Genome-wide Simple Sequence Repeat Research Articles

Genome-wide simple sequence repeat analysis and specific molecular marker development of rye

BackgroundRye (Secale cereale L.) is the most widely used related species in wheat genetic breeding, and the introduction of its chromosome fragments into the wheat genome through distant hybridization is essential for enriching the genetic diversity of wheat. Rapid and accurate detection of rye chromatin in the wheat genome is important for distant hybridization. Simple sequence repeats (SSRs) are widely distributed in the genome, and SSRs of different species often exhibit species-specific characteristics.ResultsIn this study, genome-wide SSRs in rye were identified, and their characteristics were outlined. A total of 997,027 SSRs were selected, with a density of 115.97 SSRs/Mb on average. There was no significant difference in the number of SSRs on each chromosome. The number of SSRs on 2R was the highest (15.29%), and the number of SSRs on 1R was the lowest (13.02%). The number of SSRs on each chromosome is significantly correlated with chromosome length. The types of SSR motifs were abundant, and each type of SSR was distributed on 7 chromosomes of rye. The numbers of mononucleotide simple sequence repeats (MNRs), dinucleotide simple sequence repeats (DNRs), and trinucleotide simple sequence repeats (TNRs) were the greatest, accounting for 46.90%, 18.37%, and 22.64% of the total number, respectively. Among the MNRs, the number of G/C repeats and the number of 10 bp motifs were the greatest, accounting for 26.24% and 31.32% of the MNRs, respectively. Based on the SSR sequences, a total of 657 pairs of primers were designed. The PCR results showed that 119 pairs of these primers were rye-specific and could effectively detect rye chromatin in the wheat genome. Moreover, 86 pairs of the primers could also detect one or more specific rye chromosomes.ConclusionThese results lay a foundation for both genomic evolution studies of rye and molecular breeding in wheat.

Genetic diversity and population structure of Philippine strawberry germplasm based on genome-wide simple sequence repeat markers

Abstract. Elec VH, Cadorna CAE, Tad-Awan BA, Basquial DA, Dumaslan MR, Rey JD. 2024. Genetic diversity and population structure of Philippine strawberry germplasm based on genome-wide simple sequence repeat markers. Biodiversitas 25: 2969-2979. Strawberry is a globally recognized fruit due to its health benefits and economic significance. Determining the level of genetic diversity and the relationship between germplasm resources involved in the breeding program is critical for effective crop improvement strategies. This study used 197 Simple Sequence Repeat (SSR) markers to assess the genetic diversity and population structure of 24 strawberry germplasm from the Benguet State University (BSU) collection. In this study, 70% of the markers used were polymorphic with reproducible fragments. A total of 792 alleles were detected, with an average of six per marker. The diversity indices indicated an intermediate level of diversity among the germplasm based on the Jaccard coefficient. Results from cluster analysis generated three groups, separating the Red Milky Way and Summer Princess cultivars from the rest of the germplasm evaluated, while a third cluster was further divided into six subgroups. Furthermore, identified clustering patterns of the evaluated genotypes showed independence from the region of origin, coinciding with Principal Component Analysis (PCA) and population structure analysis results. This study reveals the genetic diversity level and population structure of the strawberry cultivars in the Philippines using SSR markers, facilitating accurate identification and informed parental selection for breeding objectives.

Development of Novel Genomewide Simple Sequence Repeat Markers for Acer truncatum Bunge and Assessment of Their Transferability to Other Closely Related Species

Acer truncatum Bunge is a versatile woody tree species with high economic and medicinal value in the production of bioactive substances and unsaturated fatty acids (especially nervonic acid). However, the exploitation and evaluation of A. truncatum germplasm resources are limited owing to a lack of sound molecular marker systems. In this study, a large set of genomewide simple sequence repeat (SSR) markers of A. truncatum was developed based on its whole-genome sequences. A total of 462,331 SSR loci were identified in the genome sequences, 99.3% (459,193) of which were located on 13 chromosomes. The chromosome length was significantly positively correlated with the number of SSR loci on the chromosome (r = 0.977, p < 0.001). The (A/T)n, (AT/TA)n, and (AAT/ATT/TAA/TTA/TAT/ATA)n were the most frequent motifs for mono-, di-, and trinucleotide repeat motifs, respectively, showing A/T-base bias. After BLASTN and electronic polymerase chain reaction (PCR) analyses, 199,990 loci with specific physical positions were screened. Most of the SSR loci were located in the intergenic regions and fewest in the coding sequences (CDSs). The frequency of loci with tri- and hexanucleotide repeat motifs was the highest in the CDSs, potentially serving to maintain the stability of gene function and structure. In randomly selected 105 SSR markers, 82 (78.1%) showed allelic polymorphism, with polymorphism information content (PIC) values of 0.032–0.926 (0.481 on average). The SSRs in the noncoding regions exhibited significantly higher PIC values than those in the CDSs. The transferability of the 105 markers was 48.6%–59.0% to seven other Acer species. The large set of valid SSR markers provides a powerful tool for studies on population genetics, conservation genetics, linkage mapping, comparative genomics, and marker-assisted breeding of the genus Acer.

Population genetic variation and geographic distribution of suitable areas of Coptis species in China.

The rhizomes of Coptis plants have been used in traditional Chinese medicine over 2000 years. Due to increasing market demand, the overexploitation of wild populations, habitat degradation and indiscriminate artificial cultivation of Coptis species have severely damaged the native germplasms of species in China. Genome-wide simple-sequence repeat (SSR) markers were developed using the genomic data of C. chinensis. Population genetic diversity and structure of 345 Coptis accessions collected from 32 different populations were performed based on these SSRs. The distribution of suitable areas for three taxa in China was predicted and the effects of environmental variables on genetic diversity in relation to different population distributions were further analyzed. 22 primer pairs were selected as clear, stable, and polymorphic SSR markers. These had an average of 16.41 alleles and an average polymorphism information content (PIC) value of 0.664. In the neighbor-joining (N-J) clustering analysis, the 345 individuals clustered into three groups, with C. chinensis, C. chinensis var. brevisepala and C. teeta being clearly separated. All C. chinensis accessions were further divided into four subgroups in the population structure analysis. The predicted distributions of suitable areas and the environmental variables shaping these distributions varied considerably among the three species. Overall, the amount of solar radiation, precipitation and altitude were the most important environmental variables influencing the distribution and genetic variation of three species. The findings will provide key information to guide the conservation of genetic resources and construction of a core reserve for species.

Development and validation of genome-wide SSR molecular markers of Tapes dorsatus.

Tapes dorsatus is an economically important benthic animal in the Beibu Gulf of China. However, the deficiency of microsatellite markers has hindered the study of its genetics. The development of microsatellite markers will provide useful tools for genetic improvement, variety identification, phylogenetic analysis and resource conservation. Within the genome sequence, 145,008 simple sequence repeats (SSRs) were identified, and 29,691 primer pairs were designed successfully. A total of 100 primer pairs were randomly synthesized for testing, and 93 primers yielded products. Sixty highly polymorphic primers were used to reveal the genetic diversity of 50 T. dorsatus individuals. The average number of alleles (Na) of the population was 10.40; the average number of effective alleleswas 6.16, the average expected heterozygosity (He) was 0.82, and the average polymorphic information contentwas 0.80. The genetic structure of the population was detected, by which the population could be divided into three subpopulations. We identified 145,008 SSRs in the genome of T. dorsatus and designed 29,691 primer pairs in this study. Of 100 synthesized primers, 60 were highly polymorphic and used to reveal the genetic diversity and structure of the population. The SSR markers identified here will provide useful tools and a foundation for genetic diversity, linkage mapping and molecular marker-aided breeding in T. dorsatus.

Genome-wide identification of microsatellites for mapping, genetic diversity and cross-transferability in wheat (Triticum spp)

Wheat (Triticum aestivum L.) is a crucial global staple crop, and is consistently being improved to enhance yield, disease resistance, and quality traits. However, the development of molecular markers is a challenging task due to its hexaploid genome. Molecular marker system such as simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) are helpful for breeding, but SNP has limitations due to its development cost and its conversion to breeder markers. The study proposed an in-silico approach, by utilizing the low-cost transcriptome sequencing of two parental lines, ‘TAC 75′ and ‘WH 1105′, to identify polymorphic SSRs for mapping in a recombinant inbred line (RIL) population. This study introduces a new approach to bridge wheat genetics intricacies and next-generation sequencing potential. It presents a comprehensive genome-wide SSR distribution using IWGSC CS RefSeq v2.1 genome assembly and to identify 189 polymorphic loci through in-silico strategy. Of these, 54.76% showed polymorphism between parents, surpassing the traditional low polymorphic success rate. A RIL population screening validated these markers, demonstrating the fitness of identified markers through chi-square tests. The designed SSRs were also validated for genetic diversity analysis in a subset of 37 Indian wheat genotypes and cross-transferability in the wild/relative wheat species. In diversity analysis, a subset of 38 markers revealed 95 alleles (2.5 allele/locus), indicating substantial genetic variation. Population structure analysis unveiled three distinct groups, supported by phylogenetic and PCoA analyses. Further the polymorphic SSRs were also analyzed for SSR-gene association using gene ontology analysis. By utilizing the developing seed transcriptome data within parental lines, the study has enhanced the polymorphic SSR identification precision and facilitated in the RIL population. The undertaken study pioneers the use of transcriptome sequencing and genetic mapping to overcome challenges posed by the intricate wheat genome. This approach offers a cost-effective, less labour-intensive alternative to conventional methods, providing a platform for advancing wheat breeding research.

Construction of a genome-wide SSR marker library in Gerbera hybrida: Insights into genetic variation and germplasm resources

Gerbera hybrida is a highly sought-after cut flower globally, renowned for its diverse flower color and pattern resulting from its complex inflorescence meristem architecture. However, the scarcity of genomic information and molecular markers has impeded efforts to comprehend the genetic diversity of G. hybrida. To address this, our study aimed to construct genome-wide simple sequence repeat (SSR) markers in G. hybrida, utilizing genomic data from an inbred line. We employed 170 gerbera cultivars exhibiting distinct flower patterns and colors to analyze genetic variation and diversity. Our analyses revealed highly polymorphic SSR markers, a total of 69 alleles (Na) were amplified by 14 pairs of primers, in which polymorphic information content (PIC) ranging from 0.380 to 0.700 with an average value of 0.583. The polymorphic results of the observed heterozygosity (Ho) and Shannon's information index (I) were 0.499 and 1.195, respectively, showing a high level of genetic diversity within the enriched sequences of the G. hybrida genome. Leveraging these markers with strong differentiation, we successfully constructed a unique fingerprint map encompassing 170 gerbera germplasm lines. This study involved the construction of an SSR fingerprint library for G. hybrida germplasm resources, employing genetic analysis for screening and molecular-level identification. The findings provide valuable insights into the identification of gerbera germplasm resources and kinship studies among various groups. Moreover, this study lays a solid foundation for future advancements in molecular design breeding improvement and germplasm resource development in G. hybrida.

Development of Polymorphic Microsatellite Markers and Identification of Applications for Wild Walnut (Juglans regia L.) in Middle Asia

The common walnut (Juglans regia L.), a species of significant economic and ecological importance, along with its variant, holds developmental value. Simple sequence repeat (SSR) markers are used as an effective method for material identification due to their co-dominant inheritance. In this work, a total of 357629 SSR loci were searched based on previously published walnut genome sequences, with a density of 662.28 (SSRs/Mb). The majority of all these loci were found to be single nucleotide A/T (58.49%), followed by the dinucleotide AT (19.48%). Based on the results of genome-wide SSR site design, 22 effective primer pairs were designed and screened to analyze the genetic diversity of 48 wild walnut samples from three countries. Each locus had an average of 5.17 effective alleles (Ne), with an average polymorphism information content (PIC) of 0.71, average heterozygosity (He) of 0.52, and average genetic differentiation index (Fst) of 0.09. The analysis of 48 wild walnut samples from three countries (Kyrgyzstan, Tajikistan, and China) using the unweighted pairwise clustering method and arithmetic mean (UPGMA) along with principal coordinate analysis revealed the division of all samples into three groups. In summary, these novel SSR markers serve as a reference for the identification of wild walnut germplasm in Central Asia and for the development and utilization of wild walnut seed resources.

Exploring cross-species genetic diversity: unveiling new insights in Megalobrama through whole genome-wide simple sequence repeats

Exploring cross-species genetic diversity: unveiling new insights in Megalobrama through whole genome-wide simple sequence repeats

Genome-wide survey and characterization of microsatellites in cashew and design of a web-based microsatellite database: CMDB.

The cashew is an edible tree nut crop having a wide range of food and industrial applications. Despite great economic importance, the genome-wide characterization of microsatellites [simple sequence repeats (SSRs)] in cashew is lacking. In this study, we carried out the first comprehensive genome-wide microsatellites/SSRs characterization in cashew and developed polymorphic markers and a web-based microsatellite database. A total of 54526 SSRs were discovered in the cashew genome, with a mean frequency of 153 SSRs/Mb. Among the mined genome-wide SSRs (2-6 bp size motifs), the dinucleotide repeat motifs were dominant (68.98%) followed by the trinucleotides (24.56%). The Class I type of SSRs (≥20 bp) were 45.10%, while Class II repeat motifs (≥12-<20 bp) were 54.89% of the total genomic SSRs discovered here. Further, the AT-rich SSRs occurred more frequently in the cashew genome (84%) compared to the GC-rich SSRs. The validation of the in silico-mined genome-wide SSRs by PCR screening in cashew genotypes resulted in the development of 59 polymorphic SSR markers, and the polymorphism information content (PIC) of the polymorphic SSR markers ranged from 0.19 to 0.84. Further, a web-based database, "Cashew Microsatellite Database (CMDB)," was constructed to provide access to the genome-wide SSRs mined in this study as well as transcriptome-based SSRs from our previous study to the research community through a user-friendly searchable interface. Besides, CMDB provides information on experimentally validated SSRs. CMDB permits the retrieval of SSR markers information with the customized search options. Altogether, the genome-wide SSRs characterization, the polymorphic markers and CMDB database developed in this study would serve as valuable marker resources for DNA fingerprinting, germplasm characterization, genetic studies, and molecular breeding in cashew and related Anacardium species.

Microsatellite Characteristics of Silver Carp (Hypophthalmichthysmolitrix) Genome and Genetic Diversity Analysis in Four Cultured Populations.

Hypophthalmichthys molitrix is one of the four most important fish in China and has high breeding potential. However, simple sequence repeat (SSR) markers developed on H. molitrix genome level for genetic diversity analysis are limited. In this study, the distribution characteristics of SSRs in the assembled H. molitrix genome were analyzed, and new markers were developed to preliminarily evaluate the genetic diversity of the four breeding populations. A total of 368,572 SSRs were identified from the H. molitrix genome. The total length of SSRs was 6,492,076 bp, accounting for 0.77% of the total length of the genome sequence. The total frequency and total density were 437.73 loci/Mb and 7713.16 bp/Mb, respectively. Among the 2–6 different nucleotide repeat types, SSRs were dominated by di-nucleotide repeats (204,873, 55.59%), and AC/GT was the most abundant motif. The number of SSRs on each chromosome was positively correlated with the length. The 13 pairs of markers developed were used to analyze the genetic diversity of four cultivated populations in Hubei Province. The results showed that the genetic diversity of the four populations was low, and the ranges of alleles (Na), effective alleles (Ne), observed heterozygosity (Ho), and Shannon’s index information (I) were 3.538–4.462, 2.045–2.461, 0.392–0.450, and 0.879–0.954, respectively. Genetic variation occurs mainly among individuals within populations (95.35%). UPGMA tree and Bayesian analysis showed that four populations could be divided into two different branches. Therefore, the genome-wide SSRs were effectively in genetic diversity analysis on H. molitrix.

Development of β-carotene, lysine, and tryptophan-rich maize (Zea mays) inbreds through marker-assisted gene pyramiding

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased β-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in β-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1–BC3F1, and ICF1–ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3′TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45–88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the β-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-β1σ4-4-8-8, DBT-IC-β1σ4-9-21-21, DBT-IC-β1σ4-10-1-1, DBT-IC-β2σ5-9-51-51, DBT-IC-β2σ5-9-52-52 and DBT-IC-β2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78–99.31% for DBT-IC-β1σ4 population and 85.71–99.51% for DBT-IC-β2σ5 population) like the recurrent parents and β-carotene (14.21–14.35 μg/g for DBT-IC-β1σ4 and 13.28–13.62 μg/g for DBT-IC-β2σ5), lysine (0.31–0.33% for DBT-IC-β1σ4 and 0.31–0.34% for DBT-IC-β2σ5), and tryptophan (0.079–0.082% for DBT-IC-β1σ4 and 0.078–0.083% for DBT-IC-β2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs.

Mining and validation of novel simple sequence repeat (SSR) markers derived from coconut (Cocos nucifera L.) genome assembly

BackgroundIn the past, simple sequence repeat (SSR) marker development in coconut is achieved through microsatellite probing in bacterial artificial chromosome (BAC) clones or using previously developed SSR markers from closely related genomes. These coconut SSRs are publicly available in published literatures and online databases; however, the number is quite limited. Here, we used a locally established, coconut genome-wide SSR prediction bioinformatics pipeline to generate a vast amount of coconut SSR markers. ResultsA total of 7139 novel SSR markers were derived from the genome assembly of coconut ‘Catigan Green Dwarf’ (CATD). A subset of the markers, amounting to 131, were selected for synthesis based on motif filtering, contig distribution, product size exclusion, and success of in silico PCR in the CATD genome assembly. The OligoAnalyzer tool was also employed using the following desired parameters: %GC, 40–60%; minimum ΔG value for hairpin loop, −0.3 kcal/mol; minimum ΔG value for self-dimer, −0.9 kcal/mol; and minimum ΔG value for heterodimer, −0.9 kcal/mol. We have successfully synthesized, optimized, and amplified 131 novel SSR markers in coconut using ‘Catigan Green Dwarf’ (CATD), ‘Laguna Tall’ (LAGT), ‘West African Tall’ (WAT), and SYNVAR (LAGT × WAT) genotypes. Of the 131 SSR markers, 113 were polymorphic among the analyzed coconut genotypes. ConclusionThe development of novel SSR markers for coconut will serve as a valuable resource for mapping of quantitative trait loci (QTLs), assessment of genetic diversity and population structure, hybridity testing, and other marker-assisted plant breeding applications.

Genome-Wide Development of Polymorphic Microsatellite Markers and Association Analysis of Major Agronomic Traits in Core Germplasm Resources of Tartary Buckwheat.

Tartary buckwheat (TB; Fagopyrum tataricum Gaertn.) is an important multigrain crop and medicinal plant, but functional genomics and molecular breeding research in this species have been lacking for quite some time. Here, genome-wide screening was performed to develop simple sequence repeat (SSR) markers associated with six major agronomic traits and the rutin contents of 97 core germplasm resources. A total of 40,901 SSR loci were identified; they were uniformly distributed throughout the TB genome, with a mean distance of 11 kb between loci. Based on these loci, 8,089 pairs of SSR primers were designed, and 101 primer pairs for polymorphic SSR loci were used to genotype the 97 core germplasm resources. The polymorphic SSR loci showed high genetic variation in these core germplasm resources, with an average polymorphic information content (PIC) value of 0.48. In addition, multiple SSR markers, such as SXAU8002 [100-grain weight (HGW)] and SXAU8006 [stem diameter (SD)], were found to be associated with agronomic traits in the two environments. Finally, based on gene functional annotation and homology analysis, a candidate gene, FtPinG0007685500, that may affect the node number and SD of the main stem by participating in lignin synthesis was identified. This study reports the mining of genome-wide SSR loci and the development of markers in TB, which can be used for molecular characterization of the germplasm in its gene pool. In addition, the detected markers and candidate genes could be used for marker-assisted breeding and functional gene cloning in TB.

Association Mapping for Yield Attributing Traits and Yellow Mosaic Disease Resistance in Mung Bean [Vigna radiata (L.) Wilczek

Mung bean [Vigna radiata (L.) Wilczek] is an important short-duration grain legume widely known for its nutritional, soil ameliorative, and cropping system intensification properties. This study aims at evaluating genetic diversity among mung bean genotypes and detecting genomic regions associated with various yield attributing traits and yellow mosaic disease (YMD) resistance by association mapping. A panel of 80 cultivars and advanced breeding lines was evaluated for 10 yield-related and YMD resistance traits during kharif (monsoon) and summer seasons of 2018–2019 and 2019–2020. A total of 164 genome-wide simple sequence repeat (SSR) markers were initially screened, out of which 89 were found polymorphic which generated 317 polymorphic alleles with an average of 3.56 alleles per SSR locus. The number of alleles at each locus varied from 2 to 7. The population genetic structure analysis grouped different genotypes in three major clusters and three genetically distinct subpopulations (SPs) (i.e., SP-1, SP-2, and SP-3) with one admixture subpopulation (SP-4). Both cluster and population genetic structure analysis categorized the advanced mung bean genotypes in a single group/SP and the released varieties in other groups/SPs, suggesting that the studied genotypes may have common ancestral history at some level. The population genetic structure was also in agreement with the genetic diversity analysis. The estimate of the average degree of linkage disequilibrium (LD) present at the genome level in 80 mung bean genotypes unveiled significant LD blocks. Over the four seasons, 10 marker-trait associations were observed significant for YMD and four seed yield (SY)-related traits viz., days to flowering, days to maturity, plant height, and number of pods per plant using the mixed linear model (MLM) method. These associations may be useful for marker-assisted mung bean yield improvement programs and YMD resistance.

Genome wide simple sequence repeats development and their application in genetic diversity analysis in wax gourd (Benincasa hispida)

AbstractWax gourd is one of the most important winter vegetables of the Cucurbitaceae family. There are only limited markers available for this crop and the draft genome of wax gourd provides a powerful tool for simple sequence repeats (SSR) marker development. In this study, we developed genome‐wide SSR markers from wax gourd genome and characterized their distribution and frequency of different motifs and repeats. A total of 52,431 microsatellites from wax gourd genome were identified, of which 39,319 SSR markers were developed. The 1152 non‐wax gourd SSR markers were selected from cucumber, melon, watermelon and pumpkin to test their transferability in wax gourd. The 580 SSR markers could be transferable in wax gourd, and 42 of them were detected with polymorphic in 11 tested accessions of wax gourd. In addition, 11 good polymorphic transferrable SSR markers and 21 SSR markers of wax gourd were selected to investigate the genetic diversity and population structure of 129 wax gourd accessions. One hundred twelve alleles were detected by these 32 SSR markers. The result of population structure showed that the 129 wax gourd accessions were divided into two main populations, and the genetic diversity analysis separated them into two clusters. The large number of wax gourd SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker‐assisted selection (MAS) in wax gourd.

Development and application of a duplex simple sequence repeat panel for outcrossing fertility evaluation in red clover (Trifolium pratense) under open-pollination conditions†

Context Red clover (Trifolium pratense L.) is an important legume forage in temperate agricultural zones. Evaluation of self- and cross-pollination fertility is important for setting up an effective breeding-program scale. However, the outcrossing rate of red clover under open-pollination conditions is not certain. Development of a reliable and time-saving marker system is needed to quantify and characterise outcrossing rates. Aim We aimed to develop a duplex PCR-based protocol based on a genome-wide simple sequence repeat (SSR) screen, and to determine the outcrossing rate of red clover under open-pollination environments. Methods We screened 209 SSR markers with pooled DNA samples of 60 plants from 20 red clover accessions, and selected 185 SSR markers that produced clear scorable bands for testing with 24 individual DNA samples to determine polymorphism. We selected 70 primer pairs, and then assembled a core set of 24 loci into 12 sets of duplex markers, which were used for outcrossing behaviour analysis of 60 maternal parents and their respective 22 half-sib progenies. Key results Mean polymorphic information content (PIC) for the 70 markers was 0.490 (range 0.117–0.878). Minimum, mean and maximum PIC values for the 24 markers constituting the 12 duplexes were 0.226, 0.594 and 0.781, respectively. The outcrossing rate was identified as 99.4% for red clover in a natural environment. Conclusion We successfully developed a duplex SSR-based PCR protocol consisting of 24 markers. This SSR system was applied to determine the outcrossing rate of red clover in a natural environment.

Genome-wide development and utilization of Simple Sequence Repeats in Chinese cabbage (&lt;i&gt;Brassica rapa&lt;/i&gt; L. ssp. &lt;i&gt;pekinensis&lt;/i&gt;)

With the complementation of whole-genome sequencing of Chinese cabbage, it is necessary to develop genome-wide Simple Sequence Repeat (SSR) markers and analyse their characteristics, which will bring a revolution in the molecular marker-assisted breeding of Chinese cabbage. In this study distribution and characteristics of SSR loci in the genome of Chinese cabbage 'Chiifu' was analyzed. Finally, a total of 173,892 SSR markers that occurred in 10 chromosomes were identified, and Perfect Repeat Motifs (P type) which is a main form of SSR loci with a mean distance of 1.89 kb/SSR was identified. Among them, mono-, di-, and tri-nucleotide repeat motifs were equally distributed across chromosomes compared with tetra-, penta-, and hexa-nucleotide repeat motifs. The largest proportion of SSR loci consisted of mono- and dinucleotide repeat motifs, which accounted for 93.04% of the total SSR loci. A/T and AT/TA were the most abundant motifs, accounting for 97.61% and 64.05% of the mono- and dinucleotide repeats, respectively. Furthermore, 200 SSR markers were used for the amplification of 12 Chinese cabbage germplasm, in which 30 were highly polymorphic. These SSR markers were further used for the association analyses of flowering time of 190 Chinese cabbage accessions. The results revealed that 9 and 10 alleles were associated with bolting and flowering time, respectivley. The results show that development of SSR markers is feasible and useful in marker assisted selection of Chinese cabbage.

Genome-wide simple sequence repeat markers in potato: abundance, distribution, composition, and polymorphism.

Simple sequence repeats (SSRs) are important sources of genetic diversity and are widely used as markers in genetics and molecular breeding. In this study, we examined four potato genomes of DM1-3 516 R44 (DM) from Solanum phureja, RH89039-16 (RH) from Solanum tuberosum, M6 from Solanum chacoense and Solanum commersonii to determine SSR abundance and distribution and develop a larger list of polymorphic markers for a potentially wide range of uses for the potato community. A total of 1,734,619 SSRs were identified across the four genomes with an average of 433,655 SSRs per genome and 2.31kb per SSR. The most abundant repeat units for mono-, di-, tri-, and tetra-nucleotide SSRs were (A/T)n, (AT/AT)n, (AAT/ATT)n, and (ATAT/ATAT)n, respectively. The SSRs were most abundant (78.79%) in intergenic regions and least abundant (3.68%) in untranslated regions. On average, 168,069 SSRs with unique flanking sequences were identified in the four genomes. Further, we identified 16,245 polymorphic SSR markers among the four genomes. Experimental validation confirmed 99.69% of tested markers could generate target bands. The high-density potato SSR markers developed in this study will undoubtedly facilitate the application of SSR markers for genetic research and marker-pyramiding in potato breeding.

Development and preliminary application of novel genomewide SSR markers for genetic diversity analysis of an economically important bio-control agent Platygaster robiniae (Hymenoptera: Platygastridae)

Platygaster robiniae Buhl and Duso (Hymenoptera: Platygastridae) is an egg-larvae parasitoid of the black locust gall midge (Obolodiplosis robiniae) (Haldeman) (Diptera: Cecidomyiidae) which is a serious invasive pest in China, where it attacks an important hardwood species, the black locust tree, Robini pseudoacacia L. (Fabales: Fabaceae). Despite the use of P. robiniae as an effective biocontrol agent, the absence of sequence data and other molecular markers have limited its genetic applications for pest management in forests. Simple-sequence repeats (SSRs) are valuable molecular markers for population genetic structure studies. In the present study, we identified 14,123 SSRs, of which 7799 SSR primer pairs were successfully designed. Subsequently, 240 SSR were chosen and tested with 48 P. robiniae accessions from two geographically separated populations in north and south China. Of these, 34 were polymorphic, with an average of three alleles (Na) and four genotypes (NG) each. The average values of observed heterozygosity (Ho) was 0.3514, expected heterozygosity (He) 0.4167, Shannon's information index (I) 0.7143, and polymorphism information content (PIC) 0.3558, respectively. Neighbour joining analysis (bootstrap 1000) revealed that Chengdu (CD) and Dangdong (DD) popluations clustered into two main divisions, and some individuals from two popluations clustered together as the third devision, which indicated the gene flow and genetic differentiation were present between two populations. Our finding indicates that these SSR markers will be useful for further studies on the genotype identification and genetic mapping of the genus Platygaster.