Sequence Tagged Microsatellite Site Markers 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.

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.

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.

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.

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.

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.

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.

Variety identification and genetic diversity analysis in rice (Oryza sativa L.) using STMS markers

In the present study, 14 rice specific sequence tagged microsatellite site markers (STMS) were used to developfingerprints and to determine the genetic relatedness among 48 rice genotypes/cultivars. Ten of the 14 SSR primerpairs were found to be polymorphic, generating a total of 39 distinct reproducible alleles with an average of 2.79alleles per primer pair and 22 (56.41%) of the 39 bands amplified emerged as polymorphic. The number of alleles perprimer ranged from one to six. The polymorphic information content (PIC) value ranged from 0.03 to 0.7 with anaverage of 0.35. Cluster analysis based on Jaccard’s similarity coefficient using unweighted pair group methods forarithmetic mean (UPGMA) grouped the 48 rice cultivars into five major clusters with an average similarity index of0.87. A diagrammatic representation of DNA fingerprints of the rice cultivars based on 10 informative SSR markerswas developed for ready reference, which are expected to be key in varietal identification as well as provide IPR tohalt biopiracy.

CHARACTERISATION OF FALL-BLOOMING CAMELLIAS AS REVEALED BY SEQUENCE TAGGED MICROSATELLITE SITE MARKERS AND MORPHOLOGICAL TRAITS

Genetic resources can be described as the total genetic diversity of cultivated species and their wild relatives. Their effective conservation and use depend on the availability of information and on the extent and distribution of genetic diversity in species of interest. In recent years, numerous molecular techniques have been developed to provide markers. Among the several classes of DNA-based markers, the Sequence Tagged Microsatellite Sites (STMS) are highly polymorphic, multi-allelic, frequently co-dominant, highly reproducible, selectively neutral, and widely and randomly distributed in the genome. In order to improve the genetic knowledge in ornamentals and to solve taxonomical issues, STMSs were used to investigate the Camellia genus. A set of 44 cultivars belonging to C. sasanqua L., C. × hiemalis Nakai, C. hybrid, and C. × vernalis Makino were characterised by means of 4 STMSs and 15 morphological traits referring to flower and leaf morphology, colour, and fragrance. STMS markers successfully amplified all the accessions, and 58 alleles were scored. The distribution of the genetic variation highlighted genetic overlapping among C. sasanqua cultivars and the hybrids of C. × vernalis, C. × hiemalis, and C. hybrid. The Principal Coordinate Analyses (PCoA) based on joined molecular and morphological data sets showed a distribution of all accessions in agreement with their taxonomic classification. Taken as a whole, results demonstrated that this set of microsatellite loci are suitable for describing genetic diversity in fall-blooming camellias, and thus constitute an appropriate tool for germplasm characterization and management.

Genetic Similarity Analysis in Lentil Using Cross‐Genera Legume Sequence Tagged Microsatellite Site Markers

ABSTRACTMolecular markers have emerged as useful tools to assess the genetic diversity across crops. In lentil, molecular markers are limited. The objective of the study was to explore cross‐genera transferability of sequence tagged microsatellite site (STMS) markers from related legumes and assess their utility in lentils. Thirty lentil (Lens culinaris Medik. subsp. culinaris) accessions were evaluated for genetic similarity analysis using cross‐genera STMS markers. Thirty‐nine STMS markers amplified 68 alleles with an average of 1.74 alleles per locus. Twenty lentil‐specific STMS markers produced a total of 36 amplicons, of which 90% (18) markers were polymorphic. A maximum of four alleles were obtained with primers SSR13 and SSR19. Of 47 STMS markers from other legume genera, only 19 markers produced 32 scorable amplicons, and only 58% (11) of the amplified markers exhibited polymorphism. The polymorphism information content values observed with lentil specific markers ranged from 0.02 to 0.99, while for transferrable markers it ranged from 0.06 to 0.84. Maximum genetic similarity was observed between ‘NDL1’ and ‘LH84‐8’ (0.942) and minimum between ‘PL234’ and ‘Precoz’ (0.709). The dendrogram based on Jaccard's similarity coefficients showed limited genetic variability among the cultivars included in the present study. A combination of lentil‐specific and transferrable STMS markers was successfully used for identification of genetic similarity in lentil germplasm.

Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus

Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus. For simple sequence repeat (SSR) isolation, a genomic library enriched for (GA)(n) repeats was constructed from C. roseus 'Nirmal' (CrN1). In addition, GTMs were also designed from 12 genes of the TIA (terpenoid indole alkaloid) pathway - the medicinally most significant pathway in C. roseus. An F(2) mapping population was also generated by crossing two diverse accessions of C. roseus CrN1 (Nirmal)×CrN82 (Kew). A new set of 314 STMS markers and 64 GTMs were developed in this study. A segregating F(2) mapping population consisting of 111 F(2) individuals was generated. For generating the linkage map, a set of 423 co-dominant markers (378 newly developed and 45 published earlier) were screened for polymorphism between the parental genotypes, of which 134 were identified to be polymorphic. A total of 114 markers were mapped on eight linkage groups that spanned a 632·7 cM region of the genome with an average marker distance of 5·55 cM. Further, the mechanism of hypervariability at the gene-targeted loci was investigated at the sequence level. For the first time, a large array of STMS markers and GTMs was generated in the model medicinal plant C. roseus. Moreover, the first microsatellite marker-based linkage map was described in this study. Together, these will serve as a foundation for future genomics studies related to quantitative trait loci analysis and molecular breeding in C. roseus.