Polymorphic Microsatellite Markers Research Articles

Equivalent mating system parameters in post‐mining and undisturbed native plant populations confirms restitution of bird‐pollinator function

Abstract The restoration of diverse self‐sustaining ecosystems requires re‐establishment of functional interactions among species. For plant communities, pollinators are usually essential for pollination, seed set and seed quality. A common assumption in ecological restoration for plants pollinated by animals is one of ‘build it and they will come’, which is rarely tested. Beyond seed set, there may be negative genetic consequences for seed quality if pollinators and their behaviour do not reflect those in reference populations. Here, we conduct an ecological genetic assessment of seed quality via mating system parameters in Lambertia multiflora (Proteaceae), a species dependent on nectar‐feeding birds for pollination. Four populations of L. multiflora in disturbed sites that were rehabilitated following mineral sands mining were compared with four native reference populations, near Eneabba, Western Australia. In each population, approximately 10 offspring from each of 10 maternal plants were genotyped with 11 highly polymorphic microsatellite markers. From these data, genetic diversity and mating system parameters were assessed, and found to be equivalent across all populations. Mean allelic diversity and heterozygosity across loci were very high. All populations were completely outcrossing with no bi‐parental inbreeding. Mean correlated paternity, sibship and effective population size estimates for restored and natural populations were not significantly different and reflected uniformly high paternal diversity and wide outcrossing. Equivalent genetic results for restored and natural reference populations indicate successful restitution of bird‐pollinator services for L. multiflora in these post‐mining rehabilitation sites. Synthesis and applications. Reviewing our results with other published studies to date suggests a resilience of bird‐pollinator services in restored plant communities. These findings provide some reassurance to restoration practitioners working in these global south systems where bird pollination is a feature, at least for similar landscape scenarios. Our study also highlights the global contribution of ecological genetics to the objective assessment of functional species interactions in ecological restoration, an increasingly important goal of land managers and regulators seeking to improve restoration standards.

Development of Twenty Novel Polymorphic Microsatellite Markers and Their Application in Population Genetic Studies of Konosirus punctatus

Development of Twenty Novel Polymorphic Microsatellite Markers and Their Application in Population Genetic Studies of Konosirus punctatus

Is promiscuity the key? Multiple paternity in the garden dormouse (Eliomys quercinus)

AbstractMany mammals have a promiscuous mating system with multiple sired litters. Promiscuity can increase the genetic variability, reduce the risk of inbreeding, and increase the effective population size, and is therefore crucial for preventing genetic loss and maintaining adaptability. This is particularly true for small and threatened populations. The garden dormouse (Eliomys quercinus) is a threatened species, which exhibited a drastic decline over the last 20–30 years. The aim of this study was therefore to investigate the mating system of the garden dormouse in mountain forest habitat by parentage analyses using 5 polymorphic microsatellite markers combined with morphometric data and information about the nesting behavior. Genetic parentage analyses revealed that 64% (9 of 14) of the litters were sired by at least two males, suggesting that garden dormice have a promiscuous mating system. The genetic findings were further supported by indirect indicators of promiscuity, such as testes, that were nearly four times larger than predicted for a rodent of its body mass and only slight male biased sexual size dimorphism. The finding of a promiscuous mating system in garden dormice should be taken into account in future conservation efforts. Due to its habitat preferences and limited dispersal potential garden dormice are vulnerable to forest fragmentation. The connection of suitable habitats facilitates dispersal and promotes access to potential mating partners, which could be especially important for populations colonizing new habitats. Access to potential mates may also reduce inbreeding, loss of genetic variability which is crucial for populations viability and survival.

Combining biometrics and genetics to distinguish two subspecies of the Great Cormorant Phalacrocorax carbo carbo and P. c. sinensis at an inland lake in southeast Norway

Cover photo: Adult Great Cormorant of the subspecies Phalacrocorax carbo sinensis in breeding plumage. Photo: Frode Falkenberg. Great Cormorants Phalacrocorax carbo are now regularly seen in inland watercourses in southeast Norway, after the P. c. sinensis subspecies first established breeding colonies in coastal south Norway in 1996. Although both the sinensis and carbo subspecies occur, the ratio between them is unknown. We tested the accuracy of subspecific identification based on biometrics and genetic analyses in 75 Great Cormorants that drowned in fishing nets in a lake in southeast Norway during the period 2009-2020. Primarily based on the gular pouch angle (GPA), we classified 40 individuals to the carbo subspecies and 35 individuals to the sinensis subspecies. Eight of the carbo individuals and 14 of the sinensis individuals were within the overlapping range of GPA and were therefore classified to subspecies by supplementary measurements (bill depth minimum and bill length). Genetic analyses were based on seven polymorphic microsatellite markers. Assuming one panmictic population, we performed Structure analyses to separate the genotypes into two assumed genetic clusters. The two clusters, carbo and sinensis, could only be separated when adding information about morphological subspecies identities for the carbo, sinensis and carbo/sinensis groups. The sinensis and the carbo/sinensis groups belonged almost entirely to one genetic cluster, whereas the carbo group consisted of individuals with varying proportions of mixed ancestry. Furthermore, we found significant genetic differentiation between the carbo and sinensis groups, and between the carbo and the carbo/sinensis groups, but no significant differentiation between the sinensis and the carbo/sinensis groups. Our results suggest that gene flow is more common from sinensis into carbo than vice-versa, and that the use of GPA < 73° has limitations for the identification of Great Cormorant subspecies in areas where both forms occur. We conclude that the numbers of carbo vs. sinensis individuals in our sample were 32 and 43, respectively.

Molecular identification of Sinonovacula constricta, Sinonovacula rivularis and their interspecific hybrids using microsatellite markers

The razor clam Sinonovacula constricta, is one of the most commercially important cultured bivalves in China and Southeast Asia, while S. rivularis is its closer relatives discovered more than a decade ago. In order to obtain offspring with faster growth rate and stronger salt tolerance of S. constricta and S. rivularis, interspecific hybrids were produced, and the hybridity of the interspecific hybrids was confirmed by microsatellite markers. Microsatellite markers exhibit a high potential for transfer through cross-amplification in related species, and the transferability of 48 pairs of microsatellite marker primers from S. constricta were assessed in S. rivularis. Here, 24 universal microsatellite markers were successfully amplified in S. rivularis, of which 18 were polymorphic with the allele number from 2 to 5. The genetic diversity of two razor clams evaluated by 18 polymorphic microsatellite markers indicated that two species were both above the middle level, with a relatively higher genetic diversity, while S. constricta showed higher genetic diversity than S. rivularis according to the genetic parameters of Na, Ho, He and PIC. Furthermore, a total of two species-specific microsatellite markers were screened, which could be used for quick genetic identification of S. constricta, S. rivularis and their hybrids. The results suggest the induced interspecific hybrids are true hybrids between S. constricta and S. rivularis, which provide a basis for breeding, subsequent protection, and germplasm resources utilization of the razor clams.

Development of polymorphic microsatellite markers for genetic stock identification of green chromide, Etroplus suratensis using next generation sequencing technology

Development of polymorphic microsatellite markers for genetic stock identification of green chromide, Etroplus suratensis using next generation sequencing technology

Population genetics of gharial Gavialis gangeticus in the Chambal River, India, using novel polymorphic microsatellite markers

The gharial Gavialis gangeticus is a Critically Endangered crocodylian endemic to the Indian subcontinent. The species has experienced a 95% population decline over the past 2 centuries. The largest self-sustaining population inhabits the protected National Chambal Sanctuary (NCS) in north India and represents >80% of extant gharials globally. We developed de novo a panel of polymorphic gharial-specific microsatellites, using whole genome information and microsatellite search tools. These 15 new markers have multiple numbers of polymorphic alleles that are more informative than those obtained from previous studies. Analyses of 93 scute samples collected across age classes from wild gharials residing in the NCS facilitated accurate assessments of genetic diversity and inbreeding coefficient and identified a historical bottleneck event. Estimates of the observed and expected heterozygosities were lower than those reported earlier. The inbreeding coefficient was low, and the population did not deviate significantly from Hardy-Weinberg equilibrium. The calculated M ratio and 2 heterozygosity tests detected a genetic bottleneck, which is consistent with historic sharp declines in population size, followed by recent recovery. These new gharial microsatellite markers are statistically robust and provide an improved means to assess the population genetics of the largest self-sustaining wild gharial population. This study will facilitate additional investigations on the genetic diversity of other extant gharial populations—not only the few remaining wild populations but also those in zoos and rearing facilities. Additional genetic studies of gharial in the NCS are warranted to inform management strategies.

Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei by Illumina MiSeq sequencing

BackgroundEuropean vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations.Methods and resultsGenomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei).ConclusionsOur study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

Cryptic sexual reproduction in an emerging Eucalyptus shoot and foliar pathogen

AbstractEucalyptus scab and shoot malformation is an emerging disease and a serious threat to the global plantation forestry industry. The disease appeared in North Sumatra (Indonesia) in the early 2010s and the causal agent was recently described as a novel species, Elsinoe necatrix. Nothing is known regarding its possible origin or why it emerged rapidly to cause a serious local epidemic. To investigate its population biology, we developed 15 polymorphic microsatellite markers as well as mating‐type markers using genome sequences for two E. necatrix isolates. Isolates of the pathogen were collected from different host varieties at four locations in the Lake Toba region of North Sumatra and characterized using these markers. A high level of genotypic diversity was observed for all populations with little to no genetic differentiation between sampling areas. Discriminant analysis of principal components, genotype networks and analysis of molecular variance all showed a lack of population structure and a high level of gene flow among sampling regions. Mating‐type ratios and linkage disequilibrium analyses suggest that sexual recombination is likely to be occurring, although a sexual state has not been found for the pathogen. The results of this study highlight the fact that new genotypes of E. necatrix, probably arising from cryptic sexual recombination, will challenge efforts to manage the disease, and that breeding and selection for tolerance will require substantial host genetic diversity.

Polly: An R package for genotyping microsatellites and detecting highly polymorphic DNA markers from short-read data.

Highly polymorphic markers, such as microsatellites, are invaluable for the study of natural populations. However, contemporary methods for genotyping highly polymorphic variants have serious drawbacks that impede their efficiency. We created Polly, an R package with C++ source code that uses Illumina short-read data to genotype microsatellites, detect highly polymorphic variants and identify clusters of highly polymorphic SNPs, indels and microsatellites. We tested Polly on short-read data from Xiphophorus birchmanni (Teleostei: Poeciliidae) and Arabidopsis thaliana, finding it to be efficient and accurate both for microsatellite genotyping and polymorphic marker detection. This program can be applied to any diploid population for which there exists short-read data and at least one scaffolded reference genome.

Genetic diversity of three consecutive selective breeding generations in Pseudobagrus vachellii (Actinopterygii: Siluriformes: Bagridae)

Pseudobagrus vachellii (Richardson, 1846) is a commercially important freshwater fish species in China. To understand the effects of artificial breeding on the genetic diversity of three consecutive P. vachellii breeding populations (F1, F2, and F3) since 2012, a genetic analysis was conducted using polymorphic microsatellite markers. The mean allele number, expected heterozygosity, observed heterozygosity, and the polymorphic information content from generation F1 to F3 decreased from 7.75 to 5.63, from 0.77 to 0.63, from 0.83 to 0.77, and from 0.72 to 0.58, respectively. Analysis of molecular variance showed greater genetic divergence within the three generations (93.67%) than that among the generations (6.33%), and the overall differentiation level was moderate. Additionally, the lowest genetic differentiation was between F2 and F3 (Fst = 0.0484), and the highest was between F1 and F3 (Fst = 0.12864). Inbreeding occurred in each generation and was the highest in generation F3. Structural analysis showed that the three P. vachellii generations were most likely divided into two different genetic clusters. Although genetic diversity declined slightly in the mass selection lines after three breeding generations, overall genetic diversity was maintained at a relatively high level. To minimize the loss of genetic diversity and inbreeding in the subsequent breeding process, a moderate number of parents can be used for each generation. Information regarding the genetic diversity and structure of the selective P. vachellii breeding generations obtained in this study will be useful for future broodstock management and selective breeding programs.

Population genetic structure of tropical bed bug (Hemiptera: Cimicidae) populations and their breeding pattern in Iraq.

A study was conducted to investigate the population genetic structure and breeding pattern of 140 tropical bed bugs, Cimex hemipterus (F.) (Hemiptera: Cimicidae), collected from 14 infested sites in major cities in Iraq. The samples were genotyped using a set of 7 polymorphic microsatellite markers. High genetic variety was seen among populations, with an average of 2-9 alleles per locus. The number of alleles across 7 microsatellite loci was between 6 and 18. There was a notable disparity in the alleles per loci when comparing the overall population to those within it. The overall population exhibited an average observed heterozygosity of 0.175 and an average expected heterozygosity of 0.730. Among the population, the average observed heterozygosity was 0.173, while the average expected heterozygosity was 0.673. Analysis of molecular variance (AMOVA) revealed that 93% of the genetic variability was within the populations, and 7% was among them. The genetic differentiation coefficient (FST = 0.045), indicates a low degree of genetic differentiation and a high degree of inbreeding (FIS = 0.761), as indicated by notably significant positive inbreeding coefficients. Admixed individuals were revealed using STRUCTURE and neighbor-joining phylogenetic trees, demonstrating moderate gene flow between populations and a lack of genetic structure in the regional groups. Thus, both active dispersion and human-mediated dispersion possess the potential to influence the low population genetic structure of tropical bed bug C. hemipterus populations in Iraq, which can have implications toward tropical bed bug and management strategies.

Population Genetic Structure of the Rubber Tree Powdery Mildew Pathogen (Erysiphe quercicola) from China.

In order to manage agricultural pathogens, it is crucial to understand the population structure underlying epidemics. Rubber tree powdery mildew, caused by Erysiphe quercicola, is a serious threat to rubber plantations worldwide, especially in subtropical environments including all rubber tree-growing regions in China. However, the population structure of the pathogen is uncertain. In this study, 16 polymorphic microsatellite markers were used to genotype powdery mildew samples from the main rubber tree-growing regions including Yunnan (YN), Hainan (HN), western Guangdong (WG), and eastern Guangdong (EG). YN had higher genotypic diversity (Simpson's indices), genotypic evenness, Nei's gene diversity, allelic richness, and private allelic richness than the other regions. Cluster analysis, discriminant analysis of principal components, pairwise divergence, and shared multilocus genotype analyses all showed that YN differed significantly from the other regions. The genetic differentiation was small among the other three regions (HN, WG, and EG). Analysis of molecular variance indicated that the variability among regions accounted for 22.37% of the total variability. Genetic differentiation was significantly positively correlated (Rxy = 0.772, P = 0.001) with geographic distance. Linkage equilibrium analysis suggested possible occurrence of sexual recombination although asexual reproduction predominates in E. quercicola. The results suggested that although significant genetic differentiation of E. quercicola occurred between YN and the other regions, pathogen populations from the other three regions lacked genetic differentiation.

Unfolding genomic regions and identification of novel marker-trait associations for therapeutic traits flavonoid and anthocyanin content in rice (Oryza sativa L.)

The genetic diversity, structure analysis, and marker-trait association for total flavonoid content (TFC) and anthocyanin content (AC) were studied in rice using 79 germplasm lines with 210 polymorphic microsatellite markers. Among the landraces studied, RG83 (RPHP 93) had the highest level of TFC (227.78 QE/100g) and RG126 (Kallimadayan) contained the highest level of AC (0.416 CE/100g). The STRUCTURE analysis grouped the studied landraces into three genetic structure groups which nearly matched the geographical distance. The results of the marker-trait associations study using MLM with PCA and kinship indicated that the SSR markers RM287 and RM19358 were closely associated with AC while another two markers RM452 and RM 2197 were closely linked with TFC and these two markers were found to be in the genomic regions related to the candidate genes for TFC and AC biosynthesis. In addition, the markers RM10961, RM5963, and RM559 were also found to have significant marker-trait associations with both AC and TFC.

Anthropogenic and natural disturbances increase local genetic diversity in an early spring geophyte (Ficaria verna Huds)

AbstractThe tetraploid Ficaria verna is a common spring geophyte in central Europe and is considered invasive in the USA and Canada. It is considered an almost seed‐sterile taxon, relying on vegetative reproduction by underground tubers and aerial bulbils. Recent studies have revealed high levels of population genetic diversity in F. verna, raising the question of how genetic diversity is maintained and which factors may be responsible for the observed patterns. Polymorphic nuclear microsatellite markers were established to define multi‐locus genotypes (MLGs), to analyze fine‐scale spatial genetic structure (SGS) using grid and cross‐sampling schemes, and to quantify genetic diversity within and between nine populations with different disturbance regimes in central Germany. In total, 115 MLGs were identified among a total of 347 samples. The G/N ratio varied between 0.16 and 0.70 among populations, and in each population several unique MLGs occurred. Genotypes were highly intermingled within populations, suggesting a “guerrilla” dispersal strategy. Significant SGS (negative regression slope of kinship coefficients against inter‐individual distances) was found in four out of nine populations in fine‐scale cross‐sampling (up to 4 m) and in only one population in grid sampling (up to 14.6 m). No single MLG was found in more than one population, while many alleles were shared between populations. Within‐population clonal and allelic diversity increased with greater exposure to both anthropogenic and natural disturbances. Regular gap openings, facilitated propagule establishment, and propagule dispersal by water and mowing machines are likely important factors explaining the positive effects of disturbance on local genetic diversity of F. verna.

Does the geographic distance affect the genetic differentiation among bilberry populations sampled in Bosnia and Herzegovina?

Isolation-by-distance (IBD) pattern among bilberry (Vaccinium myrtillus L.) populations has previously been reported for this species in northern Europe. However, the number of molecular studies conducted on bilberry, using everything from isoenzymes, RAPDs to microsatellite markers, are very few and far between. Considering that Bosnia and Herzegovina (B&H) is a country rich with diverse fruit genetic resources, conducting a genetic characterization of the naturally occurring V. myrtillus populations could yield valuable data for the conservation and utilization of this resource. This study entailed genotyping samples collected from three bilberry populations located in Fojnica, Kladanj, and Srebrenica municipalities using seven polymorphic microsatellite or SSR (simple sequence repeats) markers. The obtained molecular data was used to calculate the correlation between the physical distance of the individual B&H populations and a parameter of the genetic differentiation (pairwise Fst). The results of the correlation analyses revealed an absence of a significant isolation-by-distance pattern among the three examined B&H bilberry populations. In addition, the most pronounced genetic differentiation was detected between the Srebrenica and each of the two remaining B&H populations. At the same time, the values for pFst were significant, albeit much lower, between the Fojnica and Kladanj populations. Bilberries from the sampled Srebrenica population appear to be distinct from the other B&H populations, possibly due to the different genetic origin of this population.

Major Histocompatibility Complex Region and Diversity of the Local Chicken Populations In Niger

Introduction: The major histocompatibility complex (MHC) of chicken is highly polymorphic, and it is linked to several disease resistance or susceptibility traits. Therefore, the current study aimed to analyze the genetic diversity in the MHC region of Nigerien local chicken (Dourgou, Goggori, Kolonto, Tchagara, Gouzou-gouzou, and Popular) using a high polymorphic microsatellite marker named LEI0258 to determine the diversity of chickens kept at the four agroecological zones in Niger. Materials and methods: A total of 601 chickens from six local Nigerien chickens were sampled. By capillary electrophoresis using LEI0258 marker, 403 samples with different fragment sizes were randomly chosen and sequenced. Results: The findings indicated 80 different alleles ranging in size from 181 to 474 bp. A total of 22 new alleles and 39 private alleles (that existed in only one breed) were detected. The alleles 309, 295, and 193 were the most predominant in the Nigerien local chicken population. Nine polymorphisms were observed along the LEI0258 sequence, including three in the upstream (one indel and two Single Nucleotide Polymorphism [SNP]), one in the repeat region at the last R12 (SNP), and five in the downstream (two indels and three SNPs). Conclusion: The chickens are not clustering according to their agroecological zone of origin. They are randomly distributed across the four investigated agroecological zones. The information found in this study is invaluable in breeding and conservation programs associated with several disease resistance or susceptibility traits.

Genotyping blastoderms of avian eggs

AbstractUndeveloped eggs occur frequently in birds and are often considered infertile, and discarded. However, the majority of undeveloped eggs may in fact have been fertilised and embryos might have died at an early stage. Such eggs contain valuable information, for example about offspring sex and paternity, and level of inbreeding. Obtaining such information may also give insight into the patterns and causes of early embryo mortality. Here we describe a simple technique for removing embryo cells from the blastoderm to obtain DNA to genotype the offspring and unequivocally ascertain fertilisation status, while retaining the overlying perivitelline layer (PVL) for sperm counts over the entire membrane. We tested this method on freshly collected eggs (high‐quality material), as well as on eggs from abandoned clutches and unhatched eggs (potentially deteriorated material) of blue tits (Cyanistes caeruleus). We sampled a total of 707 eggs from a wild population of blue tits, extracted DNA from the eggs' blastoderm using a Qiagen kit, and genotyped the samples with 14 polymorphic microsatellite markers, plus one sexing marker. Overall, we successfully genotyped 97% of all eggs. Our study is the most extensive dataset of genotyped undeveloped eggs to date and demonstrates that one can reliably genotype undeveloped fertile eggs as well as retain the PVL for observations of sperm and embryo cells.

Population Genetic Structure of a Viviparous Sand Lizard, the Phrynocephalus forsythii in the Tarim Basin, Xinjiang of China

The desert ecosystem occupies an important position in the composition of global biodiversity. The Tarim Basin is located in south Xinjiang of China and has the world’s second largest mobile desert, the Taklamakan Desert. As an endemic species in this region, Phrynocephalus forsythii has been demonstrated to have a potentially high extinction risk due to climate change. In order to understand the overall genetic status and provide accordant conservation strategies for the species, we investigated the genetic diversity and population structure of P. forsythii from 15 sites in the Tarim Basin using 21 highly polymorphic microsatellite markers. We found significant genetic structure across the study region. We also revealed generally low levels of gene flow between the 25 sites, suggesting individual dispersal and migration may be restricted within populations. In addition, geographical distance and ambient temperature might be important factors in explaining the observed genetic structure. Our results will provide a scientific basis for the future protection of P. forsythii in this area, as well as an important reference for the conservation and management of biodiversity in desert ecosystems.

Evaluation for Parental Polymorphism and Identification of Microsatellites Linked to Drought Tolerance in Rice (Oryza sativa L.)

Aims: To identify the ready-to-use polymorphic microsatellite markers associated with drought tolerance for marker-assisted backcross breeding through a polymorphism survey between Jyothi and Chuvannamodan rice varieties.
 Place and Duration of Study: Centre for plant biotechnology and molecular biology, Thrissur, Kerala, India, during January to May 2023.
 Methodology: The genomic DNA of Jyothi and Chuvannamodan was isolated by following CTAB (Cetyltrimethyl Ammonium Bromide) method with modification. Isolated DNA from both varieties was subjected to PCR amplification using 208 Simple Sequence Repeats (SSRs) primer pairs distributed in 12 chromosomes. The amplified PCR products were electrophoresed in 3% agarose gel and separated fragments were visualized and documented in the Gel Documentation System. Different allele sizes produced by the same SSRs primers between two varieties are identified as polymorphic markers. Polymorphism per-cent was calculated, and frequency distribution and chromosome distribution of polymorphic markers were analyzed.
 Results: A total of 208 SSR primers were surveyed for the parental polymorphism. Out of which, 85 SSR primers exhibited clear polymorphism between Jyothi and Chuvannamodan and the remaining 123 were monomorphic primers. The amplicon size ranged from 83bp (RM430) to 495bp (RM18919) among the different primers. The survey revealed maximum parental polymorphism on chromosome 4 (69.23%), followed by chromosome 5 (64.28%), and minimum polymorphism on chromosome 8 (21.73%). The average per cent of polymorphism between the parents was 40.86%. Among the 85 polymorphic markers, 66 had dinucleotide repeats, 17 had trinucleotide repeats, and 1 had tetranucleotide repeats.
 Markers with dinucleotide repeats showed higher level of polymorphism. A group of 28 polymorphic markers were identified to be linked with traits including root-related traits, grain yield, leaf rolling and leaf drying under drought conditions.
 Conclusion: The polymorphic markers identified in the present study form the basis for tagging drought-tolerant QTLs/genes, fine mapping of those genes, and subsequently in marker-assisted breeding programs. The polymorphic markers linked with the QTLs for grain yield under drought can be pyramided in Jyothi through marker-assisted backcross breeding.