as per the agro-climatic zones. Unique markers for the accession IND 07-1460, IND 07-1463 and IND 07-1482 were identified which could help in maintaining germplasm identity. Accessions from arid and semi-arid zones were grouped separately into different clusters. These accessions were collected from the regions where drought and salinity were prevalent hence could be the potential sources for developing climate resilient sugarcane varieties with drought and salinity tolerance.

Narcea\u2014an unknown, ancient cultivated rose variety from northern Spain

The present work reports the discovery and the complete characterisation of an ancient cultivated rose variety found growing in a private garden in the southwest of the Principality of Asturias (northern Spain). The variety is here given the name Narcea. The majority of roses currently cultivated belong to the so-called group of ‘Modern Roses’, all of which were obtained after 1867 via artificial crosses and improvement programmes. All are destined for ornamental use. Until the 19th century, the great majority of the many ancient cultivated roses in Europe were used in perfumery and cosmetics, or had medicinal uses. Rosa damascena and Rosa centifollia are still grown and used by the French and Bulgarian perfume industries. The Asturian Massif of the Cantabrian Mountain Range provides a natural habitat for some 75% of the wild members of the genus Rosa, but until now there was no evidence that this area was home to ancient cultivated roses. A complete botanical description is here provided for a discovered ancient rose. It is also characterised according to a series of sequence tagged microsatellite sites, and its agronomic features are reported. In addition, a histological description (optical and scanning electronic microscope studies) of the petals is offered, along with an analysis of the volatile compounds present in these organs as determined by solid phase microextraction and gas chromatography-mass spectroscopy. The results reveal the uniqueness of this ancient type of rose and suggest it may be of interest to the perfume industry.

GLADS: A gel-less approach for detection of STMS markers in wheat and rice.

Sequence tagged microsatellite site (STMS) are useful PCR based DNA markers. Wide genome coverage, high polymorphic index and co-dominant nature make STMS a preferred choice for marker assisted selection (MAS), genetic diversity analysis, linkage mapping, seed genetic purity analysis etc. Routine STMS analysis involving low-throughput, laborious and time-consuming polyacrylamide/agarose gels often limit their full utility in crop breeding experiments that involve large populations. Therefore, convenient, gel-less marker detection methods are highly desirable for STMS markers. The present study demonstrated the utility of SYBR Green dye based melt-profiling as a simple and convenient gel-less approach for detection of STMS markers (referred to as GLADS) in bread wheat and rice. The method involves use of SYBR Green dye during PCR amplification (or post-PCR) of STMS markers followed by generation of a melt-profile using controlled temperature ramp rate. The STMS amplicons yielded characteristic melt-profiles with differences in melting temperature (Tm) and profile shape. These characteristic features enabled melt-profile based detection and differentiation of STMS markers/alleles in a gel-less manner. The melt-profile approach allowed assessment of the specificity of the PCR assay unlike the end-point signal detection assays. The method also allowed multiplexing of two STMS markers with non-overlapping melt-profiles. In principle, the approach can be effectively used in any crop for STMS marker analysis. This SYBR Green melt-profiling based GLADS approach offers a convenient, low-cost (20–51%) and time-saving alternative for STMS marker detection that can reduce dependence on gel-based detection, and exposure to toxic chemicals.

Saturation of genomic region implicated in resistance to Fusarium oxysporum f. sp. ciceris race 5 in chickpea

Fusarium oxysporum f. sp. ciceris (Foc) is the major soilborne fungus affecting chickpea and race 5 (Foc5) is the most important in the Mediterranean basin. A gene controlling resistant reaction to Foc5 has been located on LG2 of the chickpea genetic map forming a cluster with resistance genes to other Foc races. The sequence-tagged microsatellite site (STMS) marker TA59 is tightly linked to this genomic region. In the current study, our aim was to look for candidate genes related to resistance to Foc5 starting from the physical position of TA59 and taking advantage of the whole chickpea genome sequence information. We selected a set of markers, covering a region of around 25 Mbp that were genotyped in near-isogenic lines and recombinant inbred lines. Joining data of different plant materials, it was possible to define an area of approximately 820 Kbp. One of the 26 genes annotated in the selected region (LOC101511605) could be considered a candidate gene for its possible implication in resistance reactions. Single-nucleotide polymorphism (SNP) markers selected in the target region were used to screen a collection of 32 genotypes differing in their reactions to Foc5. A cluster analysis showed four main groups, three of them grouping the resistant genotypes and the fourth one, the susceptible lines. The comparison between the haplotypes of representative accessions for each cluster allowed the identification of six SNPs coincident among resistant lines and different from the susceptible lines. These SNPs could be used in marker-assisted selection.

STMS (sequence tagged microsatellite site) molecular markers as a valuable tool to confirm controlled crosses in chickpea (Cicer arietinum L.) breeding programs

Chickpea (Cicer arietinum L.) is an annual, diploid and autogamous grain legume. Most breeding processes developed in this crop start with complementary crosses, but artificial hybridization is a tedious operation probably due to the injury of the pistil caused during emasculation due to the small size of the flowers. The success in pod formation after hybridization together with the possibility of testing the hybrid nature of F1 is a key point to optimize chickpea breeding. Morphological characters are not always adequate to distinguish between progeny, consequently molecular markers are needed to verify hybrid nature. In this study, we show our experience in crossing programs and the use of molecular markers to test hybridization rate in three different cases (A) obtaining advanced lines combining resistance to blight and fusarium wilt; (B) developing segregating populations for Orobanche foetida and (C) providing suitable materials to do genetic studies involving double pod mutants. A total of 2041 pollinated flowers produced 21.7, 16.8 and 8.7% of pods in each one of the three cases analyzed. Hybridization nature in F1 seeds was tested with STMS (sequence tagged microsatellite site) markers and ranged from 86 to 91% for the three cases. STMS markers were a valuable tool to detect hybrids, being simple to visualize and having a low cost. The confirmation of hybrid nature at the initial development stages of F1 plants is critical for reducing time and costs in breeding programs.

Genetic diversity and population structure among 133 elite genotypes of sugarcane (Saccharum spp.) for use as parents in sugarcane varietal improvement

A measure of genetic diversity of genotypes to be used as parents is imperative to use them prudently in crop improvement. In this study, genetic diversity and population structure of 133 sugarcane hybrid derivatives were quantified using 20 sequence-tagged microsatellite sites (STMS) primers. The number of alleles ranged from 9 to 27 with the average of 17.95 alleles per primer, while the polymorphism information content values of the primers ranged from 0.29 to 0.78. Cophenetic correlation coefficient value observed as 0.84 by STMS markers revealed that the cluster result was acceptable for the calculation of genetic similarity matrix. Principal component analysis showed that 133 genotypes fell in two groups, first and second components associated 8.34 and 3.22% with eigen values of 5.61 and 2.17, respectively. Similar trend was observed with principal coordinate analysis, wherein, the first and second component accounted to 8.34 and 3.22% with eigen values of 741.29 and 286.11. The similarity index values ranged from 0.50 to 0.87 for the possible 8778 combinations from 133 genotypes, of which 8069 combinations exhibited less/moderate genetic similarity indicating the availability of sufficient genetic diversity in the experimental material and hence their value in the genetic improvement of sugarcane. Dissimilarity analysis using DARwin of 133 genotypes could distinguish two major clusters and into five subclusters and the results matched with those of the population structure which also showed five subpopulations. The bigger group SP1 was predominantly comprised of clones developed at the main sugarcane-breeding place in India, located at Coimbatore. The subpopulation SP4 was formed largely with clones from research stations other than at Coimbatore and interspecific hybrids, while SP5 comprised of clones of early origin. These observations were similar to the radial tree based on the DARwin software in that 81.95% of the genotypes of each cluster were similar in the two analyses. The results thus showed that location and time of origin were two major factors that contributed to diversity. Based on analysis of molecular variance, subpopulations SP2 and SP4 were more variable from the rest. SP2 (comprising of Co 99008, Co 99006, Co 94012, Co 93023, CoC 671, Co 89034, Co 91003, Co 06022, Co 98017, Co 87044, Co 06018, Co 89003, Co 98014, and Co 86032) exhibited maximum genetic variation, the least gene flow, and the lowest heterozygosity value and would serve as the best group for utilization in genetic improvement. Graphical genotyping (GGT) image of each genotype was distinctly different, indicating the genetic uniqueness of sugarcane genotypes under study as revealed through STMS technology. A core set of 40 genotypes was identified using GGT 2.0 software program for the easiness of harnessing the available genetic diversity of 133 genotypes, through hybridization in sugarcane improvement programs.

High resolution melting (HRM)-based STMS marker analysis for rapid identification of radiation induced mutants of sugarcane

Molecular marker based selection has become the method of choice to find undiscoverable natural variations and induced mutations in plants. Routine gel based molecular marker screening procedures involve complex steps which require considerable investment of time, money and efforts. Most often, genomic configuration of a plant species complicates the ultimate output of such screening programme. Therefore, a reliable and rapid high-throughput method is required to facilitate ease in identification of variants in a large number of samples. Here we demonstrate the application of a high-resolution melting (HRM) method to detect gamma radiation induced mutants of sugarcane (Saccharum spp.) which is a complex aneu-polyploidy plant. We have screened nine distinct mutants using sequence-tagged microsatellite site (STMS) markers and the analyses based on gel electrophoresis and HRM curve were compared. All the six STMS microsatellite markers coupled with HRM analysis were found highly informative and generated a unique melting curve for each of the marker tested. Of these, two markers NKSCSSR 22 and NKSCSSR 42 exhibited differential melting curve among the mutants and these results were well correlated with the gel based banding pattern. Taken together these results highlighted potential of HRM based marker screening and demonstrated reliability and robustness in detecting induced mutations in a complex polyploidy plant like sugarcane.

Marker Aided Incorporation of Saltol, a Major QTL Associated with Seedling Stage Salt Tolerance, into Oryza sativa 'Pusa Basmati 1121'.

Pusa Basmati 1121 (PB1121), an elite Basmati rice cultivar is vulnerable to salinity at seedling stage. A study was undertaken to impart seedling-stage salt tolerance into PB1121 by transferring a quantitative trait locus (QTL), Saltol, using FL478 as donor, through marker assisted backcrossing. Sequence tagged microsatellite site (STMS) marker RM 3412, tightly linked to Saltol was used for foreground selection. Background recovery was estimated using 90 genome-wide STMS markers. Systematic phenotypic selection helped in accelerated recovery of recurrent parent phenome (RPP). A set of 51 BC3F2 lines homozygous for Saltol were advanced to develop four improved near isogenic lines (NILs) of PB1121 with seedling stage salt tolerance. The background genome recovery in the NILs ranged from 93.3 to 99.4%. The improved NILs were either similar or better than the recurrent parent PB1121 for yield, grain and cooking quality and duration. Biochemical analyses revealed significant variation in shoot and root Na+ and K+ concentrations. Correlation between shoot and root Na+ concentration was stronger than that between root and shoot K+ concentration. The effect of QTL integration into the NILs was studied through expression profiling of OsHKT1;5, one of the genes present in the Saltol region. The NILs had significantly higher OsHKT1;5 expression than the recurrent parent PB1121, but lower than FL478 on salt exposure validating the successful introgression of Saltol in the NILs. This was also confirmed under agronomic evaluation, wherein the NILs showed greater salt tolerance at seedling stage. One of the NILs, Pusa1734-8-3-3 (NIL3) showed comparable yield and cooking quality to the recurrent parent PB1121, with high field level seedling stage salinity tolerance and shorter duration. This is the first report of successful introgression of Saltol into a Basmati rice cultivar.

Genetic diversity analysis among selected short duration chickpeacultivars and breeding lines based on STMS markers.

Genetic diversity among 30 chickpea genotypes was evaluated using simple sequence repeat (SSR) molecular markers. The studies using Sequence Tagged Microsatellite Site (STMS) markers markers revealed that among the primers used across the genotypes produced a total of 35 alleles representing 21 SSR loci with frequencies ranging from one to two (mean 1.66) alleles per locus. Polymorphic Information Content (PIC) ranged from 0.098 to 0.500 (CAM0443, CAM0446). These primers might be an effective and useful tool to determine the genetic differences among chickpea genotypes and to study the phylogenetic relationships. Polymorphic percentage was 96.42. Hierarchical neighbour-joining UPGMA cluster analysis based on simple matching similarity matrix resolved the 30 genotypes into seven clusters. Based on STMS markers highest similarity index 0.850 was observed between BGD 72 and Annigeri-1whereas BGD 9920 and ICC 92944 showed the lowest similarity index 0.214 between them. The STMS clustering pattern indicated the presence of wide genetic diversity between the genotypes. Overall, the study ascertained that SSRs provide powerful marker tools in revealing genetic diversity and relationships in chickpeas, thereby proving useful for selection of parents in breeding programs and also for DNA fingerprinting for identification of cultivars.

Molecular characterization of chickpea (Cicer arietinum L.) genotypes using sequence tagged microsatellite site (STMS) markers

Chickpea (Cicer arietinum L.) commonly also known as gram, Chana, Bengal gram and Garbanzo beans is the second most important pulse crop of the world mainly grown in arid and semi-arid regions. Assessment of genetic variability in the base population is the first step in any breeding programme for selection of genetically divergent parents and their use in the crop improvement programme. In the present investigation 20 genotypes of chickpea were characterized using a specific set of 15 numbers of Sequence tagged microsatellite site (STMS) markers. The number of alleles, allelic distribution and their frequency was estimated and found that the 36 alleles amplified with 15 STMS loci having an average of 2.4 alleles per locus. The number of alleles amplified varied from 1 to 4. The Polymorphic information content value ranged from 0 to 0.965 with an average of 0.373 indicated the considerable efficiency of markers for studying the polymorphism level. All primer showed higher polymorphism among the genotypes except two primers namely, TAA59 and GA105 which were monomorphic in nature. Genetic similarity based on UPGMA clustering the dendrogram grouped the 20 genotypes in three clusters, cluster I, II, III comprised of 2, 4, 14 genotypes, respectively. The maximum similarity was found between genotypes ICRISAT-4183 and ICRISAT- 7722 (0.972). The present study provided an insight of the inter-relationship among the genotypes and highlights the genetic distance by STMS markers. The genetic diversity revealed in this study could be exploited for selective breeding programme of chickpea improvement.

Leaf crinkle disease in urdbean (Vigna mungo L. Hepper): An overview on causal agent, vector and host.

Urdbean leaf crinkle disease (ULCD) is an economically significant widespread and devastating disease resulting in extreme crinkling, puckering and rugosity of leaves inflicting heavy yield losses annually in major urdbean-producing countries of the world. This disease is caused by urdbean leaf crinkle virus (ULCV). Urdbean (Vigna mungo L. Hepper) is relatively more susceptible than other pulses to leaf crinkle disease. Urdbean is an important and useful crop cultivated in various parts of South-East Asia and well adapted for cultivation under semi-arid and subtropical conditions. Aphids, insects and whiteflies have been reported as vectors of the disease. The virus is also transmitted through sap inoculation, grafting and seed. The loss in seed yield in ULCD-affected urdbean crop ranges from 35 to 81%, which is dependent upon type of genotype location and infection time. The diseased material and favourable climatic conditions contribute for the widespread viral disease. Anatomical and biochemical changes take place in the affected diseased plants. Genetic variations have been reported in the germplasm screening which suggest continuous screening of available varieties and new germplasm to search for new traits (new genes) and identify new sources of disease resistance. There are very few reports on breeding programmes for the development and release of varieties tolerant to ULCD. Mostly random amplified polymorphic DNA (RAPD) as well as inter-simple sequence repeat (ISSR) molecular markers have been utilized for fingerprinting of blackgram, and a few reports are there on sequence-tagged micro-satellite site (STMS) markers. There are so many RNA viruses which have also developed strategies to counteract silencing process by encoding suppressor proteins that create hindrances in the process. But, in the case of ULCV, there is no report available indicating which defence pathway is operating for its resistance in the plants and whether same silencing suppression strategy is also followed by this virus causing leaf crinkle disease in urdbean. The antiviral principles (AVP) present in leaf extracts of several plants are known to inhibit infection by many viruses. Many chemicals have been reported as inhibitors of virus replication in plants. Raising the barrier crops also offers an effective solution to control the spread of virus.

Transferability of Sequence Tagged Microsatellite Site (STMS) Markers FromVigna Unguiculata(L.)Walp to Vigna Radiata(L.) Wilczek

The transferability of genome-specific sequence tagged microsatellite Site (STMS) markers from Cowpea [Vigna unguiculata (L.) Walp] to Mungbean [Vigna radiata (L.)Wilczek] was examined. The amplification of 19 cowpea STMS markers was carried out with 12 varieties of mungbean. Overall, Cowpea STMS primers amplified products in most of the accessions. Among the 19 pairs of VM series cowpea microsatellite primers used 17 were found to be useful, which were then checked for homozygosity. As a result only one of them was found to be polymorphic. There are no records found earlier regarding the microsatellite markers in mungbean. The transferability of these STMS primers indicates a high level of sequence conservation among cowpea and mungbean. The polymorphic SSR markers identified in this study would be useful in the analysis of genetic diversity, genetic mapping and other aspects of genetic studies in mungbean.

STMS marker assay based diversity in selected hybrid clones of sugarcane (Saccharum officinarumL.)

Molecular genetic diversity among 35 selected clones of sugarcane assayed using 30 sugarcane specific sequence tagged microsatellite site (STMS) primers. Present investigation revealed that the markers analyzed were highly informative by virtue of their PIC (polymorphic information content) values. Jaccard's similarity coefficient value estimated was ranged from 0.37 to 0.89 with an average of 0.63 indicating the existence of appreciable genetic diversity among the clones studied which opened the avenue for further improvement of the clones through hybridization. UPGMA clusters analysis detected six clusters of which, Cluster III was largest with 26 genotypes. There was poor correspondence between pedigree and molecular markers as the two genotypes evolved from crosses involving different parentages were also clustered together in addition to clustering together of clones with at least one common parent and the clones evolved from the same cross showed more diversity suggesting that involvement of genotypes derived from same parental combination need not always lead to close breeding.

Fine mapping for double podding gene in chickpea.

For the first time, fine mapping for sfl locus was carried out using a battery of new STMS and SNP markers. The target region was delimited to 92.6Kb where seven annotated genes were found that could be candidate genes for the simple/double podding trait in chickpea. Four recombinant inbred populations (RIP-1, RIP-7, RIP-11, and CPR-01) were used to map the double podding gene (sfl) in chickpea. In RIP-1, the gene was initially mapped on linkage group (LG) 6 between the two sequence-tagged microsatellite site (STMS) markers TA120 and TR1. Eight new STMS markers were added onto LG6 in the target region and sfl locus was finally located between CAGM27819 and CAGM27777 markers within an interval of 2cM. Seven out of the eight markers were mapped in RIP-7 and its reciprocal RIP-11 confirming the location of the sfl locus to a 4.8cM interval flanked by TR44 and CAGM27705. Furthermore, using a high-density single nucleotide polymorphism (SNP) map of CPR-01, sfl was mapped to the same genomic region in a 5.1cM interval between TR44 and the SNP scaffold1646p97220. Five pairs of near isogenic lines (NILs) and eight recombinant inbred lines (RILs) were used to refine this region in the chickpea physical map. Combining data from linkage analysis in four RIPs, marker physical positions and recombination events obtained in both pairs of NILs and selected RILs, sfl could be placed within a genomic window of 92.6Kb. Seven annotated genes were extracted from this region. The regulator of axillary meristem-predicted gene could be a candidate gene for the simple/double podding gene. This study provides additional set of markers flanking and tightly linked to sfl locus that are useful for marker-assisted selection.

Detection of a new QTL/gene for growth habit in chickpea CaLG1 using wide and narrow crosses

A recombinant inbred line population (RIP-9) derived from an interspecific cross (ILC72 × Cr5-10) was evaluated for growth habit during 2 years (2003 and 2004). This RIP was used to develop a pair of near isogenic lines (NILs) for erect vs prostrate growth habit in chickpea. Molecular characterization of the identified pair of NILs was performed using 52 sequence tagged microsatellite site markers distributed over different chickpea linkage groups (CaLG) of the genetic map. It revealed polymorphic markers in CaLG1 and CaLG3. Starting from a previous data base simple linear regression was applied to detect association between markers and growth habit. The RAPD (random amplified polymorphic DNA) marker OPAD091053 mapped on CaLG1 explained the highest percentage (maximum 15.4 %) of the total phenotypic variation for growth habit and it was used to develop a SCAR (sequence characterized amplified region) marker (SCAD091053). New markers were developed from sequences surrounding SCAD091053 in the physical map. QTL (quantitative trait loci) analysis revealed a new QTL (QTLHg2) in CaLG1. The Indel marker (deletion/insertion) Indel 3 and the predicted gene Ca_07000 (14,5 Mb of Ca1) and (15,3 Mb of Ca1) had the highest LOD values explaining 24.6 and 23.4 % of the phenotypic variation in years 2003 and 2004, respectively. To confirm these results, another RIP (RIP-5) derived from an intraspecific cross (WR315 × ILC3279) and segregating for erect vs semi-erect growth habit was employed. RIP-5 allowed mapping the gene (Hg2/hg2) on CaLG1 that was flnaked by two Indel markers (Indel 1 and Indel 2) in the range of 12,3 and 16,2 Mb. So, Hg2/hg2 gene corresponds to QTLHg2 region. The annotated genes Ca_07000 and Ca_06999 were homologues to predicted zinc finger genes in Glycine max and Pisum sativum, respectively. Hence, they could be considered as possible candidate genes.