Microsatellite markers help to assess genetic diversity among Opuntia ficus indica cultivated genotypes and their relation with related species

Opuntia spp. belong to the Cactaceae family and are native to Central America. The most economically important species is O. ficus indica, cultivated both for fruits and cladodes. The genus includes other important edible species (from diploid to octoploid) that occur worldwide as either wild or cultivated species in many arid or semiarid areas (e.g., the Mediterranean region). Several accessions are cultivated in different growing regions, but little is known about their ancestries and levels of genetic diversity. The aim of this study was to investigate the level of intraspecific genetic diversity among O. ficus indica cultivated varieties and some related species. Specifically, six highly polymorphic simple sequence repeats (SSR) and two expressed sequence tag (EST)-SSR loci were investigated in 62 wild and cultivated genotypes belonging to 16 Opuntia species. The clusters identified by the distance and model-based analyses clearly separated the wild opuntias from the cultivated ones. However, the O. ficus indica accessions did not cluster separately from other arborescent cactus pear species, such as O. amyclaea, O. megacantha, O. streptacantha, O. fusicaulis, and O. albicarpa, indicating that their current taxonomical classifications do not fit with their genetic variability. In general, the genotypes cultivated in Mexico showed high levels of diversity, whereas most of the spineless accessions collected in other countries had a very narrow genetic base. This study increases our knowledge of the variability among some of the most diffused Opuntia cultivated accessions. This study also points to the inconsistencies of previous taxonomical genotype assignments that were based solely on morphological characteristics.     

Utility of a multidisciplinary approach for genome diagnostics of cultivated and wild germplasm resources of medicinal Withania somnifera, and the status of new species, W. ashwagandha, in the cultivated taxon

Realizing the inconsistencies that exist in the extent and nature of differentiation in the Withania somnifera genetic resources in India, the 21 cultivated and wild accessions, and the two hybrids (cultivated?×?wild accessions and vice versa) were investigated for morphological, cytogenetical, chemical profiling, and crossability features. Their nuclear and chloroplast genomes were also assayed at the nucleotide sequence level, and by use of DNA markers. Chloroplast DNA diversity and somatic chromosome number (2n?=?48) were not helpful in identifying the differences. Other approaches, on the other hand, especially restriction endonuclease digests, types and sequence length composition of ITS 1 and ITS 2 of nuclear ribosomal DNA, AFLP fingerprinting, and crossability barriers unambiguously provided startling discrete differences between the cultivated and wild accessions, indicating a clear division of W. somnifera into two distinct lineages. These data, therefore, are indicative of the fact that because of the unique characteristics of its nuclear genome, and strong crossability barriers vis-à-vis wild accessions of W. somnifera, the cultivated accessions should be relegated to the rank of the separate species, W. ashwagandha.     

Population Structure and Diversity in Finger Millet (Eleusine coracana) Germplasm

A genotypic analysis of 79 finger millet accessions (E. coracana subsp. coracana) from 11 African and five Asian countries, plus 14 wild E. coracana subsp. africana lines collected in Uganda and Kenya was conducted with 45 SSR markers distributed across the finger millet genome. Phylogenetic and population structure analyses showed that the E. coracana germplasm formed three largely distinct subpopulations, representing subsp. africana, subsp. coracana originating from Africa and subsp. coracana originating from Asia. A few lines showed admixture between the African and Asian cultivated germplasm pools and were the result of either targeted or accidental intercrossing. Evidence of gene flow was also seen between the African wild and cultivated subpopulations, indicating that hybridizations among subspecies occur naturally where both species are sympatric. The genotyping, combined with phylogenetic and population structure analyses proved to be very powerful in predicting the origin of breeding materials. The genotypic study was complemented by a phenotypic evaluation. The wild and cultivated accessions differed by a range of domestication-related characters, such as tiller number, plant height, peduncle length, seed color and grain yield. Significant differences in plant architecture and yield were also identified between the Asian and African subpopulations. The observed population structure within cultivated finger millet is consistent with the theory that, after the introduction of finger millet from Africa into India via the trade routes some 3000 years ago, the two germplasm pools remained largely isolated until recent times. The significantly lower diversity present within the Asian subpopulation also suggests that it arose from a relatively small number of founder plants.     

Nucleotide Polymorphism in the Adh1 Locus of Pearl Millet (Pennisetum Glaucum) (Poaceae)

We investigated nucleotide polymorphism in the Adh1 locus of pearl millet (Pennisetum glaucum) (Poaceae) by determining the DNA sequence of 20 alleles from 10 individuals. The individuals were sampled from throughout pearl millet's indigenous range and represent both wild and cultivated accessions. Our results indicated that there is little nucleotide polymorphism in the Adh1 locus. Estimates of per site nucleotide polymorphism did not differ significantly between cultivated and wild millet accessions. We compared nucleotide polymorphism in pearl millet Adh1 with nucleotide polymorphism in maize (Zea mays) Adh1 and conclude that the maize Adh1 sample is more polymorphic. Increased polymorphism in maize Adh1 may be attributable, in part, to faster substitution rates in the maize lineage. Analysis suggests that substitution rates in the maize Adh1 lineage are ~1.7 times faster than substitution rates in the millet Adh1 lineage.     

Origins and Domestication of Cultivated Banana Inferred from Chloroplast and Nuclear Genes

Background

Cultivated bananas are large, vegetatively-propagated members of the genus Musa. More than 1,000 cultivars are grown worldwide and they are major economic and food resources in numerous developing countries. It has been suggested that cultivated bananas originated from the islands of Southeast Asia (ISEA) and have been developed through complex geodomestication pathways. However, the maternal and parental donors of most cultivars are unknown, and the pattern of nucleotide diversity in domesticated banana has not been fully resolved.

Methodology/Principal Findings

We studied the genetics of 16 cultivated and 18 wild Musa accessions using two single-copy nuclear (granule-bound starch synthase I, GBSS I, also known as Waxy, and alcohol dehydrogenase 1, Adh1) and two chloroplast (maturase K, matK, and the trnL-F gene cluster) genes. The results of phylogenetic analyses showed that all A-genome haplotypes of cultivated bananas were grouped together with those of ISEA subspecies of M. acuminata (A-genome). Similarly, the B- and S-genome haplotypes of cultivated bananas clustered with the wild species M. balbisiana (B-genome) and M. schizocarpa (S-genome), respectively. Notably, it has been shown that distinct haplotypes of each cultivar (A-genome group) were nested together to different ISEA subspecies M. acuminata. Analyses of nucleotide polymorphism in the Waxy and Adh1 genes revealed that, in comparison to the wild relatives, cultivated banana exhibited slightly lower nucleotide diversity both across all sites and specifically at silent sites. However, dramatically reduced nucleotide diversity was found at nonsynonymous sites for cultivated bananas.

Conclusions/Significance

Our study not only confirmed the origin of cultivated banana as arising from multiple intra- and inter-specific hybridization events, but also showed that cultivated banana may have not suffered a severe genetic bottleneck during the domestication process. Importantly, our findings suggested that multiple maternal origins and a reduction in nucleotide diversity at nonsynonymous sites are general attributes of cultivated bananas.     

Genetic Relationships Among Wild and Cultivated Accessions of Curry Leaf Plant (Murraya koenigii (L.) Spreng.), as Revealed by DNA Fingerprinting Methods

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard’s similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.     

Diversity and Species Relationship in Pearl Millet (Pennisetum typhoides) and Related Species

Thirteen accessions of pearl millet (Pennisetum typhoides (L) Leeke) collected from different states of India and eight wild species of the genus Pennisetum across the world were analyzed for genetic diversity using AFLP markers. A combined analysis of eight primer combinations showed 35% polymorphism among P. typhoides accessions while analysis with five primer combinations showed 99% polymorphism among the wild species. The dendrogram constructed for the P. typhoides accessions based on the UPGMA method revealed two major clusters with samples from Gujarat forming a separate cluster from the rest of the samples. Principal component analysis of the same data set revealed similar results with the first principal component accounting for 65% of the total variation. The percentage of rare and common alleles contributing to the diversity in the sample was analyzed using the Shannon Weiner diversity index. The SW index revealed that the samples from Gujarat contributed significantly to the overall diversity among the accessions. Among accessions of each geographical region, considerable variation was revealed by SW index with samples from Tamil Nadu being most polymorphic. The genetic diversity in the accessions could be utilized for future breeding work. The dendrogram constructed for the wild species revealed the extent of genetic diversity among them. Analysis with one primer combination showed P. typhoides being closer to P. mollissimum than to the other analyzed species.     

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

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

Phylogenetic diversity of Mesorhizobium in chickpea

Crop domestication, in general, has reduced genetic diversity in cultivated gene pool of chickpea (Cicer arietinum) as compared with wild species (C. reticulatum, C. bijugum). To explore impact of domestication on symbiosis, 10 accessions of chickpeas, including 4 accessions of C. arietinum, and 3 accessions of each of C. reticulatum and C. bijugum species, were selected and DNAs were extracted from their nodules. To distinguish chickpea symbiont, preliminary sequences analysis was attempted with 9 genes (16S rRNA, atpD, dnaJ, glnA, gyrB, nifH, nifK, nodD and recA) of which 3 genes (gyrB, nifK and nodD) were selected based on sufficient sequence diversity for further phylogenetic analysis. Phylogenetic analysis and sequence diversity for 3 genes demonstrated that sequences from C. reticulatum were more diverse. Nodule occupancy by dominant symbiont also indicated that C. reticulatum (60%) could have more various symbionts than cultivated chickpea (80%). The study demonstrated that wild chickpeas (C. reticulatum) could be used for selecting more diverse symbionts in the field conditions and it implies that chickpea domestication affected symbiosis negatively in addition to reducing genetic diversity.     

Uncovering the genetic diversity of yams (Dioscorea spp.) in China by combining phenotypic trait and molecular marker analyses

Yam is an important edible tuber and root plant worldwide; China as one of the native places of yams has many diverse local resources. The goal of this study was to clarify the genetic diversity of the commonly cultivated yam landraces and the genetic relationship between the main yam species in China. In this study, 26 phenotypic traits of 112 yam accessions from 21 provinces in China were evaluated, and 24 simple sequence repeat (SSR) and 29 sequence‐related amplified polymorphism (SRAP) markers were used for the genetic diversity analysis. Phenotypic traits revealed that Dioscorea opposita had the highest genetic diversity, followed by D. alata, D. persimilis, D. fordii, and D. esculenta. Among the 26 phenotypic traits, the Shannon diversity indexes of leaf shape, petiole color, and stem color were high, and the range in the variation of tuber‐related traits in the underground part was higher than that in the aboveground part. All accessions were divided into six groups by phenotypic trait clustering, which was also supported by principal component analysis (PCA). Molecular marker analysis showed that SSR and SRAP markers had good amplification effects and could effectively and accurately evaluate the genetic variation of yam. The unweighted pair‐group method with arithmetic means analysis based on SSR‐SRAP marker data showed that the 112 accessions were also divided into six groups, similar to the phenotypic trait results. The results of PCA and population structure analysis based on SSR‐SRAP data also produced similar results. In addition, the analysis of the origin and genetic relationship of yam indicated that the species D. opposita may have originated from China. These results demonstrate the genetic diversity and distinctness among the widely cultivated species of Chinese yam and provide a theoretical reference for the classification, breeding, germplasm innovation, utilization, and variety protection of Chinese yam resources.     

Neutral and functional marker based genetic diversity in kodo millet (<Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L.)

Kodo millet (Paspalum scrobiculatum L.) is known for its high nutritive value, dietary fiber, antioxidant activity, as well as for drought tolerance. It is primarily grown as a grain in India and in Africa it is either cultivated or harvested in wild. Neutral—ISSR (inter simple sequence repeat) as well as functional—SCoT (start codon targeted) and SRAP (sequence-related amplified polymorphism) markers were employed for genetic diversity studies in 96 accessions of kodo millet collected from diverse regions of India. The genetic diversity parameters like average bands per primer, Polymorphic information content, Nei’s gene diversity and Shannon’s information index of 11.22, 9.69; 0.12, 0.11; 0.15 ± 0.14, 0.13 ± 0.13 and 0.26 ± 0.21, 0.22 ± 0.19 was observed with neutral and functional markers respectively. Neutral markers were showing higher values as compared to functional markers for the genetic diversity parameters as discussed. Structure based analysis placed all the accessions into four sub-groups not strictly according to their geographical locations. The accessions from Bihar followed by Karnataka were showing high diversity based on both the marker systems useful for designing exploration, conservation and germplasm enrichment strategies. Further, the set of diverse accessions selected based on these markers would serve as potential sources of unique alleles and may be exploited in future for enhancement and utilization of kodo millet germplasm. Usage of African gene pool and wild species for broadening the genetic base of Indian kodo millet was also suggested based on the present studies.     

Single Nucleotide Polymorphisms in HSP17.8 and Their Association with Agronomic Traits in Barley

Small heat shock protein 17.8 (HSP17.8) is produced abundantly in plant cells under heat and other stress conditions and may play an important role in plant tolerance to stress environments. However, HSP17.8 may be differentially expressed in different accessions of a crop species exposed to identical stress conditions. The ability of different genotypes to adapt to various stress conditions resides in their genetic diversity. Allelic variations are the most common forms of genetic variation in natural populations. In this study, single nucleotide polymorphisms (SNPs) of the HSP17.8 gene were investigated across 210 barley accessions collected from 30 countries using EcoTILLING technology. Eleven SNPs including 10 from the coding region of HSP17.8 were detected, which form nine distinguishable haplotypes in the barley collection. Among the 10 SNPs in the coding region, six are missense mutations and four are synonymous nucleotide changes. Five of the six missense changes are predicted to be deleterious to HSP17.8 function. The accessions from Middle East Asia showed the higher nucleotide diversity of HSP17.8 than those from other regions and wild barley (H. spontaneum) accessions exhibited greater diversity than the cultivated barley (H. vulgare) accessions. Four SNPs in HSP17.8 were found associated with at least one of the agronomic traits evaluated except for spike length, namely number of grains per spike, thousand kernel weight, plant height, flag leaf area and leaf color. The association between SNP and these agronomic traits may provide new insight for study of the gene''s potential contribution to drought tolerance of barley.     

High-Density SNP Genotyping of Tomato (Solanum lycopersicum L.) Reveals Patterns of Genetic Variation Due to Breeding

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F _st supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F _st outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization.     

Diversity of wild and cultivated pearl millet accessions (Pennisetum glaucum [L.] R. Br.) in Niger assessed by microsatellite markers

Genetic diversity of crop species in sub-Sahelian Africa is still poorly documented. Among such crops, pearl millet is one of the most important staple species. In Niger, pearl millet covers more than 65% of the total cultivated area. Analyzing pearl millet genetic diversity, its origin and its dynamics is important for in situ and ex situ germplasm conservation and to increase knowledge useful for breeding programs. We developed new genetic markers and a high-throughput technique for the genetic analysis of pearl millet. Using 25 microsatellite markers, we analyzed genetic diversity in 46 wild and 421 cultivated accessions of pearl millet in Niger. We showed a significantly lower number of alleles and lower gene diversity in cultivated pearl millet accessions than in wild accessions. This result contrasts with a previous study using iso-enzyme markers showing similar genetic diversity between cultivated and wild pearl millet populations. We found a strong differentiation between the cultivated and wild groups in Niger. Analyses of introgressions between cultivated and wild accessions showed modest but statistically supported evidence of introgressions. Wild accessions in the central region of Niger showed introgressions of cultivated alleles. Accessions of cultivated pearl millet showed introgressions of wild alleles in the western, central, and eastern parts of Niger.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Cedric Mariac and Viviane Luong have contributed equally to this work.     

Study of genetic diversity and relationships among accessions of foxtail millet [Setaria italica (L.) P. Beauv.] in Korea, China, and Pakistan using SSR markers

In this study, 28 simple sequence repeat (SSR) primer sets were used to analyze the genetic diversity, population structure, and genetic relationships among 37 accessions of foxtail millet from Korea, China and Pakistan. A total of 298 alleles were detected with an average allele number of 10.6 per locus among 37 foxtail millet accessions. The number of alleles per locus ranged from 2 (b226) to 20 (b236). Of the 298 alleles, 138 alleles (46.3%) were rare (frequency < 0.05), 152 alleles (51.0%) were detected at an intermediate frequency (range, 0.05?C0.50), and eight alleles (2.7%) were abundant (frequency > 0.50), respectively. The average gene diversity values were 0.652, 0.692, and 0.491 and polymorphic information content values were 0.621, 0.653, and 0.438, for accessions from Korea, China, and Pakistan, respectively. The accessions from China showed higher SSR diversity than those from Korea and Pakistan. A phylogenetic tree constructed using the un-weighted pair group methods with arithmetic mean algorithm revealed three major groups of accessions that were not congruent with geographical distribution patterns with a few exceptions. The lack of correlation between the accession clusters and their geographic location indicates that the diffusion of foxtail millet from China to Korea might have occurred through multiple routes. Our results provide support for the origin and diffusion route of foxtail millet in East Asia. This SSR-based assessment of genetic diversity, genetic relationships, and population structure among genetic resources of foxtail millet landraces will be valuable to foxtail millet breeding and genetic conservation programs in Korea.     

On the origin of six-rowed barley with brittle rachis, agriocrithon [Hordeum vulgare ssp. vulgare f. agriocrithon (Aberg) Bowd.], based on a DNA marker closely linked to the vrs1 (six-row gene) locus

The origin of six-rowed cultivated barley has been revealed to be more complex since the discovery of agriocrithon, a six-rowed barley with brittle rachis. The present study investigates whether such six-rowed brittle barley is wild or hybrid in nature, by analyzing genetic diversity at the cMWG699 marker locus, which is closely linked to the vrs1 (six-row gene) locus. DNA sequence analysis for 42 accessions showed only three types in six-rowed brittle barleys; in contrast, nine sequence types were found in ten wild barleys, ssp. spontaneum, in our previous study. Nucleotide diversities for the six-rowed brittle barley were 2.8–4.5 times lower than that for the ssp. spontaneum at this marker locus. The three sequence types found in the six-rowed brittle barley also appeared in the six-rowed cultivated barley. A cross-allelism test confirmed that the six-rowed character of the six-rowed brittle barley was controlled by the vrs1 locus. The nucleotide diversity and genealogy demonstrated that f. agriocrithon does not have the same level of diversity as found in wild barley, ssp. spontaneum. Consequently, f. agriocrithon does not appear to represent genuinely wild populations, but more probably originated from hybridization between ssp. spontaneum and six-rowed cultivated barley.     

Nucleotide Diversity and Molecular Evolution of the <Emphasis Type="Italic">ALK</Emphasis> Gene in Cultivated Rice and its Wild Relatives

Gelatinization temperature (GT), an important parameter for rice cooking quality, is mainly regulated by the ALK gene encoding starch synthase IIa. Here, we reported the nucleotide diversity of the ALK gene in 122 cultivated accessions and 199 wild rice accessions that were collected around the Pearl River Basin in China. A total of 93 single nucleotide ploymorphisms (SNPs) were identified, with an average of one SNP per 40 bp. Tajima D statistics revealed that the DNA sequences covering the last exon have probably evolved under balancing selection. Based on two functional SNPs (an A to G substitution at 4198 bp and a GC to TT dinucleotide substitution at 4330/4331 bp), three haplotypes, G/GC, G/TT, and A/GC, were identified in both wild and cultivated accessions, with the G/GC haplotype being predominant. Interestingly, the A/GC haplotype was exclusively found in the wild accessions from Guangdong province, while the G/TT haplotype was only present in the wild accessions from Jiangxi province and Hainan Island. This suggests that the G/TT and A/GC variants may have arisen independently and undergone balancing selection on separate haplotypes in multiple populations. Our result supports earlier hypothesis that cultivated rice was independently domesticated from multiple domestication events in China. Our study aids in the understanding of the domestication process that led to the improvement of rice grain quality.     

Evaluation of Genetic Diversity in Chinese Wild Apple Species Along with Apple Cultivars Using SSR Markers

China, one of the primary centers of genetic diversity for the genus Malus, is very rich in wild apple germplasm. In this study, genetic diversity in 29 Malus accessions, including 12 accessions from 7 Chinese Malus species, 4 Chinese landraces, and 13 introduced apple cultivars, was assessed using a set of 19 single-locus simple sequence repeat (SSR) markers distributed across all 17 linkage groups of the apple genome. The number of alleles detected at each locus ranged from 2 to 11, with an average of 5.3 per SSR marker. In some accessions, 16 unique alleles were identified. Ten out of these 16 unique alleles (62.5%) were detected exclusively in wild species, indicating that these Chinese wild apple species have considerable genetic diversity and can be used in breeding programs to increase the genetic diversity of apple cultivars. Using 19 SSRs, an unweighted pair-group method with arithmetic average cluster analysis was conducted, and the resulting dendrogram revealed that all cultivars, except for E??peMeBckoe, were clustered together in the same group. The Russian cultivar E??peMeBckoe was closely related to the Chinese crabapple Baihaitang (M. prunifolia), with a high similarity coefficient value of 0.94. Of the two M. sieversii accessions used, one accession showed a close relationship to apple cultivars, while the other accession was closely related to wild apple species, suggesting the presence of a wider genetic diversity in Chinese M. sieversii species. The influence of SSR marker selection on genetic diversity analysis in this Malus collection was also discussed.