Polymorphic Chloroplast Microsatellite Research Articles

A novel set of polymorphic chloroplast microsatellite markers for northern red oak (Q. rubra L.)

AbstractOaks are model species due to their importance in various ecosystems, worldwide distribution, high economic value and emerging genomic resources. As such, knowledge on population differentiation across their distribution range is of high importance for sustainable forest management. As in most angiosperms, chloroplasts are maternally (via seeds) inherited in oak species and acorns are dispersed over comparably short distances. Consequently, chloroplast markers reveal comparatively high differentiation between populations, making them highly viable for the analysis of historic migration patterns and the certification of reproductive material. Despite the existence of various white oak (section: Quercus) chloroplast markers, red oak (section: Lobatae) chloroplast markers remain limited. Northern red oak is one of the most important North American oak species and a widespread non-native plantation tree species in European forests. We took advantage of chloroplast genomes of Q. rubra L. and related oak chloroplast genomes to develop a set of robust and easy-to-use chloroplast microsatellite primers for northern red oak. Furthermore, we tested transferability of those novel red oak primer pairs to Q. robur L. and Q. petraea Matt. Liebl. The new set of fifteen polymorphic chloroplast microsatellite markers revealed three additional northern red oak haplotypes after screening 80 northern red oak individuals representing seven haplotypes, identified based on formerly available markers. Therefore, they provide a higher resolution of haplotypes as compared to currently available markers.

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.

Chloroplast microsatellites reveal genetic diversity and population structure in natural populations of Himalayan Cedar (Cedrus deodara (Roxb.) G. Don) in India

Abstract Himalayan cedar (Cedrus deodara) is one of the most important temperate timber species of Western Himalayas and is considered to be among the endangered conifer species in the region. Knowledge of genetic diversity and population structure will help guide gene conservation strategies for this species. Ten polymorphic chloroplast microsatellites (cpSSR) were used to study genetic diversity and population structure in twenty one natural populations of C. deodara throughout its entire distribution range in Western Himalayas. When alleles at each of the 10 loci were jointly analysed, 254 different haplotypes were identified among 1050 individuals. The cpSSRs indicate that C. deodara forests maintain a moderately high level of genetic diversity (mean h = 0.79 ). AMOVA analysis showed that most of the diversity in C. deodara occurs within populations. Bayesian analysis for population structure (BAPS) revealed spatial structuration of the variation (22 % of the total variation) and substructuring captured nineteen genetic clusters in the entire divisions of the populations. Most of the populations were clustered independently with minor admixtures. The distribution of genetic diversity and sub-structuring of C. deodara may be due to restricted gene flow due to geographic isolation, genetic drift, and natural selection. These findings indicated existence of genetically distinct and different high diversity and low diversity clusters, which are potential groups of populations that require attention for their conservation and management. The results are interpreted in context of future conservation plans for C. deodara.

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.

Development of chloroplast microsatellite markers for identification of Glycyrrhiza species

AbstractLicorice (Glycyrrhiza glabra) is an important medicinal herb and has long been used in traditional medicine for the treatment of several diseases worldwide. Understanding the genetic diversity within Glycyrrhiza species is important for the efficient conservation of these medicinal herbs. In this study, we have developed 20 polymorphic chloroplast microsatellite (cpSSR) markers using the chloroplast genome of G. lepidota. The cpSSR markers were tested on a total of 27 Glycyrrhiza individual plants. The number of alleles per locus ranged from two to eight among the Glycyrrhiza accessions. Overall, the Shannon index (I) for each cpSSR ranged from 0.315 to 1.694, the diversity indices (h) were 0.140–0.793 and the unbiased diversity indices (uh) were 0.145–0.825. In addition, the cpSSR markers were successfully divided and classified the 27 Glycyrrhiza individuals into four groups. The cpSSR markers developed in this study could be used in the assessment of genetic diversity and rapid identification of Glycyrrhiza species.

Whole chloroplast genome sequences of the Japanese hemlocks, Tsuga diversifolia and T. sieboldii, and development of chloroplast microsatellite markers applicable to East Asian Tsuga

ABSTRACTThe genus Tsuga is an ecologically important forest tree genus of Japan with two endemic species, the subalpine Tsuga diversifolia and the temperate Tsuga sieboldii. Despite dominating some of the most important remaining natural forests in Japan, the genetic diversity and structure of these species is unknown with genetic studies hindered by a lack of genetic resources. This study reports the complete chloroplast genome sequences of both T. diversifolia and T. sieboldii and the development of polymorphic chloroplast microsatellite markers. The chloroplast genomes of T. diversifolia and T. sieboldii are 120,802 and 120,797 bp in length, respectively and shared an identical repertoire of 35 tRNA genes, 4 rRNA genes and 72 protein-coding genes. Seven polymorphic chloroplast microsatellite loci were developed and showed an average of 2.24 alleles per locus in T. diversifolia and 3.57 in T. sieboldii. These loci were all successfully genotyped in two other East Asian species (Tsuga chinensis and Tsuga ulleungensis) with an average of 2.28 alleles per locus for both species. The high transferability of these loci, and the fact that all species contained loci with non-overlapping allele size ranges, indicates that these markers will be useful for genetic studies of all East Asian Tsuga species for investigating species boundaries, phylogeography and stand-level processes.

Development and Application of Genomic Resources in an Endangered Palaeoendemic Tree, Parrotia subaequalis (Hamamelidaceae) From Eastern China.

Parrotia subaequalis is an endangered palaeoendemic tree from disjunct montane sites in eastern China. Due to the lack of effective genomic resources, the genetic diversity and population structure of this endangered species are not clearly understood. In this study, we conducted paired-end shotgun sequencing (2 × 125 bp) of genomic DNA for two individuals of P. subaequalis on the Illumina HiSeq platform. Based on the resulting sequences, we have successfully assembled the complete chloroplast genome of P. subaequalis, as well as identified the polymorphic chloroplast microsatellites (cpSSRs), nuclear microsatellites (nSSRs) and mutational hotspots of chloroplast. Ten polymorphic cpSSR loci and 12 polymorphic nSSR loci were used to genotype 96 individuals of P. subaequalis from six populations to estimate genetic diversity and population structure. Our results revealed that P. subaequalis exhibited abundant genetic diversity (e.g., cpSSRs: Hcp = 0.862; nSSRs: HT = 0.559) and high genetic differentiation (e.g., cpSSRs: RST = 0.652; nSSRs: RST = 0.331), and characterized by a low pollen-to-seed migration ratio (r ≈ 1.78). These genetic patterns are attributable to its long evolutionary histories and low levels of contemporary inter-population gene flow by pollen and seed. In addition, lack of isolation-by-distance pattern and strong population genetic structuring in both marker systems, suggests that long-term isolation and/or habitat fragmentation as well as genetic drift may have also contributed to the geographic differentiation of P. subaequalis. Therefore, long-term habitat protection is the most important methods to prevent further loss of genetic variation and a decrease in effective population size. Furthermore, both cpSSRs and nSSRs revealed that P. subaequalis populations consisted of three genetic clusters, which should be considered as separated conservation units.

Development and characterization of 24 chloroplast microsatellite markers for two species of Eranthis (Ranunculaceae).

Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.

Characterization of Polymorphic Chloroplast Microsatellites in Prunus Species and Maternal Lineages in Peach Genotypes

Several available Prunus chloroplast genomes have not been exploited to develop polymorphic chloroplast microsatellites that could be useful in Prunus phylogenetic analysis and maternal lineage group (MLG) categorization. In this study, using available bioinformatics tools, 80, 75, and 78 microsatellites were identified from the chloroplast genome of P. persica (CPpe), P. kansuensis (CPka), and P. mume (CPmu), respectively. The genome features and polymorphism status of these microsatellites were characterized. The genomic locations and motif types of most chloroplast microsatellites were conserved in CPpe, CPka, and CPmu. Of the 67 microsatellites with primer sequences and names, 57 were polymorphic for their in silico motif, amplicon lengths, or both among the three genomes. Based on the genotyping data of eight most polymorphic microsatellites, eight unique MLGs were found among the 736 peach materials in a breeding program. Most peach cultivars (111 of 161 genotyped) belong to MLG-1, the Chinese Cling-derived group reflecting the heavy use of this germplasm in early peach development. Forty-one cultivars belong to MLG-2, the European-derived group of peaches. MLG-3 consists of ornamental accessions. MLG-4 and MLG-5 contain only ‘Flordaking’ and ‘Reliance’, respectively. MLG-6 to MLG-8 consists of selections derived from P. tangutica, P. davidiana, and P. mira, respectively. These amplicons from the representative material for each MLG were sequenced, revealing additional single nucleotide polymorphisms (SNPs) within the amplicons. With the polymorphism status and amplification reliability validated, these new polymorphic chloroplast microsatellite markers may be useful in Prunus phylogenetic analysis.

Development of chloroplast microsatellite markers for the endangered Maianthemum bicolor (Asparagaceae s.l.).

Premise of the study:Ten polymorphic chloroplast microsatellite (cpSSR) markers were developed and characterized in an endemic and endangered herb, Maianthemum bicolor (Asparagaceae s.l.), for use in conservation genetics.Methods and Results:Primer sets flanking each of the 10 cpSSR loci in noncoding regions of the chloroplast genome of M. bicolor were designed. These cpSSR markers were tested on a total of 33 adult individuals from three natural populations in South Korea. The number of alleles per locus ranged from two to three. The unbiased haplotype diversity per locus ranged from 0.061 to 0.682. All markers were successfully transferred to the congeneric species M. japonicum, M. bifolium, and M. dilatatum with polymorphisms among the species.Conclusions:The developed cpSSR markers will be useful in assessing the genetic diversity and population structure of M. bicolor and will help to infer its molecular identification, thereby providing a basis for conservation.

Combining molecular and fossil data to infer demographic history of Quercus cerris: insights on European eastern glacial refugia

AbstractAimPhylogeographical studies of Eastern Mediterranean species are rare. We aim to fill a gap in the current understanding of the role of Eastern Mediterranean glacial refugia, and their connections with other refugia across Europe. To this end, we studied the genetic diversity distribution and genetic structure of the modern population of Quercus cerris in relation to its Quaternary demographic history and to more ancient events.LocationMediterranean Basin; Italian, Balkan, Anatolian peninsulas.MethodsA total of 192 populations were genotyped with six polymorphic chloroplast microsatellites, and the genetic diversity and differentiation of the populations were evaluated. The geographical structure of genetic variation was analysed with a Bayesian clustering method using baps 5.2. The demographic history of Q. cerris was explored by an approximate Bayesian computation procedure using diyabc 2.0. To reconstruct the past distribution of Q. cerris, we also considered the chronology and geographical distribution of fossil records.ResultsThirty‐five haplotypes were found, three of which (together) were found in 71.82% of individuals. Bayesian analysis resulted in three genetically and geographically distinct clusters: a Western group, a Central group, and an Eastern group. The approximate Bayesian computation analysis, together with fossil data, showed a possible bottleneck leading to the divergence of the Eastern and Central populations in the Early Pleistocene (Gelasian). The split into two groups of populations in the Italian and Balkan Peninsulas, respectively, was probably caused by a marked population contraction during a glacial phase of the Middle Pleistocene.Main conclusionsThis study provides information on the potential role of Eastern Europe and the Near East as refugia and as a source for ancient westward range expansions in the Mediterranean region. Our study covers a remarkable gap in European oak phylogeography, showing a putative eastern origin of Q. cerris and the presence of large amounts of genetic diversity in this region.

Characterization and development of chloroplast microsatellite markers for Gossypium hirsutum, and cross-species amplification in other Gossypium species.

Cotton is an important economic crop worldwide; its fiber, commonly known as cotton lint, is the main natural source for the textile industry. Sixty chloroplast microsatellites were identified and characterized from the complete sequence of the Gossypium hirsutum chloroplast genome using a bioinformatic approach. Twenty chloroplast microsatellite loci were polymorphic in the 66 Gossypium germplasm accessions. A total of 85 alleles were detected, with allele numbers varying from 2-7 per locus. Polymorphism information content varied from 0.02-0.66, with a mean of 0.48. Additionally, transferability of the 20 polymorphic chloroplast microsatellite primers was evaluated in other 31 Gossypium species. Sixteen markers were successfully amplified across all species tested, while the remaining 4 markers cross-amplified in most species tested. These polymorphic chloroplast microsatellite markers may be useful tool for studies of individual identification, genetic diversity, evolution, conservation genetics, and molecular breeding in Gossypium.

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.

Seed‐mediated connectivity among fragmented populations of Quercus castanea (Fagaceae) in a Mexican landscape

Anthropogenic fragmentation is an ongoing process in many forested areas that may create loss of connectivity among tree populations and constitutes a serious threat to ecological and genetic processes. We tested the central hypothesis that seed dispersal mitigates the impact of fragmentation by comparing connectivity and genetic diversity of adult vs. seedling populations in recently fragmented populations of the Mexican red oak Quercus castanea. Adult individuals, established before fragmentation, and seedlings, established after fragmentation, were sampled at 33 forest fragments of variable size (0.2 to 294 ha) within the Cuitzeo basin, Michoacán state, and genotyped using seven highly polymorphic chloroplast microsatellite markers (cpSSRs). To test whether seed dispersal retains connectivity among fragmented populations, we compared genetic diversity and connectivity networks between adults and progeny and determined the effect of fragment size on these values. Seventy haplotypes were identified, 63 in the adults and 60 in the seedlings, with average within-population diversity (hS) values of 0.624 in the adults and 0.630 in the seedlings. A positive correlation of genetic diversity values with fragment size was found in the seedling populations but not in the adult populations. The network connectivity analysis revealed lower connectivity among seedling populations than among adults. The number of connections (edges) as well as other network properties, such as betweenness centrality, node degree and closeness, were significantly lower in the seedlings network. Habitat fragmentation in this landscape is disrupting seed-dispersal-mediated genetic connectivity among extant populations.

Nuclear and chloroplast SSR markers in Paeonia delavayi (Paeoniaceae) and cross‐species amplification in P. ludlowii

Microsatellite primers were developed for Paeonia delavayi and P. ludlowii (Paeoniaceae) to study their population genetics and phytogeography. Nine polymorphic nuclear microsatellite loci were isolated from an enriched library of P. delavayi and primers were designed. The number of alleles per locus ranged from two to 16; the observed and expected heterozygosities ranged from 0.014 to 0.687 and 0.042 to 0.875, respectively. Six polymorphic chloroplast microsatellite loci were identified in P. delavayi and primers were provided. The number of alleles per locus ranged from two to six and the polymorphic information content ranged from 0.08 to 0.716. Both nuclear and chloroplast primers were successfully applicable to P. ludlowii. The markers developed here will facilitate analyses of genetic diversity, population genetic structure, phytogeographical patterns, and conservation for P. delavayi and P. ludlowii.

Development of novel chloroplast microsatellite markers for Dendrobium officinale, and cross-amplification in other Dendrobium species (Orchidaceae)

Nine pairs of polymorphic chloroplast microsatellite primers were developed for Dendrobium officinale Kimura et Migo, an endangered herb. Levels of polymorphism were tested across a total of 55 individuals from four natural populations (12–15 individuals per population). Allele numbers varied from two to four per locus, while the number of haplotypes ranged from four to six per population. Transferability of the nine polymorphic chloroplast microsatellite primers was checked on an additional set of 51 Dendrobium individuals (belonging to 17 different species). Three markers could be transferable to all the species tested, while the remaining six markers successfully cross-amplified in most species tested. Moreover, polymorphism of the nine chloroplast microsatellite primers was tested across Dendrobium moniliforme (L.) Sw. and Dendrobium loddigesii Rolfe. All of them were polymorphic in D. moniliforme, while seven of which were polymorphic in D. loddigesii. These polymorphic chloroplast microsatellite primers developed for D. officinale will be a useful tool for the study of genetic diversity, population genetic structure, evolution of D. officinale and establishment of effective conservation strategies.

A novel set of highly polymorphic chloroplast microsatellite and ISSR markers for the biofuel crop Jatropha curcas

A novel set of polymorphic ISSR and chloroplast microsatellite markers were identified to mark the genetic relationship among different accessions of Jatropha curcas. A total of 12 polymorphic ISSR primers used, amplified a total of 26 fragments among ten different accessions of J. curcas. The cross-species amplification of microsatellite loci is timesaving as well as a cost-effective approach for developing locus specific markers for new species. Further, in an attempt to reveal a high degree of polymorphism and genetic variation among different accessions of J. curcas chloroplast microsatellite primer pairs developed for Acorus calamus (Acoraceace) were examined for cross-species amplification and validation among them. Out of 18 cpSSRs, six (33.33%) were identified as resulting in good amplifications and polymorphism. The number of amplified alleles per cpSSR primer pair ranged from 2 to 4 with the total number of 18 size variants as whole. The 18 size variants at 6 loci among 10 accessions were combined in 4 different haplotypes. The total chloroplast DNA diversity (HT) and the differentiation (GST) were found as 0.700 and 0.560 respectively. Whereas, Shannon's information index (I), Nei's genetic diversity among different accessions of J. curcas using ISSR and cpSSR markers was estimated at 0.33±0.28; 0.22±0.19 and 0.94±0.35; 0.55±0.18 respectively. The genetic relationship among the accessions was established using UPGMA cluster analysis, which were grouped into two major clusters denoted as cluster-I and cluster-II. The implication of the results in devising the importance of a identified novel set of markers for the genetic relationship and genetic diversity analysis in J. curcas is discussed.

Assessment of the domestication state of ackee (Blighia sapida K.D. Koenig) in Benin based on AFLP and microsatellite markers

Ackee (Blighia sapida) is a native multipurpose species important for the livelihoods of the rural populations in Benin. Trees are found in natural forests or are managed by farmers in different traditional agroforestry systems. Genetic variation at amplified fragment length polymorphism (AFLP) markers, four nuclear microsatellites (nSSRs) and one chloroplast microsatellite (cpSSR) were investigated in 279 individuals from six wild and eight cultivated populations from Benin. The AFLP data revealed moderate levels of diversity of ackee in Benin (mean diversity values are proportion of polymorphic loci = 52.8% and Nei’s gene diversity = 0.157, for 375 AFLP fragments). The mean diversity values based on nSSR-markers are expected heterozygosity = 0.286, allelic richness = 2.77. Genetic variation of wild and cultivated populations did not differ markedly. AMOVA revealed that only 7.3 and 5.2% of the variation was partitioned among populations for nSSR- and AFLP-markers, respectively. A Mantel test based on these both marker-types revealed significant correlations between population pairwise geographic distance and genetic differentiation. Differentiation among cultivated populations was higher than among wild populations. The only polymorphic chloroplast microsatellite marker (ccmp7) showed three haplotypes. Cultivated populations from northeastern Benin were fixed on one haplotype which was not observed elsewhere indicating a different origin of these populations possibly from neighboring Nigeria. Farmer-led domestication had an impact on the spatial distribution of genetic variation but did not result in significant losses of diversity within populations. Measures to conserve genetic resources of ackee in each of the three main bioclimatic zones in Benin are proposed.

Development of novel chloroplast microsatellite markers to identify species in the Agrostis complex (Poaceae) and related genera

We needed a reliable way to identify species and confirm potential interspecific and intergeneric hybrids in a landscape level study of gene flow from transgenic glyphosate-resistant Agrostis stolonifera (Poaceae) to compatible relatives. We developed 12 new polymorphic chloroplast microsatellite markers to aid in identifying species recipient of transgenic pollen both within the Agrostis complex and the related genera Polypogon.

The influence of European and American wild germplasm in hop (Humulus lupulus L.) cultivars

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops.