Genetic Diversity Study on Geographical Populations of the Multipurpose Species Elsholtzia stauntonii Using Transferable Microsatellite Markers.

Elsholtzia stauntonii Benth. (Lamiaceae) is an economically important ornamental, medicinal and aromatic plant species. To meet the increasing market demand for E. stauntonii, it is necessary to assess genetic diversity within the species to accelerate the process of genetic improvement. Analysis of the transferability of simple sequence repeat (SSR) markers from related species or genera is a fast and economical method to evaluate diversity, and can ensure the availability of molecular markers in crops with limited genomic resources. In this study, the cross-genera transferability of 497 SSR markers selected from other members of the Lamiaceae (Salvia L., Perilla L., Mentha L., Hyptis Jacq., Leonurus L., Pogostemon Desf., Rosmarinus L., and Scutella L.) to E. stauntonii was 9.05% (45 primers). Among the 45 transferable markers, 10 markers revealed relatively high polymorphism in E. stauntonii. The genetic variation among 825 individuals from 18 natural populations of E. stauntonii in Hebei Province of China was analyzed using the 10 polymorphic SSR markers. On the basis of the SSR data, the average number of alleles (N A), expected heterozygosity (H E), and Shannon's information index (I) of the 10 primers pairs were 7.000, 0.478, and 0.688, respectively. Lower gene flow (N m = 1.252) and high genetic differentiation (F st = 0.181) were detected in the populations. Analysis of molecular variance (AMOVA) revealed that most of the variation (81.47%) was within the populations. Integrating the results of STRUCTURE, UPGMA (Unweighted Pair Group Method with Arithmetic Mean) clustering, and principal coordinate analysis, the 825 samples were grouped into two clusters associated with geographical provenance (southwestern and northeastern regions), which was consistent with the results of a Mantel test (r = 0.56, p < 0.001). Overall, SSR markers developed in related genera were effective to study the genetic structure and genetic diversity in geographical populations of E. stauntonii. The results provide a theoretical basis for conservation of genetic resources, genetic improvement, and construction of a core collection for E. stauntonii.

Isolation and characterization of 20 polymorphic microsatellites loci for Xenocypris davidi based on high-throughput sequencing.

Xenocypris davidi is one of the most economically important freshwater fish in China. However, few molecular markers have been reported for this species, impeding in-depth population genetic, dispersal, and gene flow studies. In the present study, a batch of novel polymorphic microsatellites from the genome of X. davidi were isolated and characterized using high-throughput sequencing. A total of 20 microsatellite markers were isolated. Analysis of 33 individuals revealed an average of 7.35 alleles per locus, ranging from 3 to 18. The observed and expected heterozygosities ranged from 0.3 to 1 and from 0.426 to 0.93, respectively. Only one tested locus significantly deviated from Hardy-Weinberg equilibrium. 18 microsatellite loci were highly polymorphic (PIC > 0.5). These newly isolated microsatellite markers would be useful to study the population genetics and stock management of X. davidi.

Development of microsatellite markers for Lippia alba and related Lippia species.

Microsatellite primers were developed in Lippia alba complex to better understanding the origins and evolution of the species. We sought to increase the numbers of available simple sequence repeat (SSR) markers. We performed low-coverage (~ twofold) genomic DNA sequencing of a diploid accession and generated a de novo assembly comprising 175,572 contigs. Sixteen SSR loci were selected and of these 13 SSR loci were successfully amplified in 20 L. alba tetraploid accessions and in 12 other Lippia species. Only one SSR locus was monomorphic, whereas 12 loci were polymorphic, yielding one to nine alleles. The heterozygosity was similar among markers, with values of 0.274-0.485; the polymorphism information content values varied from 0.237 to 0.367. These markers were successfully amplified in related species with 85% of transferability on average. Thus, we demonstrate the utility of including a de novo assembly step to obtain SSR markers from low-coverage genomic datasets.

Seventeen new microsatellites for Tamarix gallica and cross-amplification in Tamarix species.

PREMISE: Microsatellite markers were developed for the western Mediterranean tree Tamarix gallica (Tamaricaceae) as part of a study of its genetic diversity and structure. METHODS AND RESULTS: Seventeen microsatellite markers were developed for T. gallica, 14 of which were polymorphic. These microsatellites have di-, tri-, and tetranucleotide repeats with 1-13 alleles per locus and population. Levels of observed and expected heterozygosity ranged from 0.000 to 0.900 and from 0.000 to 0.863, respectively. Six microsatellites showed significant deviations from Hardy-Weinberg equilibrium in at least one population. Cross-amplification in 19 Tamarix species showed a wide transferability to other species of the genus. CONCLUSIONS: The 14 new polymorphic microsatellite markers will be used to assess the genetic diversity and population genetic structure of T. gallica. Additionally, the successful cross-species amplification suggests their potential usefulness for investigating species delimitation and population genetics in the genus Tamarix.

Optimizing depth and type of high-throughput sequencing data for microsatellite discovery.

PREMISE: Simple sequence repeat (SSR) markers (microsatellites) are a mainstay of many labs, especially when working on a limited budget, carrying out preliminary analyses, and in teaching. Whether SSRs mined from plant genomes or transcriptomes are preferred for certain applications, and the depth of sequencing needed to allow efficient SSR discovery, has not been tested. METHODS: I used genome and transcriptome high-throughput sequencing data at a range of sequencing depths to compare efficacy of SSR identification. I then tested primers from tomato for amplification, polymorphism, and transferability to related species. RESULTS: Small assemblies (two million read pairs) identified ca. 200-2000 potential markers from the genome assemblies and ca. 600-3650 from the transcriptome assemblies. Genome-derived contigs were often short, potentially precluding primer design. Genomic SSR primers were less transferable across species but exhibited greater variation (partially explained by being composed of more repeat units) than transcriptome-derived primers. DISCUSSION: Small high-throughput sequencing resources may be sufficient for identification of hundreds of SSRs. Genomic data may be preferable in species with low polymorphism, but transcriptome data may result in longer loci (more amenable to primer design) and primers may be more transferable to related species.

Development and characterization of microsatellite markers in Campomanesia adamantium, a native plant of the Cerrado ecoregions of South America.

PREMISE: A novel set of nuclear microsatellite markers was developed and characterized for Campomanesia adamantium (Myrtaceae) and tested for cross-amplification in the related species C. sessiliflora. METHODS AND RESULTS: Forty-one primer pairs were designed for simple sequence repeat loci, of which 36 successfully amplified and were polymorphic. The number of alleles ranged from two to 14, with an average of 8.14 alleles per locus. Additionally, cross-amplification was tested in C. sessiliflora; more than 55.5% of the microsatellite loci amplified, confirming the use of these microsatellite markers in a related species. CONCLUSIONS: We developed a set of microsatellite markers that will be useful for future studies of genetic diversity and population structure of C. adamantium and a closely related species, which will aid in future conservation efforts.

Development and characterization of genomic SSR markers for Tamarix chinensis (Tamaricaceae).

PREMISE OF THE STUDY: We developed a set of microsatellite markers to study the population genetic structure, mating system, and interspecific hybridization of Tamarix chinensis (Tamaricaceae), an alkali- and salt-tolerant shrub endemic to China, Korea, and Japan. METHODS AND RESULTS: Using Illumina sequencing, we developed 10 polymorphic and 11 monomorphic microsatellite primers. High levels of polymorphism were detected in four T. chinensis populations. Allele numbers ranged from two to 11, and the levels of observed and expected heterozygosity ranged from 0.182 to 0.846 and from 0.165 to 0.794, respectively. The polymorphism information content values ranged from 0.201 to 0.803. Cross-species amplification showed two to 15 alleles per locus in 24 individuals from one natural population of the congener T. ramosissima, and the levels of observed and expected heterozygosity ranged from 0.042 to 0.864 and from 0.041 to 0.892, respectively. CONCLUSIONS: These markers should be useful for exploring the population genetic structure, mating system, and gene flow of T. chinensis.

Molecular markers for Nassella trichotoma (Poaceae) to study genetic variation in New Zealand.

PREMISE OF THE STUDY: Simple sequence repeat (SSR) markers were developed for the study of genetic diversity of New Zealand Nassella trichotoma (Poaceae) and to support future studies in its native range. METHODS AND RESULTS: Genomic DNA was extracted from N. trichotoma leaf material and subjected to Roche 454 sequencing. From a total of 745 putative SSRs, 48 with di- to pentanucleotide repeats were screened, 32 primer pairs were designed, and 15 polymorphic markers were optimized for multiplex PCR on 105 N. trichotoma samples from four New Zealand regions. Each locus resulted in two to six alleles per locus, and four of the loci cross-amplified in N. tenuissima. The mean observed and expected heterozygosity ranged from 0.00 to 0.90 and 0.00 to 0.50 per locus, respectively. CONCLUSIONS: The novel SSR markers are valuable for the study of genetic diversity of N. trichotoma and might also be useful for closely related species.

Development of 15 microsatellite markers in Acer triflorum (Aceraceae) and cross-amplification in congeneric species.

PREMISE OF THE STUDY: Acer (Aceraceae) is an important genus in forest ecosystems in the Northern Hemisphere. In China, 151 species have been reported, and approximately 61 species are endemic. Thus, China is considered to host the greatest diversity of Acer, but markers are needed to evaluate the genetic structure and genetic diversity of these populations of wild Acer species. METHODS AND RESULTS: Using an enriched genomic library, we developed and characterized 15 microsatellite primers for A. triflorum, 10 of which were polymorphic. The number of alleles varied from one to nine. The levels of observed heterozygosity and expected heterozygosity per locus ranged from 0.000 to 1.000 and 0.000 to 0.826, respectively. Most primers also successfully amplified in A. ginnala, A. griseum, A. mandshuricum, A. pseudosieboldianum, A. sinopurpurascens, A. tegmentosum, and A. ukurunduense. CONCLUSIONS: These markers from A. triflorum will provide an opportunity to study genetic diversity and genetic structure in the genus Acer.

Genetic Evaluation of Natural Populations of the Endangered Conifer Thuja koraiensis Using Microsatellite Markers by Restriction-Associated DNA Sequencing.

Thuja koraiensis Nakai is an endangered conifer of high economic and ecological value in Jilin Province, China. However, studies on its population structure and conservation genetics have been limited by the lack of genomic data. Here, 37,761 microsatellites (simple sequence repeat, SSR) were detected based on 875,792 de novo-assembled contigs using a restriction-associated DNA (RAD) approach. Among these SSRs, 300 were randomly selected to test for polymorphisms and 96 obtained loci were able to amplify a fragment of expected size. Twelve polymorphic SSR markers were developed to analyze the genetic diversity and population structure of three natural populations. High genetic diversity (mean NA = 5.481, HE = 0.548) and moderate population differentiation (pairwise Fst = 0.048–0.078, Nm = 2.940–4.958) were found in this species. Molecular variance analysis suggested that most of the variation (83%) existed within populations. Combining the results of STRUCTURE, principal coordinate, and neighbor-joining analysis, the 232 individuals were divided into three genetic clusters that generally correlated with their geographical distributions. Finally, appropriate conservation strategies were proposed to protect this species. This study provides genetic information for the natural resource conservation and utilization of T. koraiensis and will facilitate further studies of the evolution and phylogeography of the species.

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB).

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC1F1, BC2F1 and BC3F1. The average recipient allele of the selected four BC1F1 plants was 80.75% which were used to produce the BC2F1 generation. BC1-P7 was the best BC1F1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC3F1 was 95.37%. Hsp gene expression analysis was carried out on BC1F1, BC2F1 and BC3F1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.

Sorghum Landrace Collections from Cooler Regions of the World Exhibit Magnificent Genetic Differentiation and Early Season Cold Tolerance.

Cold temperature is an important abiotic stress affecting sorghum production in temperate regions. It reduces seed germination, seedling emergence and seedling vigor thus limiting the production of the crop both temporally and spatially. The objectives of this study were (1) to assess early season cold temperature stress response of sorghum germplasm from cooler environments and identify sources of tolerance for use in breeding programs, (2) to determine population structure and marker-trait association among these germplasms for eventual development of marker tools for improving cold tolerance. A total of 136 sorghum accessions from cooler regions of the world were phenotyped for seedling growth characteristics under cold temperature imposed through early planting. The accessions were genotyped using 67 simple sequence repeats markers spanning all ten linkage groups of sorghum, of which 50 highly polymorphic markers were used in the analysis. Genetic diversity and population structure analyses sorted the population into four subpopulations. Several accessions distributed in all subpopulations showed either better or comparable level of tolerance to the standard cold tolerance source, Shan qui red. Association analysis between the markers and seedling traits identified markers Xtxp34, Xtxp88, and Xtxp319 as associated with seedling emergence, Xtxp211 and Xtxp304 with seedling dry weight, and Xtxp20 with seedling height. The markers were detected on chromosomes previously found to harbor QTLs associated with cold tolerance in sorghum. Once validated these may serve as genomic tools in marker-assisted breeding or for screening larger pool of genotypes to identify additional sources of cold tolerance.

Simple sequence repeat markers for kanuka (Kunzea spp.; Myrtaceae) present in New Zealand.

PREMISE OF THE STUDY: We developed simple sequence repeat (SSR) markers to facilitate population genetic studies on kanuka (Kunzea spp.; Myrtaceae). METHODS AND RESULTS: A shotgun sequencing library was constructed from leaf material of K. robusta using a Roche 454 Junior sequencer, and a total of 3174 putative SSR regions were identified. Sixteen polymorphic markers were optimized for multiplex PCR on 10 endemic New Zealand Kunzea species. Each of these loci cross-amplified in all tested species. The amplified di-, tri-, and pentanucleotide repeats resulted in eight to 24 alleles per locus for a total of 220 specimens. The mean observed and expected heterozygosity per locus ranged from 0.18 to 0.77 and 0.33 to 0.82, respectively. CONCLUSIONS: The SSR markers we produced are valuable for phylogenetic and population studies on all endemic Kunzea spp. and may also be useful for studies on closely related Kunzea species from Australia.

Short-Term Local Adaptation of Historical Common Bean (Phaseolus vulgaris L.) Varieties and Implications for In Situ Management of Bean Diversity.

Recognizing both the stakes of traditional European common bean diversity and the role farmers' and gardeners' networks play in maintaining this diversity, the present study examines the role that local adaptation plays for the management of common bean diversity in situ. To the purpose, four historical bean varieties and one modern control were multiplied on two organic farms for three growing seasons. The fifteen resulting populations, the initial ones and two populations of each variety obtained after the three years of multiplication, were then grown in a common garden. Twenty-two Simple Sequence Repeat (SSR) markers and 13 phenotypic traits were assessed. In total, 68.2% of tested markers were polymorphic and a total of 66 different alleles were identified. FST analysis showed that the genetic composition of two varieties multiplied in different environments changed. At the phenotypic level, differences were observed in flowering date and leaf length. Results indicate that three years of multiplication suffice for local adaptation to occur. The spatial dynamics of genetic and phenotypic bean diversity imply that the maintenance of diversity should be considered at the scale of the network, rather than individual farms and gardens. The microevolution of bean populations within networks of gardens and farms emerges as a research perspective.

Transcriptomic resources and marker validation for diploid and polyploid Veronica (Plantaginaceae) from New Zealand and Europe.

PREMISE OF THE STUDY: Polyploidy may generate novel variation, leading to adaptation and species diversification. An excellent natural system to study polyploid evolution in a comparative framework is Veronica (Plantaginaceae), which comprises several parallel, recently evolved polyploid series. METHODS: Over 105 million Illumina paired-end sequence reads were generated from cDNA libraries of leaf tissue from eight individuals representing three European and four New Zealand species. Forty-eight simple sequence repeat (SSR) and 48 low-copy nuclear (LCN) markers were developed and validated with Fluidigm microfluidic PCR and Illumina MiSeq amplicon sequencing on 48 different individuals each. RESULTS: Individual Trinity assemblies were similar regarding annotated transcripts (13,009-14,271), mean contig length (635-742 bp), N50 value (916-1133 bp), E90N50 value (1099-1308 bp), contigs with positive BLAST hits (42-63%), and gene ontology terms. Analyses of 29,738 single-nucleotide polymorphisms (8746 phylogenetically informative) mined from these transcriptomes plus two outgroups (Picrorhiza kurrooa and Plantago ovata) showed moderate to high bootstrap support for all branches and reticulation among sampled European Veronica. DISCUSSION: The transcriptome sequences themselves, as well as the validated SSR (40/48) and LCN (11/48) markers derived from them, show inter- and intraspecific genetic variation. These resources will be invaluable for future population genetic, phylogenetic, and functional genetic investigations in polyploid Veronica.

Development of microsatellite markers for buffalograss (Buchloë dactyloides; Poaceae), a drought-tolerant turfgrass alternative.

PREMISE OF THE STUDY: Buchloë dactyloides (Poaceae) is an important component of Great Plains prairies and a popular drought-tolerant turfgrass alternative in North America. This species comprises an autopolyploid series, and microsatellite primers were developed to understand the distribution of genetic variation among cytotypes and across its large geographic range. METHODS AND RESULTS: Fifteen microsatellite loci were designed and successfully amplified in six B. dactyloides populations. Within-population genetic diversity was comparatively high, consistent with B. dactyloides' life history. Allelic variation at 13 loci was consistent with the cytotype established in chromosome-counted samples. CONCLUSIONS: This variable, interpretable set of loci allows for the determination of multilocus genotype in B. dactyloides individuals of varying cytotype. Data such as these from a range-wide sample set can provide important insights for germplasm conservation and crop improvement in this ecologically and economically important species.

Estimation of loss of genetic diversity in modern Japanese cultivars by comparison of diverse genetic resources in Asian pear (Pyrus spp.).

BACKGROUND: Pears (Pyrus spp.) are one of the most important fruit crops in temperate regions. Japanese pear breeding has been carried out for over 100 years, working to release new cultivars that have good fruit quality and other desirable traits. Local cultivar 'Nijisseiki' and its relatives, which have excellent fruit texture, have been repeatedly used as parents in the breeding program. This strategy has led to inbreeding within recent cultivars and selections. To avoid inbreeding depression, we need to clarify the degree of inbreeding among crossbred cultivars and to introgress genetic resources that are genetically different from modern cultivars and selections. The objective of the present study was to clarify the genetic relatedness between modern Japanese pear cultivars and diverse Asian pear genetic resources. RESULTS: We genotyped 207 diverse accessions by using 19 simple sequence repeat (SSR) markers. The heterozygosity and allelic richness of modern cultivars was obviously decreased compared with that of wild individuals, Chinese pear cultivars, and local cultivars. In analyses using Structure software, the 207 accessions were classified into four clusters (K = 4): one consisting primarily of wild individuals, one of Chinese pear cultivars, one of local cultivars from outside the Kanto region, and one containing both local cultivars from the Kanto region and crossbred cultivars. The results of principal coordinate analysis (PCoA) were similar to those from the Structure analysis. Wild individuals and Chinese pears appeared to be distinct from other groups, and crossbred cultivars became closer to 'Nijisseiki' as the year of release became more recent. CONCLUSIONS: Both Structure and PCoA results suggest that the modern Japanese pear cultivars are genetically close to local cultivars that originated in the Kanto region, and that the genotypes of the modern cultivars were markedly biased toward 'Nijisseiki'. Introgression of germplasm from Chinese pear and wild individuals that are genetically different from modern cultivars seems to be key to broadening the genetic diversity of Japanese pear. The information obtained in this study will be useful for pear breeders and other fruit breeders who have observed inbreeding depression.

Identification and characterization of microsatellite markers in Penstemon scariosus (Plantaginaceae).

PREMISE OF THE STUDY: Penstemon scariosus var. albifluvis (Plantaginaceae) has been proposed to be federally listed as threatened due to its unique, geologically oil-rich habitat. Developing simple sequence repeat (SSR) markers to study its genetic diversity would be most useful. METHODS AND RESULTS: Using genomic reduction in combination with next-generation sequencing, we identified SSR motifs with five to 15 perfect repeats in 1067 P. scariosus contigs. After multiple qualifying tests, 16 SSRs were selected for their robust polymorphic reliability across 12 taxa with as high as 21 alleles in a given taxon. With the exception of two monomorphic loci, the observed and expected heterozygosity values ranged from 0.083 to 1.000 and 0.398 to 0.920, respectively. CONCLUSIONS: These microsatellite markers will directly aid in studies of the genetic diversity and relatedness of P. scariosus, P. comarrhenus, P. compactus, P. cyananthus var. cyananthus, P. fremontii var. fremontii, P. fremontii var. glabrescens, P. gibbensii, P. strictus, and P. subglaber.

Microsatellite markers for the Pilosella alpicola group (Hieraciinae, Asteraceae) and their cross-amplification in other Hieraciinae genera.

PREMISE OF THE STUDY: Microsatellite markers were developed for the Pilosella alpicola group (Asteraceae), comprising four closely related species distributed in subalpine areas of Europe. These species are believed to have diverged recently, but display contrasting cytogeographic patterns and variation in breeding systems, representing a promising model system for studying plant speciation, adaptation, and recent polyploidization. METHODS AND RESULTS: We developed 17 microsatellite markers for the P. alpicola group using 454 sequencing. Sixteen markers were polymorphic, with the number of alleles per locus ranging from seven to 16 and observed and expected heterozygosity ranging from 0.45 to 0.84 and 0.72 to 0.92, respectively. Ten and five loci amplified in the related species, P. echioides and P. officinarum, respectively, but only two in Andryala and one in Hieracium s. str. CONCLUSIONS: The developed microsatellite markers have high potential to become useful tools to study microevolutionary processes in the P. alpicola group and related Pilosella species.

Novel microsatellites for Calibrachoa heterophylla (Solanaceae) endemic to the South Atlantic Coastal Plain of South America.

PREMISE OF THE STUDY: Calibrachoa heterophylla (Solanaceae) is a petunia species restricted to the South Atlantic Coastal Plain of South America and presents a recent history of colonization from continental to coastal environments and diversification following the formation of the Coastal Plain during the Quaternary period. METHODS AND RESULTS: This study reports a suite of 16 microsatellite loci for C. heterophylla. The applicability of these markers was assessed by genotyping 57 individuals from two natural populations. Of the 16 described loci, 12 were found to be polymorphic. Successful cross-amplification tests were obtained using 12 Calibrachoa species. CONCLUSIONS: The development of microsatellite markers will be useful to recover the contemporary history of the colonization of the Coastal Plain and to provide information for the conservation of this endemic species.