Expressed Sequence Tag-simple Sequence Repeat Research Articles

EST-SSR markers development and application in an important medicinal plant, Schisandra chinensis (Schisandraceae)

Schisandra chinensis, classed as a climbing woody vine, is representative of the Schisandraceae family regarded as a significant plant in Chinese herbal medicine. However, the application of molecular breeding is constrained by the fact that the number of genetic markers is small for this species. In this study, transcriptome sequencing of S. chinensis was performed using the Illumina HiSeq platform to construct a library of expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 59,786 unigenes were obtained on the basis of 8.57 Gbp clean data. A total of 3460 putative SSR sites were detected with a frequency of 5.79%. The predominant type of repeat markers was dinucleotide (64.54%), followed by trinucleotide (23.87%), hexanucleotide (0.90%), tetranucleotide (0.12%) and pentanucleotide (0.40%). Besides, 50 pairs of EST-SSR primers were randomly selected for verification, among which 14 pairs (28%) showed polymorphism in 42 different types of S. chinensis accessions. The mean values of Na, Ne, PIC, Ho, He and SI were 2.71, 1.7642, 0.4295, 0.6016, 0.3934 and 0.6078, respectively. All of the 42 accessions were successfully identified and formed four major clusters, indicating that the EST-SSR markers are applicable for genetic diversity analysis and identification in S. chinensis. According to the result, the transcriptome data are an effective resource for developing SSR markers. These markers can provide a basis for the identification of S. chinensis accessions, genetic diversity analysis, the conservation and construction of genetic linkage maps as part of selective breeding and the conservation efforts for this valuable plant.

Genetic diversity and structure of Oriental and European beech populations from Iran and Europe

Abstract Genetic variation is a major component of plant development and adaptation, and recent studies have shown that genetic variation among plant species can have important ecological effects. Oriental beech (Fagus orientalis Lipsky) is a dominant tree species in the Hyrcanian forests, where it occupies approximately 18 % of the forested area. In this study, nine expressed sequence tag simple sequence repeat (EST-SSR) markers were used to determine the genetic diversity and structure of Iranian Oriental beech populations growing at different altitudes. We further compared the genetic structures of the Iranian populations to a F. orientalis and a Fagus sylvatica L. population from the hybrid zone of the two taxa in southeast Europe, and to a F. sylvatica population from central Europe. All populations showed a high genetic diversity, which was similar to other F. orientalis and F. sylvatica populations from Europe. The genetic differentiation among Iranian beech populations was very low. In contrast, distinct genetic differentiation was found between the Iranian populations and the analyzed European populations (F. orientalis and F. sylvatica). One EST-SSR was identified to differentiate (GST : 0.503) between F. orientalis and F. sylvatica with very pronounced allele frequency differences between taxa. Specifically, the Fagus sylvatica characteristic allele 189 was almost absent from all Iranian populations and present in low frequencies in F. orientalis populations from other regions. This study helps to extend the knowledge of genetic diversity and genetic structure of Iranian Oriental beech populations. It further gives insights into the genetic differentiation between F. orientalis populations from the center and the edge of the species’ distribution range as well as between Oriental and European beech.

Adaptive evolution of chestnut forests to the impact of ink disease in Spain

AbstractPhytophthora cinnamomi (Pc) is an extremely destructive soil‐borne pathogen of Asiatic origin responsible for “ink disease” in chestnut. This work assesses the adaptive potential to the impact of Pc of four Spanish populations of Castanea sativa undergoing different selection pressures. To explore the evolvability of C. sativa to Pc in the selected populations, parameters obtained from neutral and functional genetic diversity were compared with estimates of quantitative genetic variability. Nine expressed sequence tags‐simple sequence repeat (EST‐SSR) markers were selected and their transferability and polymorphism in 137 C. sativa individuals were evaluated. To test the potential of EST‐SSR markers for early selection of Pc tolerant plant material, the offspring of selected individuals were challenged with Pc. Expressed sequence tags‐simple sequence repeat markers and seedling life expectancy after Pc inoculation revealed significant different responses of C. sativa populations to Pc. The genetic variability observed within populations showed the potential response capacity of Spanish C. sativa populations to undergo fast adaptive evolution. The heritability value obtained for the “life expectancy” variable (h2 = 0.21 ± 0.11) indicated that selection for resistance to Pc is possible. Genetic patterns reflected two evolutionarily meaningful groupings of populations, corresponding to the different selective pressure of the oomycete between sites. The differentiation coefficient obtained through markers classified as under neutral selection (FST = 0.185) was lower than the quantitative genetic differentiation of “life expectancy” between C. sativa populations (QST = 0.682), providing evidence that selection acted spatially in a heterogeneous manner. A first link has been identified in trees between population structure and adaptive responses to pathogen‐induced selection. The study identified one marker under positive selection that could be used in marker assisted selection to predict resistance to Pc in non‐inoculated C. sativa trees.

De novo Assembly and Characterization of Patagonian Toothfish Transcriptome and Develop of EST-SSR Markers for Population Genetics

Patagonian toothfish (Dissostichus eleginoides) is a hefty notothenioid fish and a key species within the marine ecosystem with a high migratory capacity across sub-Antarctic and south American Pacific oceans. Transcriptome characterization and molecular markers associated with micro and macro-evolutionary studies are not available, which in turn limits the gain of knowledge about the genetic basis of this species. Therefore, in the present study, a de novo transcriptome from eight tissue and an embryonic state of Patagonian toothfish was developed, using the Illumina Hiseq 4000 platform. A total of 233,424 superTranscripts were assembled and 37,446 annotated against public databases. Moreover, we identified 71,107 expressed sequence tag-simple sequence repeats (EST-SSRs), with an average number of 0.3 SSRs per superTranscripts and one SSR per 1.12kB. The most abundant SSR type was repeated dinucleotide (53.67%), followed by trinucleotide (13.73%) repeats. From the total of EST-SSRs identified, 34,196 primer pairs were properly designed and a subset of twenty-five immune loci were selected for its evaluation as potential EST-SSR population markers. Of this subset, eleven proved to have good technical features and were evaluated in 64 animals from four Patagonian toothfish populations. A number of 63 alleles were identified, with a mean of 4.9 alleles per locus and a polymorphism information content ranging from 0.224 to 0.591, with a mean of 0.50. Significant (FST, range 0.082-0.117 and G´ST, range 0.069-0.291) genetic differentiation (P<0.05) was determined among the populations analyzed. Therefore, the results presented here represent a relevant genetic resource for biological studies on evolution, conservation, genetic diversity, population structure and genetic management of breeding stocks of this important species.

Pre-quaternary diversification and glacial demographic expansions of Cardiocrinum (Liliaceae) in temperate forest biomes of Sino-Japanese Floristic Region

The Sino-Japanese Floristic Region (SJFR) in East Asia is one of the most diverse temperate floras in the world. However, the relative influence of Neogene palaeogeographical changes and Quaternary climatic fluctuations as causal mechanisms on species diversification remains largely controversial, because most divergence time estimates were inferred from single-locus data and have limited geographic or taxonomic sampling. To evaluate these influences, we use SNP markers from restriction site-associated DNA sequencing (RAD-Seq) loci and expressed sequence tags-simple sequence repeat (EST-SSR) markers to investigate the levels of genetic variation, speciation and demographic history of the temperate-deciduous forest (TDF) endemic Cardiocrinum (Endlicher) Lindley (Liliaceae), a genus comprising three species in China (C. giganteum, C. cathayanum) and Japan (C. cordatum). Phylogenomic and population genomic coalescent-based analyses demonstrated that Late Neogene tectonic/climatic events triggered speciation of Cardiocrinum, and Pleistocene climatic fluctuations had limited influence on its divergence history. Population demographic inference using Approximate Bayesian Computation from EST-SSRs and palaeoclimatic niche models both indicated that all three Cardiocrinum species experienced population expansions during the transition from the LIG to the LGM. We also discussed the implications of these results on the conservation of montane TDF species in the SJFR under ongoing environmental change. Our results improve our understanding of how the constituents of montane TDF across the SJFR responded to previous periods of rapid climate and environmental change in terms of speciation and population demographic processes.

Genetic Diversity and Population Genetic Structure of Cinnamomum camphora in South China Revealed by EST-SSR Markers

Cinnamomum camphora is a valuable broad-leaf tree indigenous to South China and East Asia and has been widely cultivated and utilized by humans since ancient times. However, owing to its overutilization for essential oil extraction, the Transplanting Big Trees into Cities Program, and over deforestation to make furniture, its wild populations have been detrimentally affected and are declining rapidly. In the present study, the genetic diversity and population structure of 180 trees sampled from 41 populations in South China were investigated with 22 expressed sequence tag-simple sequence repeat (EST-SSR) markers. In total, 61 alleles were harbored across 180 individuals, and medium genetic diversity level was inferred from the observed heterozygosity (Ho), expected heterozygosity (He), and Nei’ gene diversity (GD), which were 0.45, 0.44, and 0.44, respectively. Among the 41 wild populations, C. camphora had an average of 44 alleles, 2.02 effective alleles, and He ranging from 0.30 (SC) to 0.61 (HK). Analysis of molecular variance (AMOVA) showed that 17% of the variation among populations and the average pairwise genetic differentiation coefficient (FST) between populations was 0.162, indicating relatively low genetic population differentiations. Structure analysis suggested two groups for the 180 individuals, which was consistent with the principal coordinate analysis (PCoA) and unweighted pair-group method with arithmetic means (UPGMA). Populations grouped to cluster I were nearly all distributed in Jiangxi Province (except population XS in Zhejiang Province), and cluster II mainly comprised populations from other regions, indicating a significant geographical distribution. Moreover, the Mantel test showed that this geographical distance was significantly correlated with genetic distance. The findings of this research will assist in future C. camphora conservation management and breeding programs.

Development and evaluation of EST-SSR markers in Sorbus pohuashanensis (Hance) Hedl. and their application to other Sorbus species

We developed and identified a new set of polymorphic EST-SSR markers in Sorbus pohuashanensis (Hance) Hedl. and generated an optimal sampling strategy. Sorbus pohuashanensis (Hance) Hedl. is indigenous to northern China and prized for its graceful appearance and seasonal variations in foliage color. However, only a few molecular biology studies have been conducted on this species. Therefore, its development and increased utilization are limited. The objective of the present study was to develop expressed sequence tag-simple sequence repeat (EST-SSR) markers based on the transcriptome data of this species. A total of 167 primer pairs were designed and 24 were randomly selected from the 55 initially screened for validation. Their polymorphism was assessed using samples of the Mount Kunyu in Shandong Province (SDKYS) population. Fifteen EST-SSR markers were identified as polymorphic, with a mean number of alleles (Na) and a polymorphism information content (PIC) of 8.2 and 0.6907, respectively. Thirty EST-SSRs (including another 15 EST-SSRs being previously developed) derived from S. pohuashanensis had high transferability (90%) to another 14 Sorbus species, and there were nine EST-SSRs applicable to Sorbus species tested. These Sorbus species were divided into two main clusters by an unweighted pair-group method with the arithmetic mean (UPGMA) clustering tree based on the nine EST-SSRs and was validated by principal coordinate analysis (PCoA). The sampling strategy showed that 25 randomly selected individuals from a population of S. pohuashanensis could represent 95% of its genetic diversity level. In all, these findings would provide a powerful theoretical basis for future studies on the genetic diversity and germplasm resources of S. pohuashanensis and its related species.

Development of EST-SSR markers in Cenchrus ciliaris and their applicability in studying the genetic diversity and cross-species transferability

Most of the grasses of the genus Cenchrus (20-25 species) and Pennisetum (80-140 species) are distributed throughout the tropical and subtropical regions of the world and reproduce both by sexual and apomictic modes. However, the relationships among the Cenchrus-Pennisetum species are not very clear yet. Molecular markers like expressed sequence tag-simple sequence repeats (EST-SSRs) have been reported to be a better choice for resolving the phylogenetic relationships and to estimate the genetic diversity. The present study describes the identification of EST-SSR markers based on the transcriptome data of Cenchrus ciliaris inflorescence and illustrates the genetic diversity and phylogenetic relationships among these species. Of the 378 primer pairs used across 33 accessions of 21 Cenchrus, Pennisetum, and related grass (Bothriochloa, Dichanthium and Panicum) species, 116 EST-SSR markers were found to be polymorphic with an average polymorphism information content (PIC) of 0.49. Fifty-one EST-SSR loci and 520 alleles showed that where the PIC value is >0.5 there the GAG repeat motif was highly polymorphic. Two EST-SSR markers, CcSSR_80 and CcSSR_102, are polymorphic among the Cenchrus species, while they are absent in Pennisetum and the allied species. Five SSR markers (CcSSR_75, CcSSR_85, CcSSR_87, CcSSR_88 and CcSSR_114) showed 100% cross-transferability among the 21 Cenchrus-Pennisetum species. Species-specific alleles could also be detected for seven species of Cenchrus, Pennisetum and Panicum across 10 SSR markers. Assay of polymorphism across these agamic complexes showed that the three SSR markers (CcSSR_26, CcSSR_97 and CcSSR_109) were associated with Cenchrus-Pennisetum complex, and one (CcSSR_47) with Bothriochloa-Dichanthium complex. Markers with high discriminating power, namely CcSSR_4, CcSSR_38, CcSSR_48, CcSSR_66, CcSSR_67 and CcSSR_70, can be used to estimate the allelic sequence divergence across the sexual and apomictic lineages. Genetic diversity analysis using neighbour-joining (NJ) and principal co-ordinate analysis (PCoA) based approaches showed six and five clusters for the 33 accessions, respectively, having congruence in the pattern of clustering. These accessions were grouped according to their mode of reproduction. Cenchrus and Pennisetum species were grouped separately within the same clade, implying monophyletic group within a 'bristle clade'. Thus, this study showed high discrimination power of microsatellite (EST-SSR) markers to resolve the phylogenetic relationships.

Gene Introgression among Closely Related Species in Sympatric Populations: A Case Study of Three Walnut (Juglans) Species

Gene introgression usually results from natural hybridization occurring among closely related species in sympatric populations. In this study, we discussed two rare and frequent gene flow phenomena between three species of Juglans plants and analyzed the possible causes for the difference. We collected 656 individuals from 40 populations of Persian walnut (Juglans regia L.), Chinese walnut (J. cathayensis Dode), and Iron walnut (J. sigillata Dode) that were genotyped at 17 expressed sequence tag simple sequence repeat (EST-SSR) loci to analyze the introgressions between J. regia and J. cathayensis, and J. regia and J. sigillata. Our study compared the spatial patterns of expected heterozygosity (HE), allelic richness (Rs), and private allele richness (PAR) so as to vividly infer the biogeographic history of related species of Juglans in the two regions. The results of the PCoA, UPGMA, and STRUCTURE analyses showed that all J. regia and J. sigillata populations clustered into one group, and the J. cathayensis populations clustered into the other group. The results of the historical gene flow analysis indicated that J. regia and J. sigillata have no genetic barriers, and the directional gene flow is mainly from J. regia to J. sigillata. For the three species of Juglans, all the above results indicated that gene flow was common among the same group of Juglans, and only rare and low-level gene flow appeared in distinct groups. Therefore, our study revealed multiple phenomena of gene flow and introgression among closely related species in sympatric populations, thereby providing a theoretical basis for the genetic evolution of the genus Juglans.

Genetic Diversity and DNA Fingerprints of Three Important Aquatic Vegetables by EST-SSR Markers

Twenty-two sacred lotus (Nelumbo nucifera), 46 taros (Colocasia esculenta) and 10 arrowheads (Sagittaria trifolia) were used as materials and combined with EST-SSR (expressed sequence tag-simple sequence repeats) primers developed by our laboratory. Core primers were screened from a large number of primers that were able to distinguish all materials with a high frequency of polymorphisms. Six pairs, twenty pairs and three pairs of core primers were screened from sacred lotus, taro, and arrowhead, respectively. The SSR fingerprints of these three important aquatic vegetables, producing 17-, 87- and 14-bit binary molecular identity cards, respectively, were separately determined by using the core primers. Since there were few core primers of sacred lotus and arrowhead, 3 and 9 primer pairs with higher polymorphic information content (PIC), respectively, were selected as candidate primers. These core and candidate primers were used to identify the purities of No.36 space lotus, Shandong 8502 taro and Wuhan arrowhead, which were 93.3% (84/90), 98.9% (89/90) and 100.0% (90/90), respectively. The fingerprints, displayed as binary molecular identification cards of three important aquatic vegetables, were obtained, and their purity was successfully determined with EST-SSR labeling technology. Phylogenetic trees were also constructed to analyze the genetic diversity of 22 sacred lotus, 46 taros and 10 arrowheads. This study classifies and identifies germplasm resources and is an important reference to test the authenticity and variety purity of other aquatic vegetables in the future.

Virulence structure and its genetic diversity analyses of Blumeria graminis f. sp. tritici isolates in China

BackgroundBlumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew severely affects yield security wheat production in China. Understanding the virulence structure and genetic variations of this pathogen is important for breeding wheat lines resistant to wheat powdery mildew. However, information related to genes controlling resistance remains elusive. This study analyzes the virulence structure and the genetic diversity of pathogenic Bgt populations isolated from northeastern (Liaoning, Heilongjiang) and northwestern (Gansu) China, two representative wheat producing areas, on 37 wheat cultivars each carrying a known powdery mildew resistance (Pm) gene.ResultsBgt isolates from northeastern China show higher frequencies of virulence genes than populations from Gansu Province. Many of the known Pm genes failed to provide resistance in this study. However, Pm21 provided 100% resistance to all isolates from all three provinces, obtained during two consecutive years, while Pm13 provided 100% resistance in Gansu. Pm13, Pm16, Pm18, and Pm22 also showed partial resistance in northeastern China, while Pm16, Pm18, Pm22, Pm5 + 6 and Pm2 + 6 +? maintained some resistance in Gansu. Genetic diversity among populations in different regions was detected by cluster analyses using expressed sequence tag-simple sequence repeat (EST-SSR). When the genetic similarity coefficient is relatively high, populations from the same regional origin are mostly clustered into one group while populations from different regions exhibit large genetic differences.ConclusionPm21 remains the best choice for breeding programs to maintain resistance to Bgt. Only 58% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and frequency of virulence gene data.

Cross-species transferability of EST-SSR markers derived from the transcriptome of kenaf (Hibiscus cannabinus L.) and their application to genus Hibiscus

The genus Hibiscus comprises approximately 300 diverse species whose genetic relationships have not been extensively clarified. In this study, we evaluated the cross-species transferability of 102 expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from the transcriptome of kenaf H. cannabinus L. and used them to assess the genetic diversity and inter-relationships of 87 accessions and 7 cultivars of 18 Hibiscus species. Among 102 markers, 100 (98%) were polymorphic in these 94 accessions/cultivars. The rates of cross-species transferability of the EST-SSR markers ranged from 83 H. trionum L. to 99% H. ponticus Rupr., with an average of 89.9%. A total of 871 alleles were detected using the 100 polymorphic EST-SSR markers; the number of alleles ranged from 2 (RBRC_Hc_ES_76) to 16 (RBRC_Hc_ES_80) and averaged 8.71. Polymorphism information content values varied from 0.15 (RBRC_Hc_ES_74) to 0.84 (RBRC_Hc_ES_3, RBRC_Hc_ES_95), with an average of 0.56. Phylogenetic and population structure analyses classified the 94 accessions/cultivars into two and two or three groups, respectively. Overall, our results demonstrate that the EST-SSR markers derived from the kenaf transcriptome were applied successfully to other Hibiscus species to provide useful information on the genetic diversity of this genus.

Development and characterization of EST-SSRs for Muscidae (Diptera) and their cross-genera transferability

EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat)markers were developed and used to examine genetic diversity of species assemblages of ten genera of Muscidae (Diptera), and specifically the genetic diversity of Phaonia Robineau species, from ten regions in China. 18 EST-SSR markers with high polymorphism and clear bands were screened out from 216 tested markers, with 219 alleles in total (95–280 bp) with an average of 6.1 alleles per locus. In various regions in China such as Tibet, Sichuan, and Yunnan, Muscidae species assemblages exhibited rich genetic diversity, with the polymorphism information content (PIC) values ranging from 0.831 to 0.934, while Hainan region showed a relatively low genetic diversity (PIC = 0.511). Low gene flow (Nm = 0.842) was found among the regions, and the average genetic differentiation coefficient (Fst) was 0.238, indicating a high degree of genetic differentiation among the ten surveyed assemblages. Accordingly, the ten genera of Muscidae exhibited high genetic diversity and genetic differentiation.

Genetic diversity of mutant napiergrass using Expressed Sequence Tag Simple Sequence Repeat (EST-SSR)

Abstract. Mansyur, Karti PMH, Abdullah L, Husni A, Lestari P. 2019. Genetic diversity of mutant napiergrass using Expressed Sequence Tag Simple Sequence Repeat (EST-SSR). Biodiversitas 20: 2403-2409. Napiergrass is one of the tropical grasses which has a very important role in developing ruminant livestock, its productivity is high and its nutritional content is quite good. Plant breeding to produce new varieties that have better productivity continues. One of them is through mutation breeding and in vitro culture. The purpose of this research was to look at the genetic diversity among napiergrasses using the Expressed Sequence Tag Simple Sequence Repeat (EST-SSR). This study used 14 SSR molecular markers. The results showed that mutant DNA of napiergrass can be clearly amplified by all the EST-SSR primers used. The average number of alleles was 4.57, the average frequency of the main allele was 42%, and the average value of gene diversity was 0.66. While the PIC average value was 0.60. There were five markers that were very informative and have PIC values ​​above 0.7, among others, namely ICMP3045, ICMP3018, PSMP2090, PSMP2209, and PSMP2019. Phylogenetic analysis shows that 37 numbers of napiergrass mutants split into two main clusters at a coefficient of 0.56. The first cluster consists of 26 lines while the second cluster consists of 11 mutants. The parent napiergrass is in the first cluster. There are two pairs of mutants that have the same diversity, namely R20-11 with R 20-20-3 and R100-1 with R100-3.

Development and Application of EST-SSR Markers for DNA Fingerprinting and Genetic Diversity Analysis of the Main Cultivars of Black Locust (Robinia pseudoacacia L.) in China

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement. More EST-SSR (Expressed sequence tag simple sequence repeat) markers of black locust can be used as a complement and improvement of Genomic-SSR markers for the identification of the function of gene and the construction of genetic map. Additionally, currently there is no simple method for identifying black locust cultivars. In this study, we obtained 2702 unigenes from 3095 expressed sequence tags (ESTs) from the National Center of Biotechnology Information (NCBI) database to identify simple sequence repeats (SSRs) in R. pseudoacacia samples. A total of 170 SSR loci were found to be distributed in 162 non-redundant sequences with a frequency of 6.29%. Dinucleotide repeats were the most predominant types among microsatellites (62.35%), followed by tri-nucleotide repeats (25.88%); the remaining SSRs accounted for less than 12%. The repeat motifs AG/TC (29.25%) and CT/GA (29.25%) were the most abundant among dinucleotides, and AAT/TTA (15.91%) was the most common among tri-nucleotides. A total of 62 primer pairs were designed to screen polymorphic and stable SSR loci. The resulting 25 EST-SSR markers capable of amplifying polymorphic, stable, and repeatable products. Eight newly developed EST-SSR markers and four published SSR markers were selected for DNA fingerprinting and genetic diversity analysis of the 123 main R. pseudoacacia cultivars in China. The 12 SSR loci amplified 102 alleles, with an average number of alleles per locus of 8.5 (range 4–15). The average polymorphism information content at the 12 SSR loci for the 123 cultivars was 0.670 (range 0.427–0.881). The 123 cultivars clustered into six main groups based on similarity coefficients, with most cultivars in one subgroup. Fingerprinting was performed using eight SSR markers; 110 black locust cultivars were distinguished. The results of this study increase the availability of EST-SSR markers in black locust and make it a simple method for checking the collection, the certification, and the correct attribution of clones and cultivars.

Development and characterization of EST-SSR markers for Saxifraga fortunei var. incisolobata (Saxifragaceae).

Premise Saxifraga fortunei (Saxifragaceae) includes several infraspecific taxa that are ecologically and morphologically distinct. To investigate the evolutionary history of phenotypic polymorphisms in this species, we developed expressed sequence tag–simple sequence repeat (EST‐SSR) markers for S. fortunei.Methods and ResultsWe developed 26 polymorphic markers based on transcriptome data obtained from Illumina HiSeq 2000. Within three populations of S. fortunei var. incisolobata, the number of alleles ranged from four to 25, and the levels of observed and expected heterozygosity ranged from 0.200 to 0.847 and from 0.209 to 0.930, respectively. Furthermore, all 26 loci showed transferability for S. fortunei var. obtusocuneata and S. fortunei var. suwoensis, and 18 loci were also successfully amplified in S. acerifolia.ConclusionsThese newly developed EST‐SSR markers will prove useful to infer the evolutionary history of S. fortunei var. incisolobata and its relatives in population genetic studies.

Development and Identification of Expressed Sequence Tag-based SSR Markers Associated with the Heterostyly Trait in Primula forbesii

Heterostylous Primula forbesii is an important ornamental flower in China because of its long-lasting flowers and winter bloom. This study aimed to develop markers of expressed sequence tag–simple sequence repeats (EST-SSRs) that are associated with heterostyly and that can be used for molecular-assisted selective breeding in P. forbesii. We investigated 114,474 unigenes and identified 25,095 SSRs in P. forbesii. Dinucleotide repeats (46.14%), mononucleotide repeats (44.65%), and trinucleotide repeats (8.27%) were the most abundant SSRs. Among the 25,095 SSRs, 10,645 SSR primer pairs were successfully designed, of which 130 primer pairs were randomly selected for further amplification validation using eight accessions of P. forbesii; 98 pairs produced clear and stable polymerase chain reaction (PCR) products, and 28 pairs showed polymorphism. Bulked segregant analysis (BSA) was conducted for the F1 population with respect to thrum style and pin style by scanning 28 polymorphic SSR primer combinations. One SSR marker, c64326, linked to the heterostyly trait at a genetic distance of ≈3.70 cM was identified. The marker c64326 was further validated in two populations with an accuracy of 97.92% and 90.63%. The novel and linked EST-SSR markers can be valuable resources for genetic diversity analysis, mapping, and marker-assisted breeding in P. forbesii.

Indications of Genetic Admixture in the Transition Zone between Fagus sylvatica L. and Fagus sylvatica ssp. orientalis Greut. &amp; Burd

Two subspecies of European beech (Fagus sylvatica L.) can be found in southeast Europe: Fagus sylvatica ssp. sylvatica L. and Fagus sylvatica ssp. orientalis (Lipsky) Greut. &amp; Burd. (Fagus orientalis Lipsky). In a previous study, based on genetic diversity patterns and morphological characters, indications of hybridization between both subspecies were found in northeastern Greece, a known contact zone of F. sylvatica and F. orientalis. Nevertheless, potential genetic admixture has not been investigated systematically before. Here, we investigated genetic diversity and genetic structure of 14 beech populations originating from Greece and Turkey as well as of two reference F. sylvatica populations from Germany based on nine expressed sequence tag-simple sequence repeat (EST-SSR) markers. Very low genetic differentiation was detected among F. sylvatica populations (mean GST: 0.005) as well as among F. orientalis populations (mean GST: 0.008), but substantial differentiation was detected between populations of the two subspecies (mean GST: 0.122). Indications for hybridization between both subspecies were revealed for one population in Greece. One of the genetic markers showed specific allele frequencies for F. sylvatica and F. orientalis and may be used as a diagnostic marker in future studies to discriminate both subspecies.

Development of EST-SSR markers in Saxifraga sinomontana (Saxifragaceae) and cross-amplification in three related species.

Premise Saxifraga sinomontana (Saxifragaceae) is a widespread alpine species in the Qinghai–Tibetan Plateau and its flanking mountains. We developed a set of expressed sequence tag–simple sequence repeat (EST‐SSR) markers to investigate the genetic diversity and evolutionary history of the species.Methods and ResultsWe initially designed 50 EST‐SSR markers based on transcriptome data of S. sinomontana. Nineteen of 50 loci (38%) were successfully amplified, 13 of which were polymorphic. These were tested on 71 individuals from four populations. Three to 18 alleles per locus were detected, and the levels of observed and expected heterozygosity ranged from 0.2817 to 0.9155 and 0.2585 to 0.8495, respectively. In addition, cross‐amplification was successful for all 13 loci in three congeneric species, S. tangutica, S. heleonastes, and S. congestiflora.ConclusionsThese EST‐SSR markers will be useful for studying the genetic diversity of S. sinomontana and disentangling the phylogenetic relationships of related species.

Comparative transcriptomic analysis reveals genetic divergence and domestication genes in Diospyros

BackgroundPersimmon (Diospyros kaki) is the most economically cultivated species belonging to the genus Diospyros. However, little is known about the interspecific diversity and mechanism of domestication, partly due to the lack of genomic information that is available for closely related species of D. kaki (DK). Here, we performed transcriptome sequencing on nine samples, including DK, a variety of DK and seven closely related species, to evaluate the interspecific genetic divergence and to identify candidate genes involved in persimmon domestication.ResultsWe obtained a total of 483,421 unigenes with N50 at 1490 bp in the nine Diospyros samples and identified 2603 orthogroups that were shared among all the samples using OrthoMCL analysis. A phylogenetic tree was established based on the tandem 2603 one-to-one single copy gene alignments, showing that DK was closely related to D. kaki var. silvestris (DKV) and that it clustered with the clade of D. deyangnsis (DD) and was farthest from the D. cathayensis (DC) species. The nonsynonymous substitutions (Ka), via synonymous substitution (Ks) ratios, was directly proportional to the genetic relationship of the different species. The higher the Ka/Ks ratios, the longer the distance was. Moreover, 31 positively selected genes (PSGs) involved in carbohydrate metabolism and phenolic metabolism were identified and isolated, and nearly all PSGs except the MATE gene had a high expression in the DK or DKV species. It was hypothesized that these genes might contribute to the domestication of the DK species. Finally, we developed the expressed sequence tag-simple sequence repeat (EST-SSR) and identified 2 unique amplicons DKSSR10 and DKSSR39: the former was absent in the DC species but was present in the other species, the latter had a long amplification product in the DJ species.ConclusionThis study presents the first transcriptome resources for the closely related species of persimmon and reveals interspecific genetic divergence. It is speculated that DK is derived from the hybridization of DD and DO species. Furthermore, our analysis suggests candidate PSGs that may be crucial for the adaptation, domestication, and speciation of persimmon relatives and suggests that DKSSR10 and DKSSSR39 could potentially serve as species-specific molecular markers.