Unbiased Haploid Diversity Research Articles

Transcriptome sequencing and microsatellite marker discovery in Ailanthus altissima (Mill.) Swingle (Simaroubaceae).

Ailanthus altissima Swingle, is a tree species native to East Asia and has a great potential in decorative, bioenergy and industrial applications in many countries. To date, despite its commercial importance, the genomic and genetic resources available for this species are still insufficient. In this study, we characterized the transcriptome of A. altissima and developed thirteen EST-SSRs (expressed sequence tag-simple sequence repeats) based on Illumina paired-end RNA sequencing (RNA-seq). Besides, we developed ten polymorphic chloroplast microsatellite (cpSSR) markers using the available chloroplast genome of A. altissima. The transcriptome data produced 87,797 unigenes, of which 64,891 (73.91%) unigenes were successfully annotated in at least one protein database. For cpSSR markers the number of detected alleles (N) per marker varied from three at cpSSR12 to twelve at cpSSR8, the unbiased haploid diversity indices (uh) varied from 0.111 to 0.485, and haploid diversity indices (h) ranged from 0.101 to 0.444 with an average unbiased haploid diversity index (uh) of 0.274. Overall, a total of 65 different cpSSR alleles were identified at the ten loci among 165 individuals of A. altissima. The allele number per locus for EST-SSRs varied from 2.143 to 9.357, and the values of observed and expected heterozygosity ranged from 0.312 to 1.000 and 0.505 to 0.826, respectively. The molecular markers developed in this study will facilitate future genetic diversity, population structure, long distance-gene transfer and pollen-based gene flow analyses of A. altissima populations from its known distribution ranges in China focusing on planted and natural forest stands.

Development of 20 chloroplast microsatellite primers in wuyao (Lindera aggregata, Lauraceae).

Premise of the StudyTo investigate the population genetics and evolutionary history of wuyao (Lindera aggregata, Lauraceae) and other Lindera species, polymorphic chloroplast microsatellite (cpSSR) primers were developed.Methods and ResultsThe complete chloroplast genome of L. glauca was used to design 74 primers for L. aggregata. Agarose gel electrophoresis and Sanger sequencing were used to select 20 polymorphic primers. These primers were then tested in 70 individuals from three populations of L. aggregata. The number of alleles ranged from two to four, and the unbiased haploid diversity index ranged from 0.457 to 0.685. Up to 17 primers successfully amplified in four other Lindera species: L. prattii, L. chunii, L. lungshengensis, and L. pulcherrima var. hemsleyana.ConclusionsThe 20 primers reported here can potentially be used for future genetic studies of L. aggregata and other Lindera species.

Using genomic data to develop chloroplast DNA SSRs for the Neotropical liana Stizophyllum riparium (Bignonieae, Bignoniaceae).

Premise of the study:We developed chloroplast microsatellite markers (cpSSRs) to be used to study the patterns of genetic structure and genetic diversity of populations of Stizophyllum riparium (Bignonieae, Bignoniaceae).Methods and Results:We used genomic data obtained through an Illumina HiSeq sequencing platform to develop a set of cpSSRs for S. riparium. A total of 36 primer pairs were developed, of which 28 displayed polymorphisms across 59 individuals from three populations. Two to 12 alleles were recorded, and the unbiased haploid diversity per locus ranged from 0.037 to 0.905. All 28 cpSSRs presented transferability to two closely related species, S. inaequilaterum and S. perforatum.Conclusions:We report a set of 28 cpSSRs for S. riparium. All markers were shown to be variable in S. riparium, indicating that these markers will be valuable for population genetic studies across S. riparium and congeneric species.

Chloroplast and mitochondrial microsatellites for Millettia pinnata (Fabaceae) and cross-amplification in related species.

Premise of the study:Chloroplast and mitochondrial microsatellites were identified to study the population genetics of Millettia pinnata (Fabaceae).Methods and Results:Based on publicly available plastid genome sequence data of M. pinnata, 42 primer pairs were developed, of which 17 displayed polymorphisms across 89 individuals from four populations. For chloroplast loci, two to six alleles were recovered and the unbiased haploid diversity per locus ranged from 0.391 to 0.857. For mitochondrial loci, two to four alleles were recovered and the unbiased haploid diversity ranged from 0.264 to 0.740. Sixteen of the 17 screened markers could be successfully amplified in the related species M. pulchra.Conclusions:The 17 microsatellite markers developed here exhibited variation in M. pinnata and 16 presented transferability in the related species M. pulchra, suggesting that these markers will be valuable for genetic studies across M. pinnata and its related species.

Chloroplast microsatellite markers for Pseudotaxus chienii developed from the whole chloroplast genome of Taxus chinensis var. mairei (Taxaceae).

Premise of the study:Pseudotaxus chienii (Taxaceae) is an old rare species endemic to China that has adapted well to ecological heterogeneity with high genetic diversity in its nuclear genome. However, the genetic variation in its chloroplast genome is unknown.Methods and Results:Eighteen chloroplast microsatellite markers (cpSSRs) were developed from the whole chloroplast genome of Taxus chinensis var. mairei and successfully amplified in four P. chienii populations and one T. chinensis var. mairei population. Of these loci, 10 were polymorphic in P. chienii, whereas six were polymorphic in T. chinensis var. mairei. The unbiased haploid diversity per locus ranged from 0.000 to 0.641 and 0.000 to 0.545 for P. chienii and T. chinensis var. mairei, respectively.Conclusions:The 18 cpSSRs will be used to further investigate the chloroplast genetic structure and adaptive evolution in P. chienii populations.

Characterization of 16 microsatellite markers for the Oreinotinus clade of Viburnum (Adoxaceae).

Premise of the study:Microsatellite loci were isolated from four species of Viburnum (Adoxaceae) to study population structure and assess species boundaries among morphologically similar South American Viburnum species of the Oreinotinus clade.Methods and Results:Using a microsatellite-enriched library and mining next-generation sequence data, 16 microsatellites were developed. Each locus was tested on two populations of V. triphyllum and one population of V. pichinchense. For nuclear loci, one to 13 alleles were recovered, expected heterozygosity ranged from 0 to 0.8975, Simpson diversity index ranged from 0.0167 to 1.000, and Shannon diversity index ranged from 0 to 2.3670 in a given population. For the mitochondrial locus, three to six alleles were recovered and unbiased haploid diversity values ranged from 0.756 to 0.853 in a given population.Conclusions:The 16 microsatellite loci developed for the Oreinotinus clade (Viburnum, Adoxaceae) will inform investigations of population structure and species boundaries within this group.

A genomic approach for isolating chloroplast microsatellite markers for Pachyptera kerere (Bignoniaceae).

Premise of the study:In this study, we developed chloroplast microsatellite markers (cpSSRs) for Pachyptera kerere (Bignoniaceae) to investigate the population structure and genetic diversity of this species.Methods and Results:We used Illumina HiSeq data to reconstruct the chloroplast genome of P. kerere by a combination of de novo and reference-guided assembly. We then used the chloroplast genome to develop a set of cpSSRs from intergenic regions. Overall, 24 primer pairs were designed, 21 of which amplified successfully and were polymorphic, presenting three to nine alleles per locus. The unbiased haploid diversity per locus varied from 0.207 (Pac28) to 0.817 (Pac04). All but one locus amplified for all other taxa of Pachyptera.Conclusions:The markers reported here will serve as a basis for studies to assess the genetic structure and phylogeographic history of Pachyptera.

Development and characterization of nuclear microsatellite markers in the endophytic fungus Epichloë festucae (Clavicipitaceae).

• Premise of the study: Microsatellite primers were developed for the endophytic fungus Epichloë festucae, which is symbiotic with Festuca rubra, to study the population genetics of the species and to compare population structures between E. festucae and its host F. rubra.• Methods and Results: We developed 14 polymorphic markers using the unplaced genomic scaffold sequences of E. festucae from GenBank. The number of alleles per locus (A) varied from four to 16, and unbiased haploid diversity (h) was 0.717 in eight populations located in the Faroe Islands, Finland, and Spain. The Spanish populations possessed a higher number of alleles and haploid diversity (on average A = 5.1 and h = 0.591, respectively) compared to northern populations (on average A = 1.5 and h = 0.199, respectively).• Conclusions: These polymorphic markers will be used by grass breeders for uses including the improvement of commercial turfgrass cultivars, and by population geneticists to study different species of the Epichloë genus.

Development of 12 chloroplast microsatellite markers in Vigna unguiculata (Fabaceae) and amplification in Phaseolus vulgaris.

• Premise of the study: Vigna unguiculata is an economically important legume, and the complexity of its variability and evolution needs to be further understood. Based on publicly available databases, we developed chloroplast microsatellite primers to investigate genetic diversity within V. unguiculata and its related species Phaseolus vulgaris.• Methods and Results: Twelve polymorphic chloroplast microsatellite markers were developed and characterized in 62 V. unguiculata individuals. The number of alleles per locus varied between two and four, the unbiased haploid diversity per locus ranged from 0.123 to 0.497, and the polymorphism information content varied from 0.114 to 0.369. In cross-species amplifications, nine of these markers showed polymorphism in 29 P. vulgaris individuals.• Conclusions: The newly developed chloroplast microsatellite markers exhibit variation in V. unguiculata as well as their transferability in P. vulgaris. These markers can be used to investigate genetic diversity and evolution in V. unguiculata and P. vulgaris.

Development of the first chloroplast microsatellite loci in Ginkgo biloba (Ginkgoaceae).

• Premise of the study: To investigate population genetics, phylogeography, and cultivar origin of Ginkgo biloba, chloroplast microsatellite primers were developed.• Methods and Results: Twenty-one chloroplast microsatellite markers were identified referring to the two published chloroplast genomes of G. biloba. Polymorphisms were assessed on four natural populations from the two refugia in China. Eight loci were detected to be polymorphic in these populations. The number of alleles per locus ranged from three to seven, and the unbiased haploid diversity per locus varied from 0.441 to 0.807.• Conclusions: For the first time, we developed 21 chloroplast microsatellite markers for G. biloba, including 13 monomorphic and eight polymorphic ones within the assessed natural populations. These markers should provide a powerful tool for the study of genetic variation of both natural and cultivated populations of G. biloba, as well as cultivars.

Microsatellite primers in the lichen symbiotic alga Trebouxia decolorans (Trebouxiophyceae).

• Premise of the study: Polymorphic microsatellite markers were developed for the symbiotic green alga Trebouxia decolorans to study fine-scale population structure and clonal diversity.• Methods and Results: Using Illumina pyrosequencing, 20 microsatellite primer sets were developed for T. decolorans. The primer sets were tested on 43 individuals sampled from four subpopulations in Germany. The primers amplified di-, tri-, and tetranucleotide repeats with three to 15 alleles per locus, and the unbiased haploid diversity per locus ranged from 0.636 to 0.821.• Conclusions: The identified microsatellite markers will be useful to study the genetic diversity, dispersal, and reproductive mode of this common lichen photobiont.

Polymorphic chloroplast microsatellite loci in Nelumbo (Nelumbonaceae)

To study population genetics, phylogeography, and hybridization of Nelumbo (Nelumbonaceae), chloroplast microsatellite markers were developed. Seventeen microsatellite loci were identified from the chloroplast genomes of N. nucifera and N. lutea. Polymorphisms were assessed in three populations of N. nucifera and one population of N. lutea. Nine loci were found to be polymorphic in N. nucifera, and all 17 loci were found to be polymorphic in N. lutea. In N. nucifera, the number of alleles per locus ranged from two to six, and the unbiased haploid diversity per locus ranged from 0.198 to 0.790. In N. lutea, the number of alleles ranged from two to four, and the unbiased haploid diversity per locus ranged from 0.245 to 0.694. The identified chloroplast simple sequence repeat markers will be useful for the study of genetic diversity, phylogeography, and identification of Nelumbo cultivars.

Chloroplast microsatellite markers in Liriodendron tulipifera (Magnoliaceae) and cross‐species amplification in L. chinense

A set of cpSSR markers were developed for the tree genus Liriodendron L. to investigate population genetic structure and phylogeographic history. Primers were designed directly from the chloroplast genome sequences of Liriodendron tulipifera. Among the 55 cpSSR markers tested, 11 polymorphic markers were identified in L. tulipifera. The number of alleles in the population tested ranged from two to five, and the unbiased haploid diversity per locus ranged from 0.074 to 0.644. Eighteen primer pairs generated polymorphic amplification in L. chinense. The number of alleles per locus ranged from two to seven, and the unbiased haploid diversity per locus was from 0.250 to 0.964. cpSSR markers developed here will be useful for phylogeography and population genetics studies of Liriodendron.