Genetic Diversity Analysis of Tibetan Wild Barley Using SSR Markers

One hundred and six accessions of wild barley collected from Tibet, China, including 50 entries of the two-rowed wild barley Hordeum vulgare ssp. spontaneum (HS), 29 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon (HA), and 27 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon var. lagunculiforme (HL), were analyzed using 30 SSR markers selected from the seven barley linkage groups for studying genetic diversity and evolutionary relationship of the three subspecies of Tibetan wild barley to cultivated barley in China. Over the 30 genetic loci that were studied, 229 alleles were identified among the 106 accessions, of which 70 were common alleles. H. vulgare ssp. spontaneum possesses about thrice more private alleles (2.83 alleles/locus) than HS (0.93 alleles/locus), whereas almost no private alleles were detected in HL. The genetic diversity among-subspecies is much higher than that within-subspecies. Generally, the genetic diversity among the three subspecies is of the order HS > HL > HA. Phylogenetic analysis of the 106 accessions showed that all the accessions of HS and HA was clustered in their own groups, whereas the 27 accessions of HL were separated into two groups (14 entries with group HS and the rest with group HA). This indicated that HL was an intermediate form between HS and HA. Based on this study and previous works, we suggested that Chinese cultivated barley might evolve from HS via HL to HA.     

Isolation and characterization of gene-derived single nucleotide polymorphism (SNP) markers in Scylla paramamosain

Single nucleotide polymorphisms (SNPs) are thought to be well suitable for genetic and evolutionary studies. In this study, we reported the first set of SNP markers in a commercially important crab species, Scylla paramamosain. A total of 12,500 base pairs high quality DNA sequences were obtained from 15 genes, and thirty-seven SNPs were identified, representing one SNP every 338 base pairs. Twenty-four SNPs were successfully genotyped in a single population. All loci had two alleles and the minor allele frequency ranged from 0.02 to 0.44. The observed and expected heterozygosity ranged from 0.04 to 0.59 and from 0.04 to 0.50, respectively. No significant departures from Hardy–Weinberg equilibrium at each locus was found. The linkage disequilibrium was detected in six loci pairs, but absent after sequential Bonferroni correction. These SNP markers will provide a useful addition to the genetic tools for genetic and evolutionary studies for S. paramamosain.     

Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae)

The Pilosocereus aurisetus group contains eight cactus species restricted to xeric habitats in eastern and central Brazil that have an archipelago-like distribution. In this study, 5–11 microsatellite markers previously designed for Pilosocereus machrisii were evaluated for cross-amplification and polymorphisms in ten populations from six species of the P. aurisetus group. The genotypic information was subsequently used to investigate the genetic relationships between the individuals, populations, and species analyzed. Only the Pmac101 locus failed to amplify in all of the six analyzed species, resulting in an 88?% success rate. The number of alleles per polymorphic locus ranged from 2 to 12, and the most successfully amplified loci showed at least one population with a larger number of alleles than were reported in the source species. The population relationships revealed clear genetic clustering in a neighbor-joining tree that was partially incongruent with the taxonomic limits between the P. aurisetus and P. machrisii species, a fact which parallels the problematic taxonomy of the P. aurisetus group. A Bayesian clustering analysis of the individual genotypes confirmed the observed taxonomic incongruence. These microsatellite markers provide a valuable resource for facilitating large-scale genetic studies on population structures, systematics and evolutionary history in this group.     

Microsatellite markers in western hemlock [Tsuga heterophylla (Raf.) Sarg]

Fifteen microsatellite markers have been isolated from western hemlock [Tsuga heterophylla (Raf.) Sarg] genomic DNA and six of these markers were optimized for use in mountain hemlock [Tsuga mertensiana (Bong) Carr.]. The mean expected heterozygosity (H_E) was 0.88 and 0.89 for western hemlock and mountain hemlock, respectively. Allelic diversity was high for both hemlock species, ranging from 7 to 15 alleles per locus for western hemlock and 5–30 alleles per locus for mountain hemlock. The allelic variation identified in this project will allow monitoring of changes in genetic variation as a result of selection pressure in breeding programs.     

Esterase Allozymes of Soybean Cyst Nematode,Heterodera glycines,from China,Japan, and the United States

Individual females from 19 populations of Heterodera glycines from China, Japan, and the United States were analyzed for esterase allozyme polymorphism. Eight esterase electrophoretic phenotypes were resolved. Four putative loci, est-1, est-2, est-3, and est-4, were identified, having one, one, two, and one allele, respectively. The four loci expressed six genotypes in the four Chinese populations. Loci est-2, est-3, and est-4 were identified in five Japanese populations and expressed five genotypes, whereas only loci est-2 and est-3 were identified in 10 populations from the United States and expressed four genotypes. Putative alleles at each locus were defined as characters for data analysis. Phylogenetic analysis using parsimony (PAUP) was utilized to determine relationships among the 19 populations. More loci and alleles in populations from China and phylogenetic similarities among populations from Japan and the United States are consistent with a founder effect resulting from dissemination of progenitor H. glycines from China to Japan and subsequent introductions of founder populations from Japan to the United States.     

Development of EST-SSR markers and investigation of genetic relatedness in tung tree

The tung tree is an important non-edible oilseed source and consists of two species Vernicia fordii and Vernicia montana native to southern China and southeast Asia. In this study, the frequency and distribution of simple sequence repeats (SSRs) in expressed sequence tags (ESTs) of V. fordii were characterized based on 2,407 available EST sequences from the National Center for Biotechnology Information database. Twenty-two EST-SSR markers were developed by screening the genomic DNAs of six individuals of V. fordii from three accessions and 120 individuals of V. montana from 30 indigenous V. montana accessions collected from different geographic areas in southwestern China and northern Laos. The 22 EST-SSR markers exhibited a moderate level of polymorphism in V. montana with an average of 3.36 alleles per locus and PIC of 0.401. Genetic relatedness investigation showed that there was not only distinct genetic differentiation among tung trees but also a distinct geographic pattern among V. montana accessions. The current study is the first report of the development of EST-SSRs and genetic relatedness investigation in tung trees. These EST-SSR markers reported here will be valuable resources for future genetic studies, like construction of linkage maps, diversity analysis, quantitative trait locus/association mapping, and molecular breeding of the tung tree. The genetic relatedness identified in V. montana would provide potential clues in choosing germplasms in interest as progenitors for cross breeding and variety improvement of V. montana in practice.     

Novel microsatellite markers for Begonia maxwelliana and transferability to 23 Begonia species of Peninsular Malaysia

Begonias are hyper-diverse and important horticultural plants. Six polymorphic microsatellite markers were developed from CT- and GT-enriched libraries of Begonia maxwelliana. The number of alleles per locus ranged from 2 to 12 and the observed heterozygosity ranged from 0.036 to 0.813. Null alleles were detected in one locus (Bma161) after Bonferroni correction. All the six markers were amplifiable in 23 selected Begonia species with the success rates of 17–100%. On average, species of the same section as B. maxwelliana (i.e. sect. Platycentrum) yielded higher transferability (91%). These markers will be useful for population genetic studies of the genus Begonia.     

Development of eighteen tetranucleotide microsatellite markers in Tibetan Macaque (Macaca thibetana) and genetic diversity analysis of captive population

In this study, eighteen tetranucleotide microsatellite loci were isolated from AAAG-enriched and GATA-enriched libraries of the Tibetan macaque, Macaca thibetana. These loci were tested on 53 individuals of M. thibetana, and most loci were proved to be highly polymorphic. A total of 109 alleles were detected with an average of 6.06 alleles per locus. The PIC values of these loci ranged from 0.192 to 0.879, with an average of 0.624. The observed and expected heterozygosities ranged from 0.170 to 0.800 and from 0.217 to 0.898, with an average of 0.583 and 0.675, respectively. 5 loci significantly deviated from Hardy–Weinberg equilibrium (HWE). Significant linkage disequilibrium (LD) was found between 9 pairs of loci. The newly identified polymorphic markers would facilitate the study of M. thibetana on the population structure and genetic diversity.     

Population genetic characterization of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) using novel microsatellite markers

The dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) has long been considered an endangered insect in South Korea; the detection of recent population increases leaves its endangered status uncertain. Population genetic analysis subsequent to development of molecular markers is essential for establishing proper conservation strategies. In this study, we developed ten microsatellite markers specifically for C. tripartitus. Sixty-eight individuals of C. tripartitus collected from six South Korean localities were genotyped to validate these markers and preliminarily assess population genetic characteristics. Per-locus observed number of alleles, observed heterozygosity (H_O), and expected heterozygosity (H_E) ranged from 5–12, 0.499–0.958, and 0.54–0.743, respectively. All populations showed higher H_O than H_E, negative values of inbreeding coefficient, and, overall, no sign of recent population bottlenecks (excluding one population, Seosan). This suggests that C. tripartitus did not suffer from genetic drift and inbreeding, which are typically severe in small, isolated populations. Nevertheless, detection of only one of the two gene pools in some populations and resultant genetic subdivision into two population groups may suggest that the population size is not enough to cover both gene pools. Thus, a more extended period of protection may be required to ensure higher genetic diversity of widespread populations and achieve the long-term conservation goal.     

Genetic structure of a QTL hotspot on chromosome 2 in sweet cherry indicates positive selection for favorable haplotypes

A genomic region of particular interest for sweet cherry (Prunus avium L.) breeding is a quantitative trait locus (QTL) “hotspot” on chromosome 2. QTLs for fruit size, firmness, sweetness, and flowering time are reported to map to this region. An understanding of genetic diversity, allele sources, linkage relationships, and historical recombinations is critical to enable the combining of favorable alleles at multiple loci. The objectives of this study were to characterize, visualize, and interpret the genetic structure of this previously identified QTL hotspot within North American sweet cherry breeding germplasm, using a pedigree-based haploblocking approach. Across the 29.4 cM (6.3 Mbp) region defined by single nucleotide polymorphism (SNP) information from the RosBREED cherry 6K SNP array v1, a total of 12 recombination events falling into six inter-marker regions were traced within the pedigree of elite and wild germplasm (n = 55). These recombinations defined five haploblocks containing 5–15 markers and exhibiting 7–11 haplotypes each. Over the entire QTL hotspot, 30 extended haplotypes were identified for which parental gametes could be determined. When the haploblocks and their haplotypes were used to explore genetic diversity, ancestry, and recombination patterns, and then integrated with previous QTL results for fruit size, the results indicated that favorable alleles at this QTL hotspot are under positive selection in breeding. The genetic framework provided by a haploblock approach and knowledge of haplotype-level diversity sets the stage for assigning breeding utility to these haplotypes.     

Discrimination and genetic diversity of cultivated and wild safflowers (Carthamus spp.) using EST-microsatellites markers

In spite of being one of the major oilseed crops, little is known about genetic diversity and relationships between species of safflower. In this study EST-SSR markers were used to evaluate and characterize 42 genotypes from six species including Carthamus tinctorius, Carthamus palaestinus, Carthamus oxyacanthus, Carthamus lanatus, Carthamus dentatus, and Carthamus boissieri. Thirty three primer pairs produced 123 polymorphic bands with 2–8 alleles per locus. The EST-SSR markers showed different level of gene diversity. The highest Polymorphism Information Content (PIC) values were observed for primers EL510507 and EL390720 (0.49 and 0.45, respectively). The highest genetic diversity and heterozygosity were observed for C. oxyacanthus. Both cluster and principal coordinate analysis (PCoA) clearly separated species into distinct groups. Within each species the accessions were clustered in different subgroups that mainly supported the known origins. The result showed that C. palaestinus had the most genetic similarity with cultivated safflower and C. oxyacanthus was next in this respect. In general, EST-SSR markers effectively revealed the genetic relationships and diversity of Carthamus species. This information is valuable for safflower improvement since C. palaestinus and C. oxyacanthus are both crossable with the cultivated species C. tinctorius.     

Assessment of molecular diversity and evolutionary relationship of kenaf (Hibiscus cannabinus L.), roselle (H. sabdariffa L.) and their wild relatives

Kenaf (Hibiscus cannabinus L.) and roselle (H. sabdariffa L.) are valuable fibre crop species with diverse end use. Phylogenetic relationship of 73 accessions of kenaf, roselle and their wild relatives from 15 countries was assessed using 44 inter-simple sequence repeat (ISSR) and jute (Corchorus olitorius L.) specific simple sequence repeats (SSR) markers. A total of 113 alleles were identified of which 61.95 % were polymorphic. Jute specific SSR markers exhibited high polymorphism and resolving power in kenaf, although ISSR markers exhibited higher resolving power than SSR markers. Number of polymorphic alleles varied from 1 to 5 for ISSR and 1 to 6 for SSR markers. Cultivated species exhibited higher allele polymorphism (57 %) than the wild species (35 %), but the improved cultivars exhibited lower genetic diversity compared to germplasm accessions. Accessions with common genetic lineage and geographical distribution clustered together. Indian kenaf varieties were distinct from cultivars bred in other countries and shared more genetic homology with African accessions. High genetic diversity was observed in the Indian (J = 0.35–0.74) and exotic kenaf germplasm collections (J = 0.38–0.79), suggesting kenaf might have been introduced in India from Africa through Central Asia during early domestication. Genetic similarity-based cluster analysis was in close accordance with taxonomic classification of Hibiscus.     

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy–Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species’ genetic diversity.     

Identification of favorable SNP alleles and candidate genes responsible for inflorescence-related traits via GWAS in chrysanthemum

Key message

81 SNPs were identified for three inflorescence-related traits, in which 15 were highly favorable. Two dCAPS markers were developed for future MAS breeding, and six candidate genes were predicted.

Abstract

Chrysanthemum is a leading ornamental species worldwide and demonstrates a wealth of morphological variation. Knowledge about the genetic basis of its phenotypic variation for key horticultural traits can contribute to its effective management and genetic improvement. In this study, we conducted a genome-wide association study (GWAS) based on two years of phenotype data and a set of 92,617 single nucleotide polymorphisms (SNPs) using a panel of 107 diverse cut chrysanthemums to dissect the genetic control of three inflorescence-related traits. A total of 81 SNPs were significantly associated with the three inflorescence-related traits (capitulum diameter, number of ray florets and flowering time) in at least one environment, with an individual allele explaining 22.72–38.67% of the phenotypic variation. Fifteen highly favorable alleles were identified for the three target traits by computing the phenotypic effect values for the stable associations detected in 2 year-long trials at each locus. Dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between highly favorable allele numbers and corresponding phenotypic performance were observed. Two highly favorable SNP alleles correlating to flowering time and capitulum diameter were converted to derived cleaved amplified polymorphic sequence (dCAPS) markers to facilitate future breeding. Finally, six putative candidate genes were identified that contribute to flowering time and capitulum diameter. These results serve as a foundation for analyzing the genetic mechanisms underlying important horticultural traits and provide valuable insights into molecular marker-assisted selection (MAS) in chrysanthemum breeding programs.

     

Assessment of genetic diversity and population structure in mungbean

This study was carried out to assess the genetic diversity and to analyze the population genetic structure for a total of 692 mungbean accessions preserved at National Agrobiodiversity Center (NAC) of the Rural Development Administration (RDA), Korea. Mungbean accessions were collected from 27 countries in nine different geographic regions, and were genotyped using 15 microsatellite markers, which were developed in our previous study. A total of 66 alleles were detected among 692 accessions at all the loci with an average of 4.4 alleles per locus. All the microsatellite loci were found to be polymorphic. The expected heterozygosity (H _E ) and polymorphism information content (PIC) ranged from 0.081 to 0.588 (mean = 0.345) and from 0.080 to 0.544 (mean = 0.295), respectively. Of the 66 alleles, 17 (25.8%) were common (frequency range between 0.05 and 0.5), 15 (22.7%) were abundant (frequency range > 0.5), and 34 (51.5%) were rare (frequency range < 0.05). Locus GB-VR-7 provided the highest number of rare alleles(eight), followed by GB-VR-91(six) and GB-VR-113(four). Country-wide comparative study on genetic diversity showed that accessions from the USA possessed the highest genetic diversity (PIC) followed by Nepal, Iran, and Afghanistan. And region-wide showed that accessions from Europe possessed the highest average genetic diversity, followed by accessions from the USA, South Asia, West Asia, and Oceania. Twenty-seven countries were grouped into seven clades by phylogenetic relationship analysis, but clustering pattern did not strictly follow their geographical origin because of extensive germplasm exchange between/among countries and regions. As a result of a model-based analysis (STRUCTURE) of microsatellite data, two distinct genetic groups were identified which shared more than 75% membership with one of the two genetic groups. However the genetic group pattern did not reflect their geographical origin. The Duncan’s Multiple Range Test among these two genetic groups and an admixed group, with a mean of 16 phenotypic traits, showed significant difference in 12 quantitative and qualitative traits on the basis of ANOVA. These 15 newly developed SSR markers proved to be useful as DNA markers to detect genetic variation in mungbean germplasm for reasonable management and crossbreeding purposes.     

Comparisons of Mitochondrial DNA from the Sibling Species Heterodera glycines and H. schachtii

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.     

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.     

New polymorphic microsatellite markers for the masu salmon (Oncorhynchus masou masou) from Korea and their application to wild and hatchery populations

Masu salmon, Oncorhynchus masou masou, is an economically important fish species in the Far East and occurs in two life history forms: sea-run migratory (anadromous) and freshwater resident (non-anadromous). The non-anadromous form has recently become a popular freshwater food and game fish during a well-known Korean winter festival. However, the genetic background of this species remains largely unknown, partly due to a lack of molecular genetic markers. In this study, we developed new polymorphic microsatellite markers for masu salmon using next-generation sequencing technology. From 40 primer sets, 11 primer sets (27.5% of the primer sets selected) were successfully amplified with 106 alleles (range 2–9) in 64 individuals from different populations: two wild and one hatchery. Observed and expected heterozygosities ranged from 0.304 to 0.947 and 0.278 to 0.865, respectively. Significant departures from the Hardy–Weinberg equilibrium were detected for four markers (OMM11, OMM17, OMM28, and OMM33) in a single population. All pair-wise F_ST values were highly significant between the wild and hatchery populations (range 0.084–0.183, P < 0.0001). We identified a set of robust microsatellite markers that worked well even in formalin-fixed samples, which will be suitable for biogeographical and population structure analyses of the masu salmon.     

Genome-wide development and utilization of novel intron-length polymorphic (ILP) markers in <Emphasis Type="Italic">Medicago sativa</Emphasis>

Alfalfa (Medicago sativa L.) is the most widely cultivated forage legume around the world. Though development and application of microsatellite markers in large-scale was reported in this species, a systematic investigation and large-scale exploitation of intron-length polymorphic (ILP) markers has not been conducted. In the present study, the RNA-Seq sequences of alfalfa were aligned with the genomic sequences of Arabidopsis to predict the position of introns and develop ILP markers in alfalfa. A total of 693 putative ILPs were identified, and 502 ILP markers were successfully developed. Furthermore, 100 ILP markers exhibited relatively high levels of transferability to leguminous (40.0%–83.0%) and non-leguminous (21.0%–22.0%) species. Polymorphisms in 40 randomly selected MsILP markers were evaluated in 21 alfalfa accessions and collectively yielded 169 alleles with an average of 4.7 alleles per locus. The polymorphism information content (PIC) ranged from 0.15 to 0.87 with an average of 0.60, which indicated a high level of polymorphism in the MsILP markers. For the first time, we developed large-scale ILP markers in alfalfa and demonstrated their utility in transferability, which will be valuable for genetic relationship assessments, comparative genomic studies and marker-assisted breeding of leguminous and non-leguminous species.