Development of genomic simple sequence repeat markers in faba bean by next-generation sequencing

Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. Development of genetic markers for faba bean is important to study diversity and for molecular breeding. In this study, we used Next Generation Sequencing (NGS) technology for the development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5 % and 20.9 % of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96 %) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5 % polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions.     

Genetic diversity analysis in sorghum germplasm as estimated by AFLP, SSR and morpho-agronomical markers

A comparison of the different methods of the estimation of genetic diversity is important to evaluate their utility as a tool in germplasm conservation and plant breeding. Amplified fragment length polymorphism (AFLP), microsatellites or SSR and morphological traits markers were used to evaluate 45 sorghum germplasm for genetic diversity assessment and discrimination power. The mean polymorphism information content (PIC) values were 0.65 (AFLPs) and 0.46 (SSRs). The average pairwise genetic distance estimates were 0.57 (morphological traits), 0.62 (AFLPs) and 0.60 (SSRs) markers data sets. The Shannon diversity index was higher for morphological traits (0.678) than AFLP (0.487) and SSR (0.539). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for AFLP and SSR markers, as well as for morphological and SSR markers were significantly related (p <0.001). However, morphological and AFLP data showed non-significant correlation (p >0.05). Both data sets from AFLP and SSR allowed all accessions to be uniquely identified; two accessions could not be distinguished by the morphological data. In summary, AFLP and SSR markers proved to be efficient tools in assessing the genetic variability among sorghum genotypes. The patterns of variation appeared to be consistent for the three marker systems, and they can be used for designing breeding programmes, conservation of germplasm and management of sorghum genetic resources.     

Assessing genetic diversity in <Emphasis Type="Italic">Gossypium arboreum</Emphasis> L. cultivars using genomic and EST-derived microsatellites

The cultivated diploid, Gossypium arboreum L., (A genome) is an invaluable genetic resource for improving modern tetraploid cotton (G. hirsutum L. and G. barbadense L.) cultivars. The objective of this research is to select a set of informative and robust microsatellites for studying genetic relationships among accessions of geographically diverse G. arboreum cultivars. From more than 1,500 previously developed simple sequence repeat (SSR) markers, 115 genomic (BNL) and EST-derived (MUCS and MUSS) markers were used to evaluate the allelic diversity of a core panel of G. arboreum accessions. These SSR data enabled advanced genome analyses. A set of 25 SSRs were selected based both upon their high level of informativeness (PIC ≥ 0.50) and the production of clear PCR bands on agarose gels. Subsequently, 96 accessions representing a wide spectrum of diversity of G. arboreum cultivars were analyzed with these markers. The 25 SSR loci revealed 75 allelic variants (polymorphisms) ranging from 2 to 4 alleles per locus. The Neighborjoining (NJ) method, based on genetic dissimilarities, revealed that cultivars from geographically adjacent countries tend to cluster together. Outcomes of this research should be useful in decreasing redundancy of effort and in constructing a core collection of G. arboreum, important for efficient use of this genetic resource in cotton breeding.     

Assessment of Genetic Diversity in <Emphasis Type="Italic">Secale cereale</Emphasis> Based on SSR Markers

The primary aim of this study was to estimate genetic diversity among Secale cereale L. accessions using 22 previously published simple sequence repeat (SSR) markers. The plant material included 367 rye accessions comprising historical and contemporary cultivars, cultivated materials, landraces, and breeding strains from the Polish breeding company Danko. The studied accessions represented a wide geographical diversity. Several methods were employed to analyze genetic diversity among the Secale cereale L. accessions and to determine population structure: principal coordinate analysis (PCoA), neighbor-joining (NJ), and Bayesian clustering. We also defined a core collection of 25 rye accessions representing over 93 % of SSR alleles. The results of these analyses showed that accessions from the rye gene bank are clearly divergent in comparison with materials received directly from European breeding companies. Our findings suggest also that the genetic pool of current rye cultivars is becoming narrower during breeding processes. The selected panel of SSR markers performed well in detection of genetic diversity patterns and can be recommended for future germplasm characterization studies in rye.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini     

Genetic diversity,population structure and association analysis in linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.)

The present investigation aimed to explore the level of genetic diversity, determine the population structure in a larger set of germplasm of linseed using microsatellite marker and identify linked markers through association mapping. A total of 168 accessions of linseed were evaluated for major agro-economic traits and SSRs markers deployed for diversity assessment. A total of 337 alleles were amplified by 50 SSRs ranging from 2 to 13 with an average of 6.74 ± 2.8 alleles per loci. The neighbor joining based clustering grouped all the accessions into three major clusters that were also confirmed by scatter plot of PCoA. While model based clustering determined four sub-populations (K = 4). Further, analysis of molecular variance analysis considering three population showed that maximum variation (79%) was within the population. We identified one putative SSR marker (Lu_3043) linked with days to 50% flowering through both GLM and MLM analysis of association mapping. The results of this preliminary study revealed genetic diversity, population structure in linseed and linked marker which could be utilized in future breeding program.     

Characterization and utilization of microsatellites in the <Emphasis Type="Italic">Coffea canephora</Emphasis> genome to assess genetic association between wild species in Kenya and cultivated coffee

Coffee is an important beverage crop in the world and has a significant contribution to Kenya’s economy. Here, we analyzed the genome-wide distribution of microsatellites in the Coffea canephora genome. A total of 159,041 SSRs were identified, with an overall density of 308 SSRs per Mb. Tetra-nucleotide repeats are the most abundant, accounting for 32 % of the total SSRs. AT-rich motifs are dominant across all SSR repeat units, while GC-rich motifs were generally rare. A set of 100 SSRs was selected to amplify 96 coffee accessions, including 10 wild accessions collected from Mt. Marsabit (Kenya). Of these SSRs, 33 % generated clear polymorphic bands among all tested accessions, with an average of 3.9 alleles per SSR locus. Wild coffee species from Mt. Marsabit showed a close genetic similarity with cultivated accessions in Kenya, suggesting that the wild species in Mt. Marsabit played an important role in the domestication of cultivated coffee in Kenya. Significantly low pairwise genetic divergence was observed between cultivated and wild accessions in Kenya, suggesting a relatively narrow level of genetic basis among coffee germplasm in Kenya. In addition, cultivated and wild coffee accessions in Kenya show a great divergence from those in other countries. Our results not only provide molecular tools for genetic studies in coffee but are also helpful for conservation and coffee breeding programs in Kenya.     

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties.

Genetic distances (GDs) based on molecular markers are important parameters for identifying essentially derived varieties (EDVs). In this context information about the variability of molecular markers within maize inbred lines is essential. Our objectives were to (1) determine the variation in the size of simple sequence repeat (SSR) fragments among different accessions of maize inbreds and doubled haploid (DH) lines, (2) attribute the observed variation to genetic and marker system-specific sources, and (3) investigate the effect of SSR fragment size differences within maize lines on the GD between maize lines and their consequences for the identification of essentially derived varieties. Two to five accessions from nine inbred lines and five DH lines were taken from different sources or drawn as independent samples from the same seed lot. Each accession was genotyped with 100 SSR markers that evenly covered the whole maize genome. In total, 437 SSR fragments were identified, with a mean of 4.4 alleles per locus. The average polymorphic information content (PIC) was 0.58. GD estimates between two accessions of the same genotype ranged from 0.00 to 0.12 with an average of 0.029 for inbred lines and 0.001 for DH lines. An average of 11.1 SSRs was polymorphic between accessions of the same inbred line due to non-amplification (8.1 SSRs), heterogeneity (4.0 SSRs) or unknown alleles (2.6 SSRs). In contrast to lab errors, heterogeneity contributed considerably to the observed variation for GD. In order to decrease the probability to be suited for infringing an EDV threshold by chance, we recommend to increase the level of homogeneity of inbred lines before applying for plant variety protection.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats.

Genetic diversity can be measured by several criteria, including phenotype, pedigree, allelic diversity at marker loci, and allelic diversity at loci controlling phenotypes of interest. Abundance, high level of polymorphism, and ease of genotyping make simple sequence repeats (SSRs) an excellent molecular marker system for genetics diversity analyses. In this study, we used a set of mapped SSRs to survey three representative groups of barley germplasm: a sample of crop progenitor (Hordeum vulgare subsp. spontaneum) accessions, a group of mapping population parents, and a group of varieties and elite breeding lines. The objectives were to determine (i) how informative SSRs are in these three sets of barley germplasm resources and (ii) the utility of SSRs in classifying barley germplasm. A total of 687 alleles were identified at 42 SSR loci in 147 genotypes. The number of alleles per locus ranged from 4 to 31, with an average of 16.3. Crop progenitors averaged 10.3 alleles per SSR locus, mapping population parents 8.3 alleles per SSR locus, and elite breeding lines 5.8 alleles per SSR locus. There were many exclusive (unique) alleles. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94. The cluster analysis indicates a high level of diversity within the crop progenitors accessions and within the mapping population parents. It also shows a lower level of diversity within the elite breeding germplasm. Our results demonstrate that this set of SSRs was highly informative and was useful in generating a meaningful classification of the germplasm that we sampled. Our long-term goal is to determine the utility of molecular marker diversity as a tool for gene discovery and efficient use of germplasm.     

Genetic assessment of safflower (Carthamus tinctorius L.) collection with microsatellite markers acquired via pyrosequencing method

A genetic evaluation of safflower germplasm collections derived from different geographical regions and countries will provide useful information for sustainable conservation and the utilization of genetic diversity. However, the molecular marker information is limited for evaluation of genetic diversity of safflower germplasm. In this study, we acquired 509 putative genomic SSR markers for sufficient genome coverage using next‐generation sequencing methods and characterized thirty polymorphic SSRs in safflower collection composed of 100 diverse accessions. The average allele number and expected heterozygosity were 2.8 and 0.386, respectively. Analysis of population structure and phylogeny based on thirty SSR profiles revealed genetic admixture between geographical regions contrary to genetic clustering. However, the accessions from Korea were genetically conserved in distinctive groups in contrast to other safflower gene pool. In conclusion, these new genomic SSRs will facilitate valuable studies to clarify genetic relationships as well as conduct population structure analyses, genetic map construction and association analysis for safflower.     

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.     

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.     

Development of SSR markers to study diversity in the genus Cymbidium

Cymbidium spp. are important potted flowers with extremely high ornamental and economic value. The present study reports the development of 14 new simple sequence repeat (SSR) markers through the construction of an enriched Cymbidium goeringii library and cross-amplification in Cymbidium sinensis and Cymbidium hybridium. Of 525, 322 (61.33%) clones had SSR motifs and among motifs di-nucleotides were predominant and followed by tri-nucleotide and tetra-nucleotide type. In polymorphic analysis using 14 newly developed SSRs, a total of 201 alleles across 96 Cymbidium accessions were detected with an average of 14.4 per locus. The average heterozygosity was 0.394. The average gene diversity and polymorphism information content values were 0.394 and 0.639, respectively. The mean genetic similarity coefficient was 0.4297, indicating a wide genetic variation among the Cymbidium accessions. These newly developed SSRs will be useful tools for genotype identification, germplasm conservation, molecular breeding, and assessments of genetic diversity and population structure in Cymbidium.     

Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats

Watermelon (Citrullus lanatus var. lanatus) is one of the most important vegetable crops in the world. Molecular markers have become the tools of choice for resolving watermelon taxonomic relationships and evolution. Increased numbers of single nucleotide polymorphism (SNP) markers together with simple sequence repeat (SSR) markers would be useful for phylogenetic analyses of germplasm accessions and for linkage mapping for marker-assisted breeding with quantitative trait loci and single genes. We aimed to construct a genetic map based on SNPs (generated by Illumina Veracode multiplex assays for genotyping) and SSR markers and evaluate relationships inferred from SNP genotypes between 130 watermelon accessions collected throughout the world. We incorporated 282 markers (232 SNPs and 50 SSRs) into the linkage map. The genetic map consisted of 11 linkage groups spanning 924.72 cM with an average distance of 3.28 cM between markers. Because all of the SNP-containing sequences were assembled with the whole-genome sequence draft for watermelon, chromosome numbers could be readily assigned for all the linkage groups. We found that 134 SNPs were polymorphic in 130 watermelon accessions chosen for diversity studies. The current 384-plex SNP set is a powerful tool for characterizing genetic relatedness and for developing medium-resolution genetic maps.     

Genetic analysis of maize germplasm in the Korean Genebank and association with agronomic traits and simple sequence repeat markers

We assessed genetic and phenotypic variation in 105 maize germplasm accessions from RDA-Genebank of Korea and performed association analyses for 11 agronomical traits and 100 simple sequence repeats (SSR). Genetic diversity (GD) analysis revealed a total of 1104 alleles at the 100 SSR loci. The average number of alleles per locus was 11.0. The average GD and polymorphic information content values were 0.73 and 0.70, respectively. The average major allele frequency was 0.41. Population structure analysis indicated that these maize accessions comprised two major groups and one admixed group based on a membership probability threshold of 0.80. The two major groups contained 35 and 46 maize accessions. A mixed linear model of association analysis revealed five marker-trait associations with a significance level of P?≤?0.01 involving five SSR markers. A general linear model showed 72 marker-trait associations involving 42 SSR markers. We confirmed the presence in the general linear model associations of the five significant marker-trait associations (SMTAs) identified in the mixed linear model. For these SMTAs, two loci were associated with stem diameter and one locus each was associated with ear row number, leaf width, and leaf length. These results should prove useful for breeding new inbred lines by selecting parental lines using molecular markers and will help to preserve maize genetic resources in Korea.     

Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers

Information about the genetic diversity and population structure in elite breeding material is of fundamental importance for the improvement of crops. The objectives of our study were to (a) examine the population structure and the genetic diversity in elite maize germplasm based on simple sequence repeat (SSR) markers, (b) compare these results with those obtained from single nucleotide polymorphism (SNP) markers, and (c) compare the coancestry coefficient calculated from pedigree records with genetic distance estimates calculated from SSR and SNP markers. Our study was based on 1,537 elite maize inbred lines genotyped with 359 SSR and 8,244 SNP markers. The average number of alleles per locus, of group specific alleles, and the gene diversity (D) were higher for SSRs than for SNPs. Modified Roger’s distance (MRD) estimates and membership probabilities of the STRUCTURE matrices were higher for SSR than for SNP markers but the germplasm organization in four heterotic pools was consistent with STRUCTURE results based on SSRs and SNPs. MRD estimates calculated for the two marker systems were highly correlated (0.87). Our results suggested that the same conclusions regarding the structure and the diversity of heterotic pools could be drawn from both markers types. Furthermore, although our results suggested that the ratio of the number of SSRs and SNPs required to obtain MRD or D estimates with similar precision is not constant across the various precision levels, we propose that between 7 and 11 times more SNPs than SSRs should be used for analyzing population structure and genetic diversity.     

Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo.

Simple sequence repeat (SSR) markers are valuable tools for many purposes such as phylogenetic, fingerprinting, and molecular breeding studies. However, only a few SSR markers are known and available in bamboo species of the tropics (Bambusa spp.). Considering that grass genomes have co-evolved and share large-scale synteny, theoretically it should be possible to use the genome sequence based SSR markers of field crops such as rice (Oryza sativa) and sugarcane (Saccharum spp.) for genome analysis in bamboo. To test this, 98 mapped SSR primers representing 12 linkage groups of rice and 20 EST-derived sugarcane SSR primers were evaluated for transferability to 23 bamboo species. Of the tested markers, 44 (44.9%) rice and 15 (75%) sugarcane SSR primers showed repeatable amplification in at least one species of bamboo and thus were successfully utilized for phylogenetic and genetic diversity analyses. Transferred SSR primers revealed complex amplification patterns in bamboo, with an average of 9.62 fragments per primer, indicating a high level of polyploidy and genetic variability in bamboo. Forty-two of these primers (34 rice and 8 sugarcane SSR primers) detected an average of 2.12 unique fragments per primer and thus could be exploited for species identification. Six bamboo SSR primers exhibited cross transferability, to varying degrees, to different bamboo species. The genetic similarity coefficient indicated a high level of divergence at the species level (73%). However, a relatively low level of diversity was observed within species (25% in 20 accessions of Dendrocalamus hamiltonii). Further, cluster analysis revealed that the major grouping was in accordance with the taxonomical classification of bamboo. Thus, the rice and sugarcane SSRs can be utilized for phylogenetic and genetic diversity studies in bamboo.     

Genetic Diversity and Characterization of a Core Collection of Malus Germplasm Using Simple Sequence Repeats (SSRs)

Simple sequence repeats (SSRs) were used to assess genetic diversity and study genetic relatedness in a large collection of Malus germplasm. A total of 164 accessions from the Malus core collection, maintained at the University of Illinois, were genotyped using apple SSR markers. Each of the accessions was genotyped using a single robust SSR marker from each of the 17 different linkage groups in Malus. Data were subjected to principal component analysis, and a dendrogram was constructed to establish genetic relatedness. As expected, this diverse core collection showed high allelic diversity; moreover, this allelic diversity was higher than that previously reported. Cluster analysis revealed the presence of four distinct clusters of accessions in this collection.