Genetic relationships among ten endod types as revealed by a combination of morphological,RAPD and AFLP markers

The genetic relationships among ten types of endod (Phytolacca dodecandra) cultivated by the Institute of Pathobiology of the Addis Ababa University to combat the disease bilharzia in Ethiopia were studied using morphology and molecular markers. A total of 18 morphological characters, 194 amplified fragment length polymorphism (AFLP) and 42 random amplified polymorphic DNA (RAPD) markers were used to determine genetic proximity between types. Genetic distance and cluster analysis of the AFLP data revealed the lack of genetic difference between E47 and E48 but relatively wider genetic difference among the other endod types. Cluster and principal component analyses performed on the AFLP and RAPD markers demonstrated the presence of distinct separation of E56 but not that of E44 from the others. The AFLP and RAPD data, thcrefore, did not support the hypothesis that the superiority of E44 in agronomic traits and molluscicidal potency is linked to its distinct genetic difference from the other endod types. Matrices correspondence tests demonstrated the presence of greater correspondence between AFLP and RAPD data (r = 0.842) but not between the morphology and that of AFLP and RAPD. This indicates the correspondence more between the two DNA markers systems than either of them with morphological traits. The cophenetic correlation coefficients also revealed poor fit for morphology (r = 0.716), good fit for RAPD (r = 0.872) and very good fit for AFLP (r = 0.975), reflecting the hyper-variability and higher resolving power of AFLP.     

Genetic diversity and genetic relationships in Hyacinthaceae in India using RAPD and SRAP markers

Genetic diversity and relationship among three genera namely Drimia, Dipcadi and Ledebouria of Hyacinthaceae in India was studied using RAPD and SRAP markers. Twenty one RAPD primers and nine SRAP were used for analyzing 41 accessions. RAPD gave an average 12.6 markers per primer, while SRAP generated 10.1 markers per primer pair. The family emerged very diverged with high polymorphism. The study resolved the three genera into monophyletic groups corresponding to three subfamilies; Urginoideae, Hyacinthoideae and Ornithogaloideae. Drimia wightii emerged a very distinct species and species specific markers were obtained with both marker systems. AMOVA analysis also revealed the genera to be quite well diverged. The two markers showed high correlation (r = 0.932) in Mantel matrix crresspondance test. The combined data also showed a very good correlation with the respective markers individually.     

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard “Coker” varieties and the diploid G. arboreum L. var. Ravi (old world cotton). Received: 12 July 1996 / Accepted: 26 July 1996     

Genetic fingerprinting of Australian cotton cultivars with RAPD markers.

RAPD (random amplified polymorphic DNA) markers generated by 30 random decamer primers were used to fingerprint 12 released cultivars and a breeding line of Gossypium hirsutum and 1 cultivar of G. barbadense presently under cultivation in Australia. Among a total of 453 developed markers, 69 (15.2%) were only present (unique) in the G. barbadense cultivar Pima S-7. Of the remaining markers, 128 (33.3%) were fixed in all 13 G. hirsutum cultivars. In pairwise comparisons of the degree of band sharing, nine closely-related cultivars showed 92.1-98.9% genetic similarity. Cluster analysis of genetic distance estimates between each of the cultivars revealed phylogenetic relationships in broad agreement with the known lineage of the cultivars. Ten of the G. hirsutum cultivars can be characterized individually based upon cultivar-specific RAPD markers, thus making it possible to differentiate closely related cultivars by molecular markers.     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

Evaluation of genetic relationships in Dalbergia species using RAPD markers

Conservation of identified germplasm is an important component forefficient and effective management of plant genetic resources. Traditionally,species identification has relied on morphological characters like growth habit,floral morphology like flower colour, and agronomic characteristics of the plant.Dalbergia species are important wind-dispersed tropicaltimber trees which exhibit high intrafruit seed abortion because of intensesibling competition for maternal resources. Studies were undertaken foridentification and genetic relationships in five species ofDalbergia and to evaluate genetic diversity withinpopulations of Dalbergia sisso, D.latifolia, D. paniculata, D.assamica and D. spinosa by using randomamplified polymorphic DNAs (RAPD) markers. Analysis was started by using 30decamer primers that allowed to distinguish five species and to select a reducedset of primers. The selected primers were used for identification and forestablishing a profiling system to estimate genetic relationships and toevaluate the genetic variability among the individuals in a population ofDalbergia species. A total of 120 distinct DNA fragments(bands), ranging from 0.3 to 4.0 kb, were amplified byusing nine selected random decamer primers. The genetic similarity was evaluated onthe basis of presence or absence of bands, which revealed a wide range ofvariability within the species. The cluster analysis indicated that five speciesof Dalbergia formed two major clusters. The first clusterconsisted of D. spinosa, D. latifolia and D.sisso. The second cluster was represented by two species, i.e.D. paniculata and D. assamica.A maximum similarity of 60% was observed in D. paniculata andD. assamica and they formed a minor cluster.Dalbergia latifolia and D. sissoformed another minor cluster with more than 50% similarity. Dalbergiaspinosa shared up to 40% similarity with D.latifolia and D. sisso. All the species sharemore than 20% similarity among themselves. The closest genetic distance existedwithin populations of different Dalbergia species. Thus,these RAPD markers have the potential for conservation of identified clones andcharacterization of genetic relatedness among the species. This is also helpful intree breeding programs and provides an important input into conservation biology.     

Genetic relationships and structure among open-pollinated maize varieties adapted to eastern and southern Africa using microsatellite markers

Molecular characterization of open-pollinated maize varieties (OPVs) is fundamentally important in maize germplasm improvement. We investigated the extent of genetic differences, patterns of relationships, and population structure among 218 diverse OPVs widely used in southern and eastern Africa using the model-based population structure, analysis of molecular variance, cluster analysis, principal component analysis, and discriminant analysis. The OPVs were genotyped with 51 microsatellite markers and the fluorescent detection system of the Applied Biosystems 3730 Capillary Sequencer. The number of alleles detected in each OPV varied from 72 to 155, with an overall mean of 127.6. Genetic distance among the OPVs varied from 0.051 to 0.434, with a mean of 0.227. The different multivariate methods suggest the presence of 2–4 possible groups, primarily by maturity groups but also with overlapping variation between breeding programs, mega-environments, and specific agronomic traits. Nearly all OPVs in group 1 and group 2 belong to the intermediate-late and early maturity groups, respectively. Group 3 consisted of mainly intermediate maturing OPVs, while group 4 contained OPVs of different maturity groups. The OPVs widely used in eastern Africa either originated from the southern African maize breeding programs, or the majority of inbred lines used as parents by the two breeding programs in developing the OPVs might be genetically related. Some of the OPVs are much older than others, but they still did not show a clear pattern of genetic differentiation as compared with the recently developed ones, which is most likely due to recycling of the best parental lines in forming new OPVs.     

Genetic diversity and relationships among Egyptian Galium (Rubiaceae) and related species using ISSR and RAPD markers

Genetic diversity and phylogenetic analyses of 24 species, representing nine sections of the genus Galium (Rubiaceae), have been made using the Inter Simple Sequence Repeats (ISSR), Randomly Amplified Polymorphic DNA (RAPD), and combined ISSR and RAPD markers. Four ISSR primers and three RAPD primers generated 250 polymorphic amplified fragments. The results of this study showed that the level of genetic variation in Galium is relatively high. RAPD markers revealed a higher level of polymorphism (158 bands) than ISSR (92 bands). Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrograms were compared. Six clades can be recognized within Galium, which mostly corroborate, but also partly contradict, traditional groupings. UPGMA-based dendrogram showed a close relationship between members of section Leiogalium with G. verum and G. humifusum (sect. Galium), and G. angustifolium (sect. Lophogalium). Principal coordinated analysis, however, showed some minor differences with UPGMA-based dendrograms. The more apomorphic groups of Galium form the section Leiogalium clade including the perennial sections Galium, Lophogalium, Jubogalium, Hylaea and Leptogalium as well as the annual section Kolgyda. The remaining taxa of Galium are monophyletic.     

Genetic relationships among melon breeding lines revealed by RAPD markers and agronomic traits

 RAPD markers and agronomic traits were used to determine the genetic relationships among 32 breeding lines of melon belonging to seven varietal types. Most of the breeding lines were Galia and Piel de Sapo genotypes, which are currently being used in breeding programmes to develop new hybrid combinations. A total of 115 polymorphic reliable bands from 43 primers and 24 agronomic traits were scored for genetic distance calculations and cluster analysis. A high concordance between RAPDs and agronomic traits was observed when genetic relationships among lines were assessed. In addition, RAPD data were highly correlated with the pedigree information already known for the lines and revealed the existence of two clusters for each varietal type that comprised the lines sharing similar agronomic features. These groupings were consistent with the development of breeding programmes trying to generate two separate sets of parental lines for hybrid production. Nevertheless, the performance of certain hybrids indicated that RAPDs were more suitable markers than agronomic traits in predicting genetic distance among the breeding lines analysed. The employment of RAPDs as molecular markers both in germplasm management and improvement, as well as in the selection of parental lines for the development of new hybrid combinations, is discussed. Received: 25 July 1997 / Accepted: 6 October 1997     

Varietal identification and assessment of genetic relationships in Hippeastrum using RAPD markers

The National Botanical Research Institute (NBRI) in Lucknow, India, maintains germplasm of Hippeastrum, a beautiful summer blooming ornamental. Germplasm collections comprise NBRI hybrids developed through selective breeding, hybrids with unknown parentage, local species, and Dutch hybrids for research purposes. Considering the importance of protecting plant breeders’ rights for commercial exploitation of hybrids, a PCR-based technique (random amplified polymorphic DNA—RAPD) was used to correctly identify known and unknown hybrids and to determine cultivar relatedness. RAPD profiles were used very successfully to trace and confirm the parentage of all the hybrids tested and to determine clear molecular relationships among varieties.     

Genetic relationships among Pistacia species using AFLP markers

This study addresses the phylogenetic relationship between Pistacia species by amplified fragment length polymorphism (AFLP). The plant materials of this study consisted of a total of 44 accessions belonging to P. vera, P. eurycarpa, P. khinjuk, all subspecies of P. atlantica (atlantica, mutica, kurdica and cabulica), three unknown genotypes and three accessions, proposed to be hybrid from P. eurycarpa × P. atlantica. The accessions were from Iran, Turkey, USA and Syria. Six AFLP primer combinations produced a total of 475 fragments, with average of 79.16 fragments per primer pair, of which, 336 bands were polymorphic. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on jaccard’s similarity coefficient matrix and also average similarity of each species. According to the results, two main clusters were developed and P. vera, P. eurycarpa, P. atlantica (subsp. atlantica, kurdica, mutica, cabulica) and the hybrid genotypes located in the first main cluster. P. khinjuk accessions from Iran and USA localized in second main cluster. The hybrid accessions located between eurycarpa and atlantica species and their hybrid nature between these two species were confirmed. One of the unknown accessions clustered with the hybrid ones and the two other were grouped closely with P. Khinjuk. According to this study, the closest species to P. vera was Eurycarpa group, followed by P. atlantica. UPGMA analysis separated P. atlantica subsp. mutica and cabulica from P. atlantica and P. eurycarpa. Subspecies mutica and cabulica were two closest genotypes; hence, P. atlantica subsp. mutica could be classified as a distinct species as P. mutica and the cabulica as a subspecies of P. mutica. This study revealed that P. eurycarpa is synonym for P. atlantica subsp. kurdica and should be considered distinct from P. atlantica; however, P. atlantica showed a closer genetic similarity to P. eurycarpa than the other species.     

Genetic mapping of mutations using phenotypic pools and mapped RAPD markers.

Genetic markers facilitate the study of inheritance and the cloning of genes by genetic approaches. Molecular markers detect differences in DNA sequence, and are thus less ambiguous than phenotypic markers, which require gene expression. We have demonstrated a molecular approach to the mapping of mutant genes using RAPD markers and pooling of individuals based on phenotype. To map genes by phenotypic pooling a strain carrying a mutation is crossed to a strain that is homozygous for the wild-type allele of the corresponding gene. A set of primers corresponding to mapped RAPDs distributed throughout the genome and in coupling phase with respect to the wild type parent is then used to amplify DNA from wild type and mutant pools of F2 individuals. Linkage between the mutant gene and the RAPD markers is visualized by the absence of the corresponding RAPD DNA bands in the mutant pool. We developed a mathematical model for calculating the probability of linkage between RAPDs and target genes and we successfully tested this approach with the model plant Arabidopsis thaliana.     

Genetic analysis of populations of threatened snake species using RAPD markers

Snakes are a particularly threatened vertebrate taxon, with distributions of many species and populations becoming increasingly fragmented. At present, little is known about the degree of genetic differentiation that exists between isolated populations even though such information may be critical to their survival and conservation. As an example of how recently developed RAPD genetic markers can be used in conservation genetics, we present preliminary results from a study which used these DNA-based markers to assess population divergence in two threatened Canadian snakes, the black rat snake ( Elaphe o. obsoleta ) and the eastern massasauga rattlesnake ( Sistrurus c. catenatus ). We present information on the levels of variation and reliability of amplification for fragments generated from five primers. We then use a recently developed analytical technique to estimate levels of nucleotide diversity within populations and sequence divergence between populations. Our results show that intrapopulation levels of divergence as estimated by the methods of Clark & Lanigan ( Molecular Biology and Evolution 1993, 10 , 1096–1111) approximate those found for mtDNA in vertebrates and that diversity between snake populations is small and non-significant when tested using randomization procedures. Thus, our study provides an example of how RAPDs can be applied to conservation genetic studies of vertebrates and suggest that the snake populations we examined have only recently become isolated and maybe considered genetically equivalent from a conservation perspective, although this conclusion needs to be confirmed with other DNA-based markers.     

Genetic diversity of ten Egyptian chicken strains using 29 microsatellite markers

In this study, we assessed the genetic diversity of three Egyptian local chicken strains (Fayoumi, Dandarawi and Sinai) and six synthetic breeds derived from Fayoumi and Sinai by intercrossing with Barren Plymouth Rock, Rhode Island Red or White Cornish. Diversity measures were based on interrogation of 29 microsatellites. We identified three main clusters of chicken populations encompassing selected Fayoumi lines and Doki-4 (cluster-1), native Dandarawi (cluster-2) and Sinai, and all six synthetic breeds (cluster-3). Dandarawi and Fayoumi lines exhibited lower intra-population genetic diversity and allelic privacy than Sinai and synthetic breeds. The global inbreeding (F(IT) ) was 0.11, among-population differentiation (F(ST) ) was 0.07, and within-population differentiation (F(IS) ) was 0.04. The between-population marker-estimated kinship was lower than within-population estimates. The cluster analysis classified the Fayoumi lines, Dandarawi and Gimmizah as clearly separated populations. The other strains were configured in mosaic admixed groups.     

Genetic variability and relationships among thirty genotypes of finger millet (Eleusine coracana L. Gaertn.) using RAPD markers

Ragi or finger millet (Eleusine coracana L.) is an important crop used for food, forage, and industrial products. It is distributed in tropical and temperate regions of the world. The germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Traditionally, species or varieties identification has relied on morphological characters like growth habit, leaf architecture or floral morphology. Investigation through RAPD (random amplified polymorphic DNA) markers was undertaken for identification and determination of the genetic variation among thirty genotypes of ragi of the family Poaceae. Thirteen selected decamer primers were used for genetic analysis. A total of 124 distinct DNA fragments ranging from 300-3000 bp was amplified by using selected random RAPD marker. The genetic similarity was evaluated on the basis of the presence or absence of bands. Cluster analysis was made by the similarity coefficient. It indicated that the 30 genotypes of ragi form two major clusters, first, a major cluster having only one genotype, i. e. Dibyasinha and a second major cluster having twenty-nine genotypes. The second major cluster again subdivides into two minor clusters. A first minor cluster has only three varieties, i. e. Neelachal, OEB-56 and Chilika. The genotypes Neelachal and OEB-56 exhibit a 86% similarity with each other and 80% similarity with Chilika. A second minor cluster has 26 genotypes and is divided into two sub-minor clusters. The first sub-minor cluster has only one genotype (VL-322). The second sub-minor cluster again subdivides into two groups. One group has one genotype and the second group again is divided into two sub-groups, one with 13 genotypes and the other with 11 genotypes. The highest similarity coefficient was detected in a genotype collected from southern India and the least from northern India. The genotypes of finger millet collected from diverse agroclimatic regions of India constitute a wide genetic base. This is helpful in breeding programs and a major input into conservation biology of cereal crop.     

Genetic mapping in Eucalyptus urophylla and Eucalyptus grandis using RAPD markers.

Two single-tree linkage maps were constructed for Eucalyptus urophylla and Eucalyptus grandis, based on the segregation of 480 random amplified polymorphic DNA (RAPD) markers in a F1 interspecific progeny. A mixture of three types of single-locus segregations were observed: 244 1:1 female, 211 1:1 male, and 25 markers common to both, segregating 3:1. Markers segregating in the 1:1 ratio (testcross loci) were used to establish separate maternal and paternal maps, while markers segregating in the 3:1 ratio were used to identify homology between linkage groups of the two species maps. An average of 2.8 polymorphic loci were mapped for each arbitrary decamer primer used in the polymerase chain reaction. The mean interval size beween framework markers on the maps was 14 cM. The maps comprised 269 markers covering 1331 cM and 236 markers covering 1415 cM, in 11 linkage groups, for E. urophylla (2n = 2x = 22) and E. grandis (2n = 2x = 22), respectively. A comparative mapping analysis with two other E. urophylla and E. grandis linkage maps showed that RAPDs could be reliable markers for establishing a consensus species map. RAPD markers were automatically and quantitatively scored with an imaging analyzer. They were classified into four categories based on their optical density. A fragment intensity threshold is proposed to optimize the selection of reliable RAPD markers for future mapping experiments. Key words : genetic linkage map, Eucalyptus urophylla, Eucalyptus grandis, random amplified polymorphic DNA, RAPD, automated data collection.     

Genetic linkage mapping in peach using morphological,RFLP and RAPD markers

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F₂ individuals derived from the self-fertilization of four F₁ individuals of a cross between New Jersey Pillar and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color () loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F₂ trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 121 or 31) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.     

应用RAFD标记研究不同生态区谷子品种的遗传差异

应用RAPD标记对19份国内不同生态区的谷子品种的遗传变异进行了研究。结果表明：分子水平上,不同生态区的谷子品种间存在一定的遗传差异,但遗传差异程度并不高。11个随机引物共扩增出54条多态性带,不同引物扩增的带数差异较大,每个引物可扩增2—8条多态性带,平均每个引物扩增出4．91条多态性带。引物1050扩增的多态性带最多(8条)。聚类结果表明,基于RAPD标记分析的遗传聚类群与生态类型有很大的一致性。     

Genetic relationships of five Indian horse breeds using microsatellite markers

The genetic relationships of five Indian horse breeds, namely Marwari, Spiti, Bhutia, Manipuri and Zanskari were studied using microsatellite markers. The DNA samples of 189 horses of these breeds were amplified by polymerase chain reaction using 25 microsatellite loci. The total number of alleles varied from five to 10 with a mean heterozygosity of 0.58 ± 0.05. Spiti and Zansakari were the most closely related breeds, whereas, Marwari and Manipuri were most distant apart with Nei's D_A genetic distance of 0.071 and 0.186, respectively. In a Nei's D_A genetic distances based neighbour joining dendrogram of these breeds and a Thoroughbred horse outgroup, the four pony breeds of Spiti, Bhutia, Manipuri and Zanskari clustered together and then with the Marwari breed. All the Indian breeds clustered independently from Thoroughbreds. The genetic relationships of Indian horse breeds to each other correspond to their geographical/environmental distribution.