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.     

Polymorphism of SSR alleles in pear cultivars grown in Belarus

Using SSR markers designed for Malus × domestica Borkh. genetic polymorphism of 43 pear accessions cultivated in Belarus was examined. A total of 217 alleles were identified with the mean number of 12.8 alleles per marker. The mean PIC value was 0.81; the mean number of informative alleles, 6.49. The heterozygosity level ranged from 0.30 to 0.84. Genetic diversity of SSR alleles in pear and apple genomes was compared. A method of identification of commercial pear cultivars using a set of six SSR markers was suggested.     

Genetic diversity,population structure,and linkage disequilibrium of elite and local apple accessions from Belgium using the IRSC array

The identification of molecular markers associated with economic and quality traits will help improve breeding for new apple (Malus × domestica Borkh.) cultivars. Tools such as the 8K apple SNP array developed by the RosBREED consortium allow for high-throughput genotyping of SNP polymorphisms within collections. However, genetic characterization and the identification of population stratification and kinship within germplasm collections is a fundamental prerequisite for identifying robust marker–trait associations. In this study, a collection of apple germplasm originally developed for plant architectural studies and consisting of both non-commercial/local and elite accessions was genotyped using the 8K apple SNP array to identify cryptic relationships between accessions, to analyze population structure and to calculate the linkage disequilibrium (LD). A total of nine pairs of synonyms and several triploids accessions were identified within the 130 accessions genotyped. In addition, most of the known parent-child relations were confirmed, and several putative, previously unknown parent-child relations were identified among the local accessions. No clear subgroups could be identified although some separation between local and elite accessions was evident. The study of LD showed a rapid decay in our collection, indicating that a larger number of SNPs is necessary to perform whole genome association mapping. Finally, an association mapping effort for architectural traits was carried out on a small number of accessions to estimate the feasibility of this approach.     

Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

Genetic Diversity and Identity of Chinese Loquat Cultivars/Accessions (<Emphasis Type="Italic">Eriobotrya japonica</Emphasis>) Using Apple SSR Markers

Loquat (Eriobotrya japonica) is an underutilized fruit crop that originated in China and for which only a small number of molecular markers are available. This number can be increased by identifying apple SSRs that are transferable to loquat cultivars/accessions to provide new insight into the level of genetic diversity within loquat and synteny with apple. We evaluated 71 apple SSR markers distributed across 17 linkage groups, and identified 39 SSRs transferable to loquat. Testing 54 loquat accessions, from Japan, Spain, four provinces in China, and two wild species gave a total of 155 different alleles with a mean value of 3.38 per locus. The mean effective number of alleles was 2.21, and the mean observed heterozygosity was 0.47. These values indicate a high degree of genetic diversity in the set of Chinese loquat accessions analyzed. Unweighted pair-group method analysis based on simple matching coefficent clustered the accessions into two groups, cultivated and wild loquat. The cultivated loquat can be subdivided into three subgroups which generally reflect their geographic origin in China. The Spanish cultivars clustered with those of the Jiangsu and Zhejiang provinces. A core set of five SSR markers could distinguish most accessions.     

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.     

Vf scab resistance of Malus

The apple production in temperate regions with spring rains, the Scab caused by the fungus Venturia inaequalis is the most important constraint. To produce spotless apples and avoid damage that develops during storage, growers apply fungicide on a regular or weather-determined basis. All major apple cultivars are highly susceptible to this disease. To limit the need for fungicide applications, apple breeders are currently introgressing disease resistance from wild Malus accessions into commercial lines. The first attempts to do this were made 100 years ago. As apples are self-incompatible, pseudo-backcrossing is used to eliminate unwanted traits from wild Malus and select new cultivars that are attractive to both producers and consumers. This process, from the first cross of a commercial cultivar with a wild, disease-resistant Malus, is extremely long due to apple’s long juvenile phase, the need for more than seven backcross steps and the high heterozygosity of this genus. Therefore, most of today’s scab-resistant cultivars rely on a single introduction of scab resistance from Malus floribunda 821, referred to as Vf. In this paper, we trace the history of Vf from its initial identification through its use in breeding and commercial production. We sum up the literature describing how and where Vf resistance has been overcome by new pathotypes of V. inaequalis. Finally, we describe the current knowledge of the genes behind Vf resistance, its mode of action and the use of Vf genes in gene technology.     

Rosaceae conserved orthologous sequences marker polymorphism in sweet cherry germplasm and construction of a SNP-based map

The Rosaceae Conserved Orthologous Set (RosCOS) provides a gene-based genome-wide set of markers that have been used in comparative analyses of peach (Prunus persica), apple (Malus × domestica), and strawberry (Fragaria spp.). In order to extend the use of these RosCOS to sweet cherry (Prunus avium L.), we identified markers that are polymorphic in breeding germplasm. Ninety-five percent (595/627) of previously designed RosCOS primer pairs amplified a product in six sweet cherry cultivars predicted to represent the range of genetic diversity in breeding germplasm. A total of 45% (282/627) RosCOS were polymorphic among the six cultivars, and allele number ranged from 2 to 6, with a genome-wide mean of 2.35. A subset of 92 genome-wide single nucleotide polymorphisms (SNPs) corresponding to 76 RosCOS was analyzed in 36 founder accessions and progeny. The expected and observed heterozygosity suggested that 83% of the RosCOS were in Hardy–Weinberg equilibrium, implying that most RosCOS behave as neutral markers. Principal coordinate analysis (PCO) identified one wild accession and two Spanish landraces that clustered differently from the other accessions. The relatively high number of unique alleles found in the three differentially clustered selections suggested that their use as parents has potential to increase the genetic diversity in future US-bred cultivars. Of the 92 RosCOS SNPs, 81 SNPs that represented 68 genome-wide RosCOS segregated in four mapping populations. These RosCOS were mapped in four F₁ populations, thereby greatly improving the genetic linkage map of sweet cherry.     

LSGermOPA,a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) germplasm fingerprinting

To deploy a high-throughput genotyping platform in germplasm management, we designed and tested a custom OPA (Oligo Pool All), LSGermOPA, for assessing the genetic diversity and population structure of the USDA cultivated lettuce (Lactuca sativa L.) germplasm collection using Illumina’s GoldenGate assay. This OPA contains 384 EST (expressed sequence tag)-derived SNP (single nucleotide polymorphism) markers selected from a large set of SNP markers experimentally validated and mapped by the Compositae Genome Project. Used for genotyping were DNA samples prepared from bulked leaves of five randomly-selected seedlings from each of 380 lettuce accessions. High-quality genotype data were obtained from 354 of the 384 SNPs. The reproducibility of automatic genotype calls was 99.8% as calculated from the four pairs of duplicated DNA samples in the assay. An unexpectedly high percentage of heterozygous genotypes at the polymorphic loci for most accessions indicated a high level of heterogeneity within accessions. Only 148 homogenous accessions, collectively comprising all five horticultural types, were used in subsequent analyses to demonstrate the usefulness of LSGermOPA. The results of phylogenetic relationship, population structure and genetic differentiation analyses were consistent with previous reports using other marker systems. This suggests that LSGermOPA is capable of revealing sufficient levels of polymorphism among lettuce cultivars and is appropriate for rapid assessment of genetic diversity and population structure in the lettuce germplasm collection. Challenges and strategies for effective genotyping and managing lettuce germplasm are discussed.     

Unravelling genetic diversity and cultivar parentage in the Danish apple gene bank collection

Characterization of apple germplasm is important for conservation management and breeding strategies. A set of 448 Malus domestica accessions, primarily of local Danish origin, were genotyped using 15 microsatellite markers. Ploidy levels were determined by flow cytometry. Special emphasis was given to pedigree reconstruction, cultivar fingerprinting and genetic clustering. A reference set of cultivars, mostly from other European countries, together with a private nursery collection and a small set of Malus sieversii, Malus sylvestris and small-fruited, ornamental Malus cultivars, was also included. The microsatellite markers amplified 17–30 alleles per loci with an average degree of heterozygosity at 0.78. We identified 104 (23%) duplicate genotypes including colour sports. We could infer first-degree relationships for many cultivars with previously unknown parentages. STRUCTURE analysis provided no evidence for a genetic structure but allowed us to present a putative genetic assembly that was consistent with both PCA analysis and parental affiliation. The Danish cultivar collection contains 10% duplicate genotypes including colour sports and 22% triploids. Many unique accessions and considerable genetic diversity make the collection a valuable resource within the European apple germplasm. The findings presented shed new light on the origin of Danish apple cultivars. The fingerprints can be used for cultivar identification and future management of apple genetic resources. In addition, future genome-wide association studies and breeding programmes may benefit from the findings concerning genetic clustering and diversity of cultivars.     

Genetic diversity of taro, Colocasia esculenta (L.) Schott, in Southeast Asia and the Pacific

The genetic diversity of 255 taro (Colocasia esculenta) accessions from Vietnam, Thailand, Malaysia, Indonesia, the Philippines, Papua New Guinea and Vanuatu was studied using AFLPs. Three AFLP primer combinations generated a total of 465 scorable amplification products. The 255 accessions were grouped according to their country of origin, to their ploidy level (diploid or triploid) and to their habitat—cultivated or wild. Gene diversity within these groups and the genetic distance between these groups were computed. Dendrograms were constructed using UPGMA cluster analysis. In each country, the gene diversity within the groups of wild genotypes was the highest compared to the diploid and triploid cultivars groups. The highest gene diversity was observed for the wild group from Thailand (0.19), the lowest for the diploid cultivars group from Thailand (0.007). In Malaysia there was hardly any difference between the gene diversity of the cultivars and wild groups, 0.07 and 0.08, respectively. The genetic distances between the diploid cultivars groups ranges from 0.02 to 0.10, with the distance between the diploid accessions from Thailand and Malaysia being the highest. The genetic distances between the wild groups range from 0.05 to 0.07. First, a dendrogram was constructed with only the diploids cultivars from all countries. The accessions formed clusters largely according to the country from which they originated. Two major groups of clusters were revealed, one group assembling accessions from Asian countries and the other assembling accessions from the Pacific. Surprisingly, the group of diploid cultivars from Thailand clustered among the Pacific countries. Secondly, a dendrogram was constructed with diploid cultivated, triploid cultivated and wild accessions. Again the division of the accessions into an Asian and a Pacific gene pool is obvious. The presence of two gene pools for cultivated diploid taro has major implications for the breeding and conservation of germplasm.     

Genetic Diversity and Phylogenetic Relationships in the Genus Lycopersicon (Tourn.) Mill. as Revealed by Inter-Simple Sequence Repeat (ISSR) Analysis

Inter-simple sequence repeat (ISSR) analysis was for the first time used to study the genetic diversity and phylogenetic relationships in 54 wild accessions and cultivars of the genus Lycopersicon. Analysis involved 14 ISSR primers homologous to microsatellite repeats and containing additional selective anchor nucleotides. In total, 318 ISSR fragments were amplified for the wild and cultivated tomato genomes. The interspecific polymorphism revealed with the ISSR primers was 95.6%. Species-specific ISSR fragments were detected for each tomato species. The highest number (more than 20) of species-specific fragments were obtained for L. esculentum sensu lato, although the intraspecific variation of ISSR patterns was low. UPGMA cluster analysis was used to construct a dendrogram and to estimate the genetic distances between the species of the genus Lycopersicon; between populations ofL. peruvianum, L. pimpinellifolium, and L. esculentum; and between tomato cultivars. The ISSR-based phylogeny was generally consistent with Lycopersicon taxonomy based on morphological and molecular evidence, suggesting the applicability of ISSR analysis for genotyping and phylogenetic studies in tomato.     

Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model‐based approach implemented in the software structure , we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars.     

Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers

The genetic variation within and between wild apple samples (Malus sylvestris) and cultivated apple trees was investigated with amplified fragment length polymorphisms (AFLP) and microsatellite markers to develop a conservation genetics programme for the endangered wild apple in Belgium. In total, 76 putative wild apples (originating from Belgium and Germany), six presumed hybrids and 39 cultivars were typed at 12 simple sequence repeats (SSR) and 139 amplified fragment length polymorphism (AFLP) loci. Principal co-ordinate analysis and a model-based clustering method classified the apples into three major gene pools: wild Malus sylvestris genotypes, edible cultivars and ornamental cultivars. All presumed hybrids and two individuals (one Belgian, one German) sampled as M. sylvestris were assigned completely to the edible cultivar gene pool, revealing that cultivated genotypes are present in the wild. However, gene flow between wild and cultivated gene pools is shown to be almost absent, with only three genotypes that showed evidence of admixture between the wild and edible cultivar gene pools. Wild apples sampled in Belgium and Germany constitute gene pools that are clearly differentiated from cultivars and although some geographical pattern of genetic differentiation among wild apple populations exists, most variation is concentrated within samples. Concordant conclusions were obtained from AFLP and SSR markers, which showed highly significant correlations in both among-genotypes and among-samples genetic distances.     

Development and Evaluation of SoySNP50K,a High-Density Genotyping Array for Soybean

The objective of this research was to identify single nucleotide polymorphisms (SNPs) and to develop an Illumina Infinium BeadChip that contained over 50,000 SNPs from soybean (Glycine max L. Merr.). A total of 498,921,777 reads 35–45bp in length were obtained from DNA sequence analysis of reduced representation libraries from several soybean accessions which included six cultivated and two wild soybean (G. soja Sieb. et Zucc.) genotypes. These reads were mapped to the soybean whole genome sequence and 209,903 SNPs were identified. After applying several filters, a total of 146,161 of the 209,903 SNPs were determined to be ideal candidates for Illumina Infinium II BeadChip design. To equalize the distance between selected SNPs, increase assay success rate, and minimize the number of SNPs with low minor allele frequency, an iteration algorithm based on a selection index was developed and used to select 60,800 SNPs for Infinium BeadChip design. Of the 60,800 SNPs, 50,701 were targeted to euchromatic regions and 10,000 to heterochromatic regions of the 20 soybean chromosomes. In addition, 99 SNPs were targeted to unanchored sequence scaffolds. Of the 60,800 SNPs, a total of 52,041 passed Illumina’s manufacturing phase to produce the SoySNP50K iSelect BeadChip. Validation of the SoySNP50K chip with 96 landrace genotypes, 96 elite cultivars and 96 wild soybean accessions showed that 47,337 SNPs were polymorphic and generated successful SNP allele calls. In addition, 40,841 of the 47,337 SNPs (86%) had minor allele frequencies ≥10% among the landraces, elite cultivars and the wild soybean accessions. A total of 620 and 42 candidate regions which may be associated with domestication and recent selection were identified, respectively. The SoySNP50K iSelect SNP beadchip will be a powerful tool for characterizing soybean genetic diversity and linkage disequilibrium, and for constructing high resolution linkage maps to improve the soybean whole genome sequence assembly.     

Genetic diversity and structure of local apple cultivars from Northeastern Spain assessed by microsatellite markers

A set of 493 old and local Spanish accessions of apple (Malus x domestica Borkh) maintained at three collections in Northeastern Spain was studied using 16 simple sequence repeats in order to estimate their genetic diversity and to identify the genetic structure and relationships among their accessions. An additional diverse set of 45 apple cultivars, including old Spanish and international cultivars, was added as reference. Genetic analyses performed by Bayesian model-based clustering revealed a very strong differentiation of two major groups. The first one clustered 159 individuals (52?% of unique genotypes) including local accessions and six old Spanish cultivars. The second major group was formed by 145 individuals, including 38 international reference cultivars and one old Spanish cultivar. Nested Bayesian clustering was applied to those two groups and two and four sub-groups were found at each one, respectively. The identification of private and unique alleles, and the remarkable differences in allelic richness among groups and sub-groups constitute further evidence of a clear genetic structure. The results obtained through the factorial correspondence and analyses of molecular variance confirmed those obtained by Bayesian analyses, revealing moderate but significant differentiation among the two major groups (F _ST?=?0.076) and the six sub-groups (F _ST?=?0.111). Our results highlight that the genetic diversity encompassed by currently cultivated apple accounts only for a small fraction of that existing within the species, and that an important part (??60?%) of the local material analyzed constitutes a good example of genetic distinctness with respect to the main cultivars used in European orchards.     

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality.     

One-step reconstruction of multi-generation pedigree networks in apple (Malus × domestica Borkh.) and the parentage of Golden Delicious

The increasing availability of genomic tools improves our ability to investigate the patterns of genetic diversity and relatedness among individuals. The pedigrees of many apple cultivars are completely unknown, often reducing the efficiency of breeding programs. Using a multilocus simple sequence repeat dataset, we applied a novel multi-generation pedigree-network reconstruction procedure based on the software FRANz in a Malus × domestica collection (101 cultivated and 22 wild apples) with partially known pedigree relationships. The procedure produced 78 parent–offspring relationships organized into three networks and showed high power for detecting real pedigree links (98.5 %) and a low false-positive rate (9.0 %). The largest reconstructed pedigree network spanned four generations and involved 65 cultivars. The availability of detailed pedigree connections confirmed that recent genealogical relationships affect population genetic structure in apple. Finally, our analysis enabled us to confirm or discard several pedigrees known only anecdotically, among which the cultivar Grimes Golden was validated as a parent of the widely grown cultivar Golden Delicious. The pedigree reconstruction protocol here described will be of broad applicability to other collections and crop species.