Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae)

? Premise of the study: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. ? Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). ? Conclusions: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.     

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

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

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae)

? Premise of the study: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. ? Methods and Results: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. ? Conclusions: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.     

SSR-based genetic mapping and QTL analysis for timing of spring bud flush,young shoot color,and mature leaf size in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Tea plant (Camellia sinensis) is a major beverage crop across the world. To uncover the genetic controls of agronomic traits and facilitate marker-assisted breeding (MAB) in tea plant, we constructed a saturated SSR-based linkage map using an F₁ population derived from the crossing of ‘Longjin43’ × ‘Baihaozao’. A total of 483 SSR markers, consisting of 117 novel loci, 129 transferred from other tea plant maps, and 237 previously mapped, were successfully integrated into a new consensus map. The map has 15 linkage groups, covering 1226.2 cM in total with an average marker distance of 2.5 cM. The 126 markers in common enabled us to align this map to the reference genetic maps of tea plant. Phenotype data were collected in 2014 and 2015 for five traits: timing of spring bud flush (TBF), young shoot color (YSC), mature leaf length (MLL), mature leaf width (MLW), and leaf shape index (LSI, i.e., MLL/MLW). QTL analyses were performed for the five traits using the new consensus map and 15 QTLs were identified. The SSR markers, linkage map, and QTLs reported here are useful resources for future QTL mining, identification of causal genes, and MAB in tea plant.     

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

Background

Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results

Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (H _E ) and observed (H _o ) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion

UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.     

Development of intron targeting (IT) markers for potato and cross-species amplification in Solanum nigrum (Solanaceae)

? Premise of the study: Intron Targeting (IT) primers were developed for potato using expressed sequence tags (EST) and NCBI database records to study genetic diversity. ? Methods and Results: Twenty-nine polymorphic intron targeting (IT) markers were generated and characterized from 30 samples of potato and 22 samples of Solanum nigrum to detect polymorphism. The number of alleles (A) per locus ranged from 2 to 7 in the analyzed populations, and the observed heterozygosity (H(O)) and expected heterozygosity (H(E)) from 0 to 0.833 and 0.750, respectively. All of the primers also amplified in the related species S. nigrum. ? Conclusions: The developed markers will provide valuable tools for genetic diversity analysis, genetic mapping, and marker-assisted breeding of potato and related Solanum species.     

Construction of fingerprinting for tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) accessions using new genomic SSR markers

As one of the most popular non-alcoholic beverage crops, the tea plant (Camellia sinensis) plays an important role in human health and lifestyle. Genetic fingerprinting based on genomic-derived markers in tea, however, is still in the initial stages, which has limited tea germplasm resource utilization and cultivar protection. In the current study, we identified whole genome-based simple sequence repeat (SSR) loci and successfully developed 36 new genomic SSR markers, which are highly polymorphic with average allele number and polymorphic information content (PIC) of 14.9 and 0.862, respectively. A phylogenetic tree for 80 tea plant accessions was subsequently constructed based on their genotypic scores for these 36 markers. The phylogenetic relationships among the 80 accessions are highly consistent with their genetic backgrounds or original places. Noteworthy, robust fingerprinting power was performed, and the overall probability of finding two random individuals sharing identical genotypes across the 36 loci was estimated to be 1.5 × 10^?56. We subsequently identified five SSR markers as a recommended core marker set for fingerprinting the tea plant cultivars or accessions. The combined PI and PIsibs of the marker set were 1.49 × 10^?9 and 2.57 × 10^?3, respectively, which allowed us to fully discriminate all 80 tea plant accessions from one another. The SSR markers developed here will provide a valuable resource for tea plant genetics and genomic studies, as well as breeding programs. The fingerprinting profiles can serve as a database that is essential for the tea industry and commercial breeding, and for tea plant cultivar identification, utilization, and protection.     

Development and characterization of 41 novel EST-SSR markers for Pisum sativum (Leguminosae)

? Premise of the study: Expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers were developed in Pisum sativum for further use in genetic studies and breeding programs. ? Methods and Results: Forty-one novel EST-SSR primers were developed and characterized for size polymorphism in 32 Pisum sativum individuals from four populations from China. In each population, the number of alleles per locus ranged from one to seven, with observed heterozygosity and expected heterozygosity ranging from 0 to 0.8889 and 0 to 0.8400, respectively. Furthermore, 53.7% of these markers could be transferred to the related species, Vicia faba. ? Conclusions: The developed markers have potential for application in the study of genetic diversity, germplasm appraisal, and marker-assisted breeding in pea and other legume species.     

Development of seven novel EST-SSR markers from Cycas panzhihuaensis (Cycadaceae)

? Premise of the study: Cycas panzhihuaensis L. Zhou & S. Y. Yang is a vulnerable gymnosperm endemic to China, where its range represents the northernmost occupation of Cycas L. We developed EST-derived SSR markers to investigate its genetic diversity and population structure. ? Methods and Results: Based on the expressed sequence tag (EST) database for Cycas rumphii Miq., seven simple sequence repeat (SSR) markers were identified and screened in 55 individuals from seven wild populations of C. panzhihuaensis. Alleles numbered 2 to 5, and their observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.6545 and from 0.0535 to 0.6966, respectively. ? Conclusions: These new EST-SSR markers will enhance further studies of the population genetics of C. panzhihuaensis, allowing researchers to design reasonable conservation and management protocols.     

Isolation and characterization of 15 microsatellite markers from wild tea plant (Camellia taliensis) using FIASCO method

Camellia taliensis is one of the most important wild tea plants in China, especially in Yunnan Province. In this study, we described the development of 15 microsatellite markers from the genome of C. taliensis using the protocol of fast isolation by AFLP of sequences containing repeats (FIASCO). Polymorphism of each locus was assessed in 24 samples collected from six wild populations of C. taliensis. The average allele number of the microsatellites was four per locus, ranging from 2 to 7. The observed and expected heterozygosities varied from 0.076 to 0.5833 and from 0.1560 to 0.6917, respectively. Cross-species amplification in other three tea plants showed eleven of them holding promise for sister species. These polymorphic SSR markers would be useful tools for population genetics studies and assessing genetic variations to establish conservation strategy, molecular identification and molecular breeding on this tea plant and its allied species and varieties in section Thea genus Camellia.     

Development of microsatellite markers for Anadenanthera colubrina (Leguminosae), a neotropical tree species

? Premise of the study: We developed and characterized nuclear microsatellite markers for Anadenanthera colubrina, a tropical tree species widely distributed in South America. ? Methods and Results: Leaf samples of mature A. colubrina trees, popularly called "angico," were collected from an area that is greatly impacted by agricultural practices in the region of Ribeir?o Preto in S?o Paulo State in southeastern Brazil. Twenty simple sequence repeat (SSR) markers were developed, 14 of which had polymorphic loci. A total of 96 alleles were detected with an average of 6.86 alleles per polymorphic locus. The expected heterozygosity, calculated at polymorphic loci, ranged from 0.18 to 0.83. Finally, we demonstrated that 18 loci were cross-amplified in A. peregrina. ? Conclusions: A total of 14 polymorphic markers suggest a high potential for genetic diversity, gene flow, and mating system analyses in A. colubrina.     

广西普通油茶种质资源遗传多样性的SSR分析

普通油茶( Camellia oleifera)是我国分布最广、产量最多的山茶属中一个重要油料树种。广西是普通油茶的重要分布区,种质资源十分丰富。为深入了解广西普通油茶种质资源的遗传变异,服务于种质保存和品种选育,该研究首先对已开发的SSR分子标记进行多态性筛选和评价,在此基础上利用多态性较高的引物,对97份广西有代表性的普通油茶种质资源进行遗传多样性分析。结果表明：(1)在已开发的10对油茶SSR分子标记中,7对能稳定扩增且表现为共显性,2对扩增不稳定,另外1对无法扩增出产物。(2)7对共显性SSR标记总共检测到33个等位基因,每对标记检测到等位基因数目的变化范围为3~6个,平均每个位点等位基因数为4.7143个,有效等位基因数目的变化范围为2.0842~4.3148,平均有效等位基因数为2.8288;基因多样性变化范围为0.5202~0.7682,平均每个位点基因多样性为0.6281。(3)参试群体中绝大多数位点未处于Hardy-Weinberg平衡,存在遗传结构;观测杂合度和期望杂合度的变化范围分别为0.4130~0.6701和0.5233~0.7724,其平均值分别为0.5698和0.6316。(4)种质资源间遗传距离变化范围为0.05~0.7917,平均遗传距离为0.3545;UPGMA聚类显示相同来源的种质资源无法聚成一类,在同一聚类分支上混有不同来源的种质资源。这表明已开发的油茶SSR分子标记适用于广西普通油茶,广西普通油茶种质资源拥有较丰富的遗传多样性。该研究结果为广西普通油茶资源的深度开发和高效利用提供了科学依据。     

Isolation and characterization of 11 microsatellite loci from Camellia sinensis in Taiwan using PCR-based isolation of microsatellite arrays (PIMA)

We report 11 novel microsatellite primer pairs for the wild tea, Camellia sinensis (L.) O. Kuntze forma formosensis Kitamura. These simple sequence repeat markers were tested in 24 samples collected from wild tea populations, and in cultivars and C. japonica. The number of alleles ranged from 4 to18. The expected (H _E) and observed (H _O) heterozygosity were 0.687–0.946 and 0.042–0.792, respectively. All loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the rare species. Significant LD was discovered in most loci. These primers may provide a tool for understanding demography and population structure in wild tea.     

Isolation and characterization of microsatellite loci in a clonal herb, Cardamine leucantha (Brassicaceae)

? Premise of the study: Microsatellite markers were developed to analyze the clonal diversity and population genetic structure of a clonal plant, Cardamine leucantha. ? Methods and Results: Microsatellite loci were isolated by using compound simple sequence repeat (SSR) primers, resulting in 25 pairs including 14 useful markers. Samples from 33, 20, and 17 ramets in three populations were identified as belonging to 23, 18, and 17 genotypic individuals, respectively. ? Conclusions: These SSR markers will be used for identifying genets and evaluating genetic diversity of the clonal plant C. leucantha.     

Simple sequence repeat DNA markers in alfalfa and perennial and annual Medicago species.

Simple sequence repeat (SSR) or microsatellite DNA markers have been shown to function well in plant and mammalian species for genetic map construction and genotype identification. The objectives of the work reported here were to search GenBank for the presence of SSR-containing sequences from the genus Medicago, to assess the presence and frequency of SSR DNA in the alfalfa (Medicago sativa (L.) L. &L.) genome, and to examine the function of selected markers in a spectrum of perennial and annual Medicago species. The screening of an alfalfa genomic DNA library and sequencing of clones putatively containing SSRs indicated approximately 19 000 (AT)n + (CT)n + (CA)n + (ATT)n SSRs in the tetraploid genome. Inheritance was consistent with Mendelian expectations at four selected SSR loci with different core motifs. Additionally, genotypes of a range of Medicago species, including 10 perennial subspecies of the M. sativa complex and other perennial and annual Medicago species, were analyzed at each of the loci to ascertain the presence, number, and size of SSR alleles at each locus in each genotype. These studies indicate that SSR markers can function in alfalfa for the construction of genetic maps and will also be useful in a range of Medicago species for purposes of assessing genetic relatedness and taxonomic relationships, and for genotype identification.     

Development and characterization of EST‐SSR markers in an East Asian temperate plant genus Diabelia (Caprifoliaceae)

A set of expressed sequence tag (EST) simple sequence repeat (SSR) markers were developed and characterized using next‐generation sequencing technology for the genus Diabelia (Caprifoliaceae). De novo assembly of RNA‐seq reads resulted in 58 669 contigs with the N50 length of 1211 bp. A total of 2746 contigs were identified to harbor SSR motifs, of which 48 primer pairs were designed and 11 were shown to be polymorphic across three morphospecies of Diabelia. When evaluated with 30 individuals, the number of alleles per locus ranged from 2 to 11 and the expected heterozygosity varied from 0.399 to 0.873, respectively. Distance‐based clustering indicated that the EST‐SSR markers can provide sufficient power to distinguish the three species (or populations). These markers will be useful for evaluating the range‐wide genetic diversity of each species and examining genetic divergence and gene flow between the three species.     

SSR标记鉴定浙江省主要无性系茶树品种的研究

为促进浙江省茶树育种的发展,利用SSR引物对浙江省茶树育成品种的遗传多样性进行了研究,筛选出可用于鉴别浙江省茶树品种的核心鉴定引物和标准品种,并进一步应用于未知茶苗身份鉴定。首先,利用35对SSR引物研究了36个茶树育成品种,并进行聚类分析;然后,根据电泳谱带和基因型筛选出核心鉴定引物和标准品种;最后,对4株未知茶苗进行了身份鉴定。结果表明:共有34对引物表现出多态性,各品种基本按遗传背景聚类,重复样本间遗传距离介于0~0.094;有10对引物确定为核心鉴定引物,8个品种为标准品种;4株未知身份茶苗中,NH-01属于乌牛早品种,另外3株并非浙江现有品种。本研究认为,核心鉴定引物在两个浙江育成品种间差异引物对≥2时,应判定为不同品种;差异引物对≤1时,应判定为相同品种或极相似品种,必要时应引入其余24对引物计算遗传距离进一步验证,遗传距离>0.140判定为不同品种,遗传距离≤0.140判定为同一品种。     

Generation and characterization of 24 novel EST derived microsatellites from tea plant (Camellia sinensis) and cross-species amplification in its closely related species and varieties

A total of 31 expressed sequence tag (EST)-derived polymorphic microsatellites from tea plant, Camellia sinensis (L.) O. Kuntze, were generated and characterized using the ESTs of the author’s EST sequencing project and other sources. A set of 40 accessions tea germplasms had been used to examine the diversity. Among the 31 microsatellite loci, 24 had two to eight polymorphic alleles. Observed heterozygosity (H _o) were relatively higher (on an average of 0.533), varying from 0.175 (primer 21) to 0.950 (primer 228). Cross-species polymorphic amplification in other four species and two varieties of section Thea (L.) Dyer genus Camellia L. was successful for the 24 loci. Contribution of the 24 novel EST-SSR primers presented here will provide necessary and powerful molecular tools for management and conservation studies on the tea germplasms in the future. Li-Ping Zhao and Zhen Liu contributed equally to this work.