Genomic Simple Sequence Repeat Markers Research Articles

Development of Genomic Simple Sequence Repeat Markers for Evaluating Resources of Armillaira ostoyae and Their Transferability to Armillaira gallica

In this study, we aimed to develop simple sequence repeat (SSR) markers for evaluating resources in Armillaria ostoyae and examine their transferability to Armillaria gallica, related species. SSR markers were developed using the released A. ostoyae whole-genome sequence (GenBank assembly accession: GCA_900157425.1). The SSR regions were analyzed using the MISA (MIcroSAtellite identification tool) program. A total of 2319 SSR loci consisting of 922 (39.76%) mononucleotide, 763 (32.90%) trinucleotide, and 517 (22.29%) dinucleotide motifs were identified. Marker design involved an arbitrary choice of 150 SSR loci, considering motif abundance. A total of 22 strains of A. ostoyae were analyzed using the developed markers, and 105 markers were successfully amplified. The mean values of major allele frequency, number of alleles, expected heterozygosity, observed heterozygosity, and polymorphism information content (PIC) values were approximately 5.89, 5.4, 0.541, 0.255, and 0.504, respectively. A. gallica was analyzed, and 52 markers (49.5%) were successfully amplified to evaluate the transferability of the developed SSR markers. When these markers were used, the mean values of major allele frequency, number of alleles, expected heterozygosity, observed heterozygosity, and PIC were calculated to be approximately 0.615, 4.3, 0.517, 0.133, and 0.502, respectively. In conclusion, SSR markers were developed using the genome of A. ostoyae, and some of these markers exhibited transferability to A. gallica. These results can be used for resource evaluation of A. ostoyae and A. gallica.

Agarose-Resolvable SSR Markers Based on ddRADSeq in Chickpea

Exploitation of genetic diversity is essential for sustainable crop production. Molecular markers have potential to reach these goals in more rapid and efficient manner. Simple sequence repeats (SSRs) are one of the most popular molecular markers in breeding studies due to their co-dominant inheritance, abundance in the genome, multi-allelic and reproducible nature. Here, we developed genomic SSR markers from chickpea with the use of ddRADSeq data. 1,396 SSR regions with an average of 530 SSR/Mb in the whole genome were successfully identified. Considering different types of repeats, dinucleotides were the most frequent type accounting for 62.03% of the total SSR regions identified, followed by trinucleotides (25.50%) and tetranucleotides (4.58%). The AT/TA motif was greatly characterized among dinucleotide repeats, and it was also the most common type in the chickpea genome accounting for 36.5% of the total SSR regions identified, followed by AG/GA (139) and TC/CT (135) among dinucleotide motifs. Considering their genomic distribution and simple visualization on agarose gels, we examined SSR regions of 10 bp and longer for identification of SSR markers. A total of 10 SSR markers were successfully designed and resulted in successful polymorphic bands among chickpea genotypes. Consequently, the results show that ddRADSeq is effective for marker development and these markers might be valuable for biodiversity studies, marker-assisted selection (MAS) and linkage map construction in chickpea.

Cross species transferability of G-SSR and EST-SSR markers to Neltuma affinis Spreng.

Aim of study: To examine the transferability of G-SSR (genomic simple sequence repeats) and EST-SSR (expressed sequence tag simple sequence repeats) markers developed for several Neltuma species to N. affinis, a species with no genomic data. Area of study: West-Center of Entre Ríos province, Argentina. The set of molecular markers here proposed can be used to analyze samples from the entire species’ distribution range. Material and methods: Twenty-five genomic G-SSRs and eleven EST-SSRs from multiple species were amplified in thirty N. affinis genotypes. Polymorphism, discrimination power and possible deviations from Hardy-Weinberg equilibrium were assessed. Main results: Seventeen highly polymorphic G-SSRs were successfully transferred to N. affinis, with a PIC (polymorphic information content) average value of 0.811 and a He (expected heterozygosity) average value of 0.694; thirteen were validated, showing very low frequencies of null alleles and no linkage disequilibrium. Additionally, seven polymorphic EST-SSRs were transferred. As expected, PIC and He average values were low. Six out of seven markers were validated, and very low frequencies of null alleles and no linkage disequilibrium were observed. Research highlights: This work provides information on the levels of microsatellites’ cross transferability to N. affinis, and its polymorphism degree. Two sets of polymorphic SSRs (genomic and expressed) to study the genetic status of the species are proposed.

Genetic diversity and structure of a core collection of Huangqi (Astragalus ssp.) developed using genomic simple sequence repeat markers

Genetic diversity and structure of a core collection of Huangqi (Astragalus ssp.) developed using genomic simple sequence repeat markers

Development and characterisation of SSR markers in the potato rot nematode Ditylenchus destructor

Summary The potato rot nematode, Ditylenchus destructor, causes serious disease limiting the production of many crops. This disease usually decreases sweet potato yield by 20-50%, and in heavily infested fields the crop may be completely lost. Although the nematode has economic importance in China, its transmission route and genetic diversity are unknown. In this study, a collection of 1761 contigs of the D. destructor genome was mined for simple sequence repeat (SSR) markers, which resulted in the identification of 9745 SSRs. A total of 150 pairs of SSR primers were further developed and used for validation of the amplification rate and assessment of the polymorphism. Nine SSR markers were finally identified and analysed using 96 individual specimens of D. destructor sampled from four provinces in China. These loci were found to be moderately polymorphic with 2-8 alleles per locus. The observed and expected heterozygosity across the four populations ranged from 0.000 to 0.833 and from 0.000 to 0.666, respectively. This is the first report of the development and characterisation of genomic SSR markers in D. destructor. Our study demonstrated the obvious gene differentiation among different populations of D. destructor in China. This suggests that D. destructor in China may have been introduced from multiple origins. Much more work is needed on this species to identify patterns of spread, and the microsatellite loci we develop here should be useful in many regions for modelling range expansion, studying the evolution of resistance, and increasing the effectiveness of pest management strategies.

Cross-species transferability of genomic SSR markers and genetic diversity among Asparagus racemosus Willd. Accessions

Asparagus racemosus Willd. is an important multipurpose plant, recognized as queen of herbs due to its important medicinal properties. The objective of this study was to contribute to the understanding of A. racemosus genetic diversity by genotyping 20 accessions from ten different agro-ecological zones of Kerala state, India using simple sequence repeat (SSR) markers. A set of 22 SSR markers previously developed for A. officinalis were evaluated for polymorphism in A. racemosus accessions and 18 primer pairs were useful and revealed 46 alleles. Transferability from A. officinalis to A. racemosus varied from 60 to 100% and 15 primers were successfully amplified in all the accessions. Thus, the transferability of SSR primers across the species increased the value of such markers as information about Expressed Sequence Tag (EST) derived markers as well as genomic SSR markers for A. racemosus are not currently available. Comprehensive and systematic surveys on A. racemosus diversity revealed 82.81% polymorphism among accessions. The polymorphic information content values of the transferred SSR markers were between 0.05 and 0.48. A set of six unique alleles detected in five accessions may be useful in genotype identification procedures. Cluster analysis placed the accessions into three main clusters. SSRs derived from garden asparagus were a good resource for genetic studies in A. racemosus.

Genome size, ploidy levels, and development of novel SSR primer to evaluate genetic diversity of Corylopsis Siebold & Zucc. germplasm collections

Corylopsis Seibold & Zucc. is a genus of flowering shrubs with few cultivars and little information on genome size, ploidy levels, and genetic relationships among taxa. The United States National Arboretum and collaborating gardens in North America maintain a diverse germplasm collection of Corylopsis including cultivated and wild-collected taxa. The objectives of this study were to determine genome size, ploidy, and genetic diversity in Corylopsis. Flow cytometry revealed a ploidy series of diploid, tetraploid, pentaploid, and hexaploid taxa in Corylopsis with 2C genome sizes ranging from 1.9 pg to 5.7 pg. Representative taxa were selected for root tip cytology, revealing diploid (C. platypetala Rehder & E.H. Wilson), tetraploid (C. gotoana Makino), pentaploid (unnamed hybrid selection), and hexaploid (C. willmottiae Rehder & E.H. Wilson) taxa. Seventeen genomic simple sequence repeat markers were developed and used to determine the genetic diversity of Corylopsis. These markers had an average of 7.4 alleles per locus with a range of three to 17 alleles. A dendrogram was generated by unweighted pair group method with arithmetic mean (UPGMA) cluster analysis using the Jaccard similarity coefficient to visualize genetic relationships and ploidy series within taxa. Information gained from this study will further breeding efforts and aid management of germplasm collections of Corylopsis.

Quantitative Trait Loci Analysis Based on High-Density Mapping of Single-Nucleotide Polymorphisms by Genotyping-by-Sequencing Against Pine Wilt Disease in Japanese Black Pine (Pinus thunbergii).

Identifying genes/loci for resistance to pine wilt disease (PWD) caused by the pine wood nematode (PWN) is beneficial for improving resistance breeding in Pinus thunbergii, but to date, genetic information using molecular markers has been limited. Here, we constructed a high-density linkage map using genotyping-by-sequencing (GBS) and conducted quantitative trait loci (QTL) analysis for PWD resistance for the self-pollinated progeny of “Namikata 73,” which is the most resistant variety among resistant varieties of P. thunbergii, following inoculation tests with PWN. An S1 mapping population consisting of the 116 progenies derived from self-pollination of the resistant variety, “Namikata 73” (resistance rank 5 to PWN), was inoculated with PWN isolate Ka-4 and evaluated for disease symptoms. To construct a high-density linkage map, we used single-nucleotide polymorphisms (SNPs) identified by GBS based on next-generation sequencing technology and some anchor DNA markers, expressed sequence tag (EST)-derived SNP markers and EST-derived simple sequence repeat (SSR) markers, and genomic SSR markers. The linkage map had 13 linkage groups (LGs) consisting of 2,365 markers including 2,243 GBS-SNP markers over a total map distance of 1968.4 centimorgans (cM). Results from QTL analysis using phenotype data and the linkage map indicated that PWD resistance is controlled by a single locus located on LG-3, as identified in a previous study. This locus showed overdominant genetic action in the present study. With the confirmation of PWD1 in two different mapping populations (present study and a previous study), the locus associated with this region is thought to be a good target for marker-assisted selection in P. thunbergii breeding programs in order to obtain high levels of resistance to PWD caused by PWN.

Genome-Wide Analysis of Simple Sequence Repeats in Cabbage (Brassica oleracea L.).

Cabbage (Brassica oleracea L. var. capitata) accounts for a critical vegetable crop belonging to Brassicaceae family, and it has been extensively planted worldwide. Simple sequence repeats (SSRs), the markers with high polymorphism and co-dominance degrees, offer a crucial genetic research resource. The current work identified totally 64,546 perfect and 93,724 imperfect SSR motifs in the genome of the cabbage ‘TO1000.’ Then, we divided SSRs based on the respective overall length and repeat number into different linkage groups. Later, we characterized cabbage genomes from the perspectives of motif length, motif-type classified and SSR level, and compared them across cruciferous genomes. Furthermore, a large set of 64,546 primer pairs were successfully identified, which generated altogether 1,113 SSR primers, including 916 (82.3%) exhibiting repeated and stable amplification. In addition, there were 32 informative SSR markers screened, which might decide 32 cabbage genotypes for their genetic diversity, with level of polymorphism information of 0.14–0.88. Cultivars were efficiently identified by the new strategy designating manual diagram for identifying cultivars. Lastly, 32 cabbage accessions were clearly separately by five Bol-SSR markers. Besides, we verified whether such SSRs were available and transferable in 10 Brassicaceae relatives. Based on the above findings, those genomic SSR markers identified in the present work may facilitate cabbage research, which lay a certain foundation for further gene tagging and genetic linkage analyses, like marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Genome survey sequencing of common vetch (Vicia sativa L.) and genetic diversity analysis of Chinese germplasm with genomic SSR markers

BackgroundCommon vetch (Vicia sativa L.) is an annual legume with excellent suitability in cold and dry regions. Despite its great applied potential, the genomic information regarding common vetch currently remains unavailable.Methods and resultsIn the present study, the whole genome survey of common vetch was performed using the next-generation sequencing (NGS). A total of 79.84 Gbp high quality sequence data were obtained and assembled into 3,754,145 scaffolds with an N50 length of 3556 bp. According to the K-mer analyses, the genome size, heterozygosity rate and GC content of common vetch genome were estimated to be 1568 Mbp, 0.4345 and 35%, respectively. In addition, a total of 76,810 putative simple sequence repeats (SSRs) were identified. Among them, dinucleotide was the most abundant SSR type (44.94%), followed by Tri- (35.82%), Tetra- (13.22%), Penta- (4.47%) and Hexanucleotide (1.54%). Furthermore, a total of 58,175 SSR primer pairs were designed and ten of them were validated in Chinese common vetch. Further analysis showed that Chinese common vetch harbored high genetic diversity and could be clustered into two main subgroups.ConclusionThis is the first report about the genome features of common vetch, and the information will help to design whole genome sequencing strategies. The newly identified SSRs in this study provide basic molecular markers for germplasm characterization, genetic diversity and QTL mapping studies for common vetch.

Novel Expressed Sequence Tag-Derived and Other Genomic Simple Sequence Repeat Markers Revealed Genetic Diversity in Ethiopian Finger Millet Landrace Populations and Cultivars.

Finger millet (Eleusine coracana (L.) Geartn.) is a self-pollinating amphidiploid crop cultivated with minimal input for food and feed, as well as a source of income for small-scale farmers. To efficiently assess its genetic diversity for conservation and use in breeding programs, polymorphic DNA markers that represent its complex tetraploid genome have to be developed and used. In this study, 13 new expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on publicly available finger millet ESTs. Using 10 polymorphic SSR markers (3 genomic and 7 novel EST-derived), the genetic diversity of 55 landrace accessions and 5 cultivars of finger millet representing its major growing areas in Ethiopia was assessed. In total, 26 alleles were detected across the 10 loci, and the average observed number of alleles per locus was 5.6. The polymorphic information content (PIC) of the loci ranged from 0.045 (Elco-48) to 0.71 (UGEP-66). The level of genetic diversity did not differ much between the accessions with the mean gene diversity estimates ranging only from 0.44 (accession 216054) to 0.68 (accession 237443). Similarly, a narrow range of variation was recorded at the level of regional states ranging from 0.54 (Oromia) to 0.59 (Amhara and Tigray). Interestingly, the average gene diversity of the landrace accessions (0.57) was similar to that of the cultivars (0.58). The analysis of molecular variance (AMOVA) revealed significant genetic variation both within and among accessions. The variation among the accessions accounted for 18.8% of the total variation (FST = 0.19; P < 0.001). Similarly, significant genetic variation was obtained among the geographic regions, accounting for 6.9% of the total variation (P < 0.001). The results of the cluster, principal coordinate, and population structure analyses suggest a poor correlation between the genetic makeups of finger millet landrace populations and their geographic regions of origin, which in turn suggests strong gene flow between populations within and across geographic regions. This study contributed novel EST-SSR markers for their various applications, and those that were monomorphic should be tested in more diverse finger millet genetic resources.

Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata)

Mungbean is an important pulse crop and it is mainly cultivated in Asia for human consumption. Its small genome and diploid nature make it a well-suited model organism among legume crops. Thus, cost-effective, reliable and highly polymorphic molecular markers distributing the whole genome are needed for diversity, mapping and functional genomics studies in this model species. The whole-genome sequence of mungbean was obtained and used as a source of identification of simple sequence repeats (SSR). The sequence reads were aligned and SSRs detection was performed using the Phobos plugin tandem repeat finder in the Geneious software program. A total of 12 mungbean genotypes were selected to validate the newly developed SSR markers. In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats were identified from the mungbean genome. The tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). We designed 1330 SSR primers based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats). Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes. The newly developed genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean breeding programs.

Allelic Diversity, Structural Analysis, and Genome-Wide Association Study (GWAS) for Yield and Related Traits Using Unexplored Common Bean (Phaseolus vulgaris L.) Germplasm From Western Himalayas.

The north-western Indian Himalayas possesses vast diversity in common bean germplasm due to several years of natural adaptation and farmer’s selection. Systematic efforts have been made for the first time for the characterization and use of this huge diversity for the identification of genes/quantitative trait loci (QTLs) for yield and yield-contributing traits in common bean in India. A core set of 96 diverse common bean genotypes was characterized using 91 genome-wide genomic and genic simple sequence repeat (SSR) markers. The study of genetic diversity led to the identification of 691 alleles ranging from 2 to 21 with an average of 7.59 alleles/locus. The gene diversity (expected heterozygosity, He) varied from 0.31 to 0.93 with an average of 0.73. As expected, the genic SSR markers detected less allelic diversity than the random genomic SSR markers. The traditional clustering and Bayesian clustering (structural analysis) analyses led to a clear cut separation of a core set of 96 genotypes into two distinct groups based on their gene pools (Mesoamerican and Andean genotypes). Genome-wide association mapping for pods/plant, seeds/pod, seed weight, and yield/plant led to the identification of 39 significant marker–trait associations (MTAs) including 15 major, 15 stable, and 13 both major and stable MTAs. Out of 39 MTAs detected, 29 were new MTAs reported for the first time, whereas the remaining 10 MTAs were already identified in earlier studies and therefore declared as validation of earlier results. A set of seven markers was such, which were found to be associated with multiple (two to four) different traits. The important MTAs will be used for common bean molecular breeding programs worldwide for enhancing common bean yield.

Genetic Diversity and Population Structure of Cannabis Based on the Genome-Wide Development of Simple Sequence Repeat Markers.

Cannabis has been used as a source of nutrition, medicine, and fiber. However, lack of genomic simple sequence repeat (SSR) markers had limited the genetic research on Cannabis species. In the present study, 92,409 motifs were identified, and 63,707 complementary SSR primer pairs were developed. The most abundant SSR motifs had six repeat units (36.60%). The most abundant type of motif was dinucleotides (70.90%), followed by trinucleotides, tetranucleotides, and pentanucleotides. We randomly selected 80 pairs of genomic SSR markers, of which 69 (86.25%) were amplified successfully; 59 (73.75%) of these were polymorphic. Genetic diversity and population structure were estimated using the 59 (72 loci) validated polymorphic SSRs and three phenotypic markers. Three hundred ten alleles were identified, and the major allele frequency ranged from 0.26 to 0.85 (average: 0.56), Nei’s genetic diversity ranged from 0.28 to 0.82 (average: 0.56), and the expected heterozygosity ranged from 0.28 to 0.81 (average: 0.56). The polymorphism information content ranged from 0.25 to 0.79 (average: 0.50), the observed number of alleles ranged from 2 to 8 (average: 4.13), and the effective number of alleles ranged from 0.28 to 0.81 (average: 0.5). The Cannabis population did not show mutation-drift equilibrium following analysis via the infinite allele model. A cluster analysis was performed using the unweighted pair group method using arithmetic means based on genetic distances. Population structure analysis was used to divide the germplasms into two subgroups. These results provide guidance for the molecular breeding and further investigation of Cannabis.

Population genetic structure and marker-trait associations in East and West African Striga hermonthica with varying phenotypic response to Fusarium oxysporum f.sp. strigae isolates Foxy-2 and FK3.

To examine the genetic basis for the variable susceptibility of Striga hermonthica from differing zones of sub-Saharan Africa to Fusarium oxysporum f.sp. strigae (Fos) isolates Foxy-2 and FK3, 10 S.hermonthica populations from Eastern and Western Africa were phenotyped for their susceptibility response to Foxy-2 and FK3, and then genotyped with 22 simple sequence repeat (SSR) markers. There is low genetic differentiation between East African and West African S.hermonthica populations (i.e. the proportion of the total genetic variance contained in the subpopulation relative to the total genetic variance, FST =0.012, P<0.05), but intermediate genetic differentiation (FST =0.143, P<0.01) underlies the S.hermonthica groups that are differentiated by their phenotypic responses to Fos isolates. An expressed sequence tag SSR (EST-SSR) marker Y53 (P<0.01) and a genomic SSR marker E1009 (P<0.05) were associated with the S.hermonthica class susceptible to Foxy-2 and FK3 (groupA). A divergent S.hermonthica class, consisting of groups with intermediate susceptibility to Foxy-2 (groupB) and susceptibility to either FK3 (groupC) or Foxy-2 (groupD), showed no marker-trait association, instead demonstrated linkage disequilibrium decay. Owing to point substitutions and insertion-deletion mutations, the unique, protein-coding nucleotide sequence at the E1009 locus in groupA was partly dissimilar to groupB, but was totally distinct from groupsC and D. These findings implied that the inconsistent effectiveness of a Fos isolate is better explained by genomic variation in S.hermonthica, rather than by S.hermonthica sampling zones.

Identification of microsatellite loci, gene ontology and functional gene annotations in Indian salmon (Eleutheronema tetradactylum) through next-generation sequencing technology using illumina platform

Abstract Whole genome sequencing was performed on three samples of four finger threadfin Eleutheronema tetradactylum (KET25, KET29 and KET30) using illumina NextSeq500 platform using 2 × 150 bp chemistry. 8,390,317, 7,085,775 and 8,461,589 high quality reads were obtained after trimming low quality reads and adapter sequence. These high quality reads obtained were used for de novo assembly and obtained a number of scaffolds. From these scaffolds of vast sequenced data, we were able to identify 60246, 46107 and 60907 Simple Sequence Repeats (SSR) markers in KET25, KET29 and KET30 respectively, which will be useful in population genetic analysis and other diversity studies in Indian salmon. The gene prediction on assembled scaffolds predicted 31,943 genes for KET25; 26,487 genes for KET29 and 31,654 genes for KET30 with average gene size of 458bp, 424bp and 459bp respectively. A total of 30,209, 25,107 and 29,943 genes were annotated against the NCBI Nr database for the samples respectively. E. tetradactylum is a commercially important fish species for many countries. This is the first report on the identification of genomic SSR markers in E. tetradactylum using NGS technology. This study provides an insight of baseline knowledge of the genome sequence of Indian salmon for future studies.

Quantitative trait loci analysis of phenolic acids contents in Salvia miltiorrhiza based on genomic simple sequence repeat markers

Phenolic acids are one of the main active components in Salvia miltiorrhiza root, which have been proved to have extensive pharmacological activities. In this study, quantitative trait loci (QTL) for phenolic acids contents were identified by a new genetic map constructed based on genomic simple sequence repeat (SSR) markers. The results demonstrated that the map comprised eight linkage groups which were in accord with the haploid chromosome number of S. miltiorrhiza (2n = 2x = 16). The map spanned 397.5 cM with average inter-marker distance as 3.6 cM. A total of six QTL were identified through interval mapping, including three salvianolic acid B (SAB) content-related QTL on linkage group (LG) 7, two lithospermic acid (LA) content markers on LG2 and LG4, as well as one rosmarinic acid (RA) content-linked QTL on LG7. These QTL could be used in marker-assisted breeding for the phenolic acids contents of S. miltiorrhiza.

Development of a Core Set of SSR Markers for Cultivar Identification and Seed Purity Tests in Oriental Melon (Cucumis melo L. var. makuwa)

Oriental melon (Cucumis melo L. var. makuwa), also called ‘Korean melon’ or ‘Chamoe’, is widely grown in East Asia, including Korea, Japan, and China. We used 521 simple sequence repeats (SSRs) to develop a core set of SSR markers as an effective tool for cultivar identification and seed purity tests in oriental melon. The polymorphism test using a subset of seven cultivars identified 20 expressed sequence tag SSR (EST-SSR) and 23 genomic SSR markers that showed high levels of polymorphism and simple segregation patterns. These SSR markers were used for DNA fingerprinting in additional 46 commercial cultivars. The average number of alleles per marker was 2.95 and 4.61 for the EST-SSR and genomic SSR markers in the 53 cultivars, respectively. For polymorphic information content (PIC), we found 0.411 to 0.816 with an average of 0.536 for the EST-SSR markers and 0.453 to 0.862 with an average of 0.568 for the genomic SSR markers. The unweighted pair group mean algorithm (UPGMA) dendrograms with the three sets of SSR markers (20 EST-SSR, 23 genomic SSR, and all 43 SSR markers) revealed that the 53 cultivars were separated into 4 to 5 major clusters. Furthermore, the set of all SSR markers was more effective at identifying the 53 cultivars relative to the other two marker sets. Therefore, the 43 SSR markers as a core set will be a valuable tool for several applications, including cultivar identification and seed purity tests in oriental melon.

Taraxacum kok-saghyz (rubber dandelion) genomic microsatellite loci reveal modest genetic diversity and cross-amplify broadly to related species

Taraxacum kok-saghyz (TKS) carries great potential as alternative natural rubber source. To better inform future breeding efforts with TKS and gain a deeper understanding of its genetic diversity, we utilized de novo sequencing to generate novel genomic simple sequence repeats markers (gSSRs). We utilized 25 gSSRs on a collection of genomic DNA (gDNA) samples from germplasm bank, and two gDNA samples from historical herbarium specimens. PCR coupled with capillary electrophoresis and an array of population genetics tools were employed to analyze the dataset of our study as well as a dataset of the recently published genic SSRs (eSSRs) generated on the same germplasm. Our results using both gSSRs and eSSRs revealed that TKS has low- to- moderate genetic diversity with most of it partitioned to the individuals and individuals within populations, whereas the species lacked population structure. Nineteen of the 25 gSSR markers cross-amplified to other Taraxacum spp. collected from Southeastern United States and identified as T. officinale by ITS sequencing. We used a subset of 14 gSSRs to estimate the genetic diversity of the T. officinale gDNA collection. In contrast to the obligatory outcrossing TKS, T. officinale presented evidence for population structure and clonal reproduction, which agreed with the species biology. We mapped the molecular markers sequences from this study and several others to the well-annotated sunflower genome. Our gSSRs present a functional tool for the biodiversity analyses in Taraxacum, but also in the related genera, as well as in the closely related tribes of the Asteraceae.

Identification and development of microsatellite markers in Hamamelis mollis (Hamamelidaceae).

Premise of the Study Hamamelis mollis (Hamamelidaceae) is a Tertiary relict species endemic to southern China. Polymorphic microsatellite markers were developed to reveal the genetic diversity of this species.Methods and ResultsThe genome of H. mollis was sequenced and de novo assembled into 642,351 contigs. A total of 72,097 paired primers were successfully designed from 80,282 simple sequence repeat (SSR) markers identified in 63,419 contigs. PCR amplification showed that 96 of the 136 synthesized primers could be successfully amplified, and 22 demonstrated polymorphism. The mean number of alleles, levels of observed heterozygosity, and levels of expected heterozygosity were 4.602 ± 0.140, 0.632 ± 0.020, and 0.696 ± 0.010, respectively. The majority of the 96 primer pairs could be amplified in at least one other Hamamelidaceae species, including Distylium myricoides (60), Loropetalum chinense (39), Exbucklandia populnea (24), and E. tonkinensis (24).ConclusionsThese microsatellite loci provide abundant genomic SSR markers to evaluate genetic diversity of this woody ornamental plant.