Nuclear Simple Sequence Repeat Loci Research Articles

Development of nuclear SSR and chloroplast genome markers in diverse Liriodendron chinense germplasm based on low-coverage whole genome sequencing

BackgroundLiriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm.ResultsWe performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79.ConclusionsThe genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

Distinctions in Fine-Scale Spatial Genetic Structure Between Growth Stages of Picea jezoensis Carr.

Conifers in northern forests, such as fir and spruce, preferably regenerate on coarse woody debris, including fallen logs, stumps, and snags. In northern Japan, the sub-boreal conifer species Picea jezoensis is completely dependent on coarse woody debris for seedling establishment. To understand the fine-scale spatial genetic structure (FSGS) of this species, a 5-ha plot was established in central Hokkaido, and 531 individual trees were categorized into four life-stages (seedling, sapling, juvenile, and mature) on the basis of age and size. The FSGS of the established seedlings and later growth stages was investigated using 11 nuclear simple sequence repeat loci. A STRUCTURE analysis of seedlings and saplings established on fallen logs revealed that genetically related individuals were spatially localized between adjacent logs. We also found a significant FSGS in early life-stages based on a decline in the kinship coefficient calculated between individuals over shorter to longer spatial distances. Furthermore, the estimation of dispersal kernels indicated the frequent occurrence of short-distance seed dispersal. These results indicated that genetically related seedlings and saplings regenerated on the same or nearby fallen logs. In contrast to the results for the early stages, mature-stage trees showed no significant FSGS. We ran a simulation to examine the hypothesis that the FSGS could be eliminated by demographic thinning during life history processes. We calculated values for simulated offspring generated under three sets of conditions; i.e., by removing (i) inbred individuals, (ii) randomly chosen individuals, and (iii) all individuals on the specific fallen logs. However, the results for the FSGS were significant for all simulated data sets. This indicated that inbreeding depression, stochastic loss, or eradication of establishment sites by local disturbances alone could not explain the lack of FSGS among mature-stage trees. Therefore, it is possible that the colonization history of mature trees present on the study site might differ from that of the current offspring.

DNA taxonomy in the timber genus Milicia: evidence of unidirectional introgression in the West African contact zone

DNA-based techniques are helpful in characterising hybridisation patterns in plant species. To be efficient in disentangling species boundaries and interspecific gene flow, it is recommended to combine various methodologies and types of markers. Here, we used different analytical tools (algorithms implemented in Structure, Tess, NewHybrids and HIest, and the haploweb approach) and three nuclear genetic markers (7 nuclear simple sequence repeat loci (SSRs), 62 single-nucleotide polymorphism loci (SNPs) and a single-copy gene region, At103) to revisit hybridisation patterns in the commercially important African tree genus Milicia. Samples were collected in the natural ranges of Milicia regia and Milicia excelsa in West Africa. Using real data sets, simulated purebreds and hybrid genotypes, we found that SNPs yielded results more consistent than SSRs; outputs from the Bayesian and maximum-likelihood analyses differed significantly using the SSRs, whereas they were perfectly congruent using SNPs. A proportion of 12.4% hybrids were detected amongst the SNP genotype samples. A haploweb analysis of At103 gene sequences confirmed the existence of interspecific hybrids. There was also a clear evidence of advanced generations of hybrids (backcrossed individuals) but only towards M. regia. Although more investigation is required for understanding the mechanisms responsible for this asymmetric introgression, we suggest that it may be due to the differences in flowering time between species and between sexes, combined with a maternal inheritance of flowering time.

Warm-cold colonization: response of oaks to uplift of the Himalaya-Hengduan Mountains.

Clarifying the relationship between distribution patterns of organisms and geological events is critical to understanding the impact of environmental changes on organismal evolution. Quercus sect. Heterobalanus is now distributed across the Himalaya-Hengduan Mountains (HHM) and warm lowland in East China, yet how the distribution patterns of this group changed in response to the HHM uplift remains largely unknown. This study examines the effect of tectonic events in the HHM region on the oaks, providing a biological perspective on the geological history of this region. Fifty-six populations of Quercus sect. Heterobalanus were genotyped using four chloroplast DNA regions and nine nuclear simple sequence repeat loci to assess population structure and diversity, supplemented by molecular dating and ancestral area reconstructions. The underlying demographic dynamics were compared using ecological niche models of the species distributions during the last glacial maximum and the present. These analyses illustrate that Quercus sect. Heterobalanus diversified as the HHM uplifted and climatic cooling during the mid-Miocene, colonizing the cold habitats from warm broadleaf mixed forests. Lineages in cold highlands and warm lowlands have diverged as a consequence of local adaptation to diverging climates since the late Miocene. Our results suggest that continuous uplift of the HHM in the late Miocene to early Pliocene accompanied by simultaneous cooling triggered the differentiation of oaks. The biogeography of Quercus sect. Heterobalanus illuminates the geological events responsible for the modern-day HHM.

Fine- and local- scale genetic structure of Dysoxylum malabaricum, a late-successional canopy tree species in disturbed forest patches in the Western Ghats, India

Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, which is the world’s most densely populated biodiversity hotspot. In this study, we used variation at ten nuclear simple sequence repeat loci to investigate genetic diversity and fine scale spatial genetic structure (FSGS) in seedlings and adults of D. malabaricum from four forest patches in the northern part of the Western Ghats. When genetic variation was compared between seedlings and adults across locations, significant differences were detected in allelic richness, observed heterozygosity, fixation index (F IS), and relatedness (P 130 cm) are present in the southern sacred forests but not in the northern forest reserves. This pattern is likely due to stronger harvesting pressure in the north compared to the south, because in the north there are no cultural taboos regulating the extraction of natural resources. The implications for forest conservation in this biodiversity hotspot are discussed.

Genetic diversity and genetic structure of black alder (Alnus glutinosa [L.] Gaertn) in the Belgium-Luxembourg-France cross-border area

Due to its beneficial effects on river ecosystems, black alder (Alnus glutinosa) is one of the tree species selected for planting on riverbanks in the cross-border area encompassing Wallonia in Belgium, Lorraine in France, and Luxembourg. The preservation of this species, however, is threatened by an invasive pathogen that particularly targets and kills young alder individuals. The objectives of this study were to characterize the genetic diversity and the genetic structure of A. glutinosa at this local level with the aim of assisting the conservation and replanting strategies and to determine if a germplasm collection comprising individuals from the same cross-border area captures the diversity present in the region. Nuclear simple sequence repeat (SSR) and chloroplastic DNA (cpDNA) markers were used to analyze four local wild populations and the germplasm collection which is representative of two river catchments and six legal provenance regions. Three populations distant from the studied area were also included. A panel of 14 nuclear SSR loci revealed high allelic diversity and very low differentiation among wild populations (mean F ST = 0.014). The germplasm collection displayed a range of alleles that were representative of the different populations, and no significant differentiation between the germplasm collection and the local wild populations was observed, making this collection, as far as allelic diversity is concerned, suitable for providing trees for riverbank replanting programs. Using SSR markers, various statistical approaches consistently indicated the lack of a significant geographical structure at the level of the river catchments or provenance regions. In contrast, two cpDNA haplotypes were detected and displayed a cross-border geographically structured distribution that could be taken into account in defining new cross-border provenance regions.

Reduced Genetic Variation in Populations of Black Cherry (Prunus serotinasubsp.serotina, Rosaceae) at Its Western Range Limit in Kansas

We compared genetic variation at five nuclear simple sequence repeat loci between three populations of Black Cherry (Prunus serotina subsp. serotina) at the edge of its western range in Kansas to four populations from within the range interior. Although within-population expected heterozygosity did not differ between edge and core populations, allelic richness was significantly lower in the edge populations. This finding is consistent with a loss of rare alleles due to genetic drift in demographically unstable edge populations.

Amazon diversification and cross‐Andean dispersal of the widespread Neotropical tree species Jacaranda copaia (Bignoniaceae)

AbstractAimThe phylogeographical history of Neotropical species can be difficult to reconstruct because of superimposed Neogene and Quaternary histories, and because of taxonomic uncertainty. We analysed range‐wide genetic diversity in a widespread pioneer tree species, Jacaranda copaia (Aubl.) D. Don, to characterize phylogeographical structure, date the evolutionary relationships among lineages, and evaluate the role of dispersal and vicariance in establishing the present geographical range.LocationGuiana Shield; central, southern and western Amazon Basin; Chocó region; Central America.MethodsWe analysed nine nuclear simple sequence repeat loci (nuSSR), eight chloroplast SSRs (cpSSR), and two cpDNA intergenic sequences in 341 adult trees. Genetic differentiation at nuSSRs was inferred using Bayesian clustering. Dating of chloroplast lineage divergence was obtained using a range of published mutation rates and Bayesian coalescence analyses. Population divergence dating was performed using an isolation‐with‐migration model for eight loci (one cp sequence and seven nuSSRs).ResultsNuclear SSR variation identified three geographically overlapping clusters (nu‐1, nu‐2, nu‐3). Twelve cpDNA haplotypes were clustered into two haplogroups (cp‐1, cp‐2) with the central Amazon harbouring the highest diversity. Molecular dating analysis suggests that cpDNA haplotype diversification started around the end of the Pliocene (2.61 Ma on average), whereas population divergence was more recent and occurred during the mid‐Quaternary (point estimates between 357 and 436 ka).Main conclusionsThe genetic variation of J. copaia in the Neotropics was shaped mainly by Pleistocene events. Chloroplast diversity did not display the expected cis/trans Andean disjunction, indicating recent dispersal. Nuclear variation revealed that separate regions share a recent history, with a centre of diversity in the central Amazon Basin. The geographical pattern of diversity is congruent with the distribution of the two subspecies, J. copaia copaia and J. copaia spectabilis, and evidence of nuSSR admixture between the two taxa supports their classification as subspecies.

Impact of negative frequency-dependent selection on mating pattern and genetic structure: a comparative analysis of the S-locus and nuclear SSR loci in Prunus lannesiana var. speciosa

Mating processes of local demes and spatial genetic structure of island populations at the self-incompatibility (S-) locus under negative frequency-dependent selection (NFDS) were evaluated in Prunus lannesiana var. speciosa in comparison with nuclear simple sequence repeat (SSR) loci that seemed to be evolutionarily neutral. Our observations of local mating patterns indicated that male-female pair fecundity was influenced by not only self-incompatibility, but also various factors, such as kinship, pollen production and flowering synchrony. In spite of the mating bias caused by these factors, the NFDS effect on changes in allele frequencies from potential mates to mating pollen was detected at the S-locus but not at the SSR loci, although the changes from adult to juvenile cohorts were not apparent at any loci. Genetic differentiation and isolation-by-distance over various spatial scales were smaller at the S-locus than at the SSR loci, as expected under the NFDS. Allele-sharing distributions among the populations also had a unimodal pattern at the S-locus, indicating the NFDS effect except for alleles unique to individual populations probably due to isolation among islands, although this pattern was not exhibited by the SSR loci. Our results suggest that the NFDS at the S-locus has an impact on both the mating patterns and the genetic structure in the P. lannesiana populations studied.

Process to extinction and genetic structure of a threatened Japanese conifer species, Picea koyamae

Since the end of the glacial age, Picea koyamae has been sparsely distributed in Japan as a relict species and is presently threatened with extinction. We investigated the population structure and genetic structure of nine populations of P. koyamae. Population size was assessed at 9–135 individuals in habitats ranging from 0.5 to 11.5 ha, and seedlings and saplings were observed in all but one particular population, which had a Sasa-type (bamboo grass) forest floor. The effective number of alleles per locus (Ne) within peripheral populations in the Yatsugatake Mountains was 1.8–2.7, much lower than that of core populations in the Akaishi Mountains (2.8–4.3) using five nuclear simple sequence repeat loci. This finding suggests that genetic variation in these populations has been reduced by isolation from other populations. The standardized genetic differentiation among populations (G′ST) was 0.410 and higher than that found in other Japanese conifers, suggesting that isolation and inbreeding have progressed in this species. In two isolated populations at the Yatsugatake Mountains, the fixation index (FIS) was 0.315–0.354, much higher than the values determined for the other populations (−0.188 to 0.263). This suggests that these two populations have survived several generations while increasing the degree of inbreeding. However, the highest seedling density was in a population with low genetic variation and high FIS. The most serious problems at present appear to be the declining number of mature trees and the deterioration of suitable environments for seedling establishment.

The genetic structure of Quercus crispula in northeastern Japan as revealed by nuclear simple sequence repeat loci

Previous studies have reached different discussions about the genetic variation and genetic structure of Quercus crispula populations in northeastern Japan. This is a common oak species in Eastern Asia. Some studies have suggested that the populations in northeastern Japan were derived from those remaining in the southwest after the last glacial maximum (LGM), whilst other studies have found evidence that populations persisted in northeastern Japan during the LGM. Using seven highly polymorphic nuclear simple sequence repeat loci, we investigated the genetic structure of 16 Q. crispula populations along a latitudinal gradient in northeastern Japan (northern Honshu and Hokkaido), spanning about half of the species' biogeographic range in the country. Although the level of population differentiation was low (F (ST) = 0.021; [Formula: see text] = 0.090), two geographically differentiated clusters were detected by STRUCTURE analysis. The first cluster included most of the populations in Hokkaido, and may indicate continued survival throughout past glacial periods. We found a significant decrease in allelic richness with latitude, so the second cluster may represent an expansion of the lineage from Honshu during the post-glacial period. These results should enhance our understanding of historical north-south migrations of this species in northeastern Japan.

Prié blanc and Legiruela: a unique grape cultivar grown in distant European regions

<p style="text-align: justify;"><strong>Aims</strong>: The aim of this study was to identify synonyms among grape cultivars growing in two distant regions, Spain and Italy, based on comparative analysis of genetic markers. The identification of synonyms and genetically-related cultivars should contribute to our understanding of their history and the preservation of biodiversity.</p><p style="text-align: justify;"><strong>Methods and results</strong>: Using a set of 9 nuclear simple sequence repeats (SSR) markers, we compared the genetic profiles of major and currently neglected grape cultivars from two laboratories in Spain and Italy. A set of approximately 700 genotypes were analyzed and several putative synonyms were discovered. Among these, a clear synonymy was identified (confirmed by ampelographic observations) between two minor varieties: the Spanish Legiruela and the Italian Prié blanc, known in the past as Agostenga. Using an additional set of 25 nuclear SSR loci, two white cultivars, Albillo Real (or Albillo de Madrid) and Luglienga bianca, from Spain and Italy, respectively, showed a genetic relationship with Prié blanc/Legiruela. To further characterize this parent/progeny kinship, chloroplast SSR analyses were performed. Several hypotheses on the origin and movements of these traditional ancient grapes are discussed.</p><p style="text-align: justify;"><strong>Conclusion</strong>: The Italian Prié blanc and the Spanish Legiruela cultivars correspond to a unique genotype. The occurrence in Spain under the name of Legiruela, a cultivar long-recognized in Italie, and the presence in both countries of very old grape varieties genetically related to this genotype, indicate that Prié blanc/Legiruela has moved from its original to actual growing area several centuries ago.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: By identifying synonyms, accurately establishing the « true to type » of traditional grapes (used for commercial trade or maintained in collections), and studying their genetic relationships, the present work affects marketing and commercial exploitations related to grape cultivars. It also contributes to the knowledge of cultivar history and movements in ancient times.</p>

Landscape genetic structure of Betula maximowicziana in the Chichibu mountain range, central Japan

We evaluated the genetic structure of 16 Betula maximowicziana populations in the Chichibu mountain range, central Japan, located within a 25-km radius; all but two populations were at altitudes of 1,100–1,400 m. The results indicate the effects of geographic topology on the landscape genetic structure of the populations and should facilitate the development of local-scale strategies to conserve and manage them. Analyses involving 11 nuclear simple sequence repeat loci showed that most populations had similar intrapopulation genetic diversity parameters. Population differentiation (FST = 0.021, G′ST = 0.033) parameters for the populations examined were low but were relatively high compared to those obtained in a previous study covering populations in a much larger area with a radius of approximately 1,000 km (FST = 0.062, G′ST = 0.102). Three populations (Iriyama, Kanayamasawa, and Nishizawa) were differentiated from the other populations by Monmonier’s and spatial analysis of molecular variance algorithms or by STRUCTURE analysis. Since a high mountain ridge (nearly 2,000 m) separates the Kanayamasawa and Nishizawa populations from the other 14 populations and the Kanayamasawa and Nishizawa populations are themselves separated by another mountain ridge, the genetic structure appears to be partly due to mountain ridges acting as genetic barriers and restricting gene flow. However, the Iriyama population is genetically different but not separated by any clear geographic barrier. These results show that the landscape genetic structure is complex in the mountain range and we need to pay attention, within landscape genetic studies and conservation programs, to geographic barriers and local population differentiation.

Genetic diversity in managed subpopulations of Norway spruce [ Picea abies (L.) Karst.

Uniparentally inherited chloroplast simple sequence repeats (cpSSRs), mitochondrial markers (mt), as well as biparentally inherited nuclear simple sequence repeats (SSRs) were used to analyse genetic diversity in different subpopulations of Norway spruce in Austria. For that purpose Norway spruce individuals of different age classes, old (70–100 years) versus young (6–10 years) trees, were fingerprinted to elucidate the genetic effects caused by human management. Three populations from different elevations were selected in a restricted area. Four hundred and fifty individuals, 50 old and 100 young trees from each population, were analysed for this study. Seven paternally inherited cpSSRs revealed no variation within and among populations. In contrast, analysis of three maternal mitochondrial markers showed variation in one locus in individuals originating from the high elevation sample set. Analysis of five polymorphic nuclear SSR loci revealed 135 different alleles. Nuclear SSRs results showed that there is generally more genetic variation and heterozygosity within populations than among populations. Populations from high elevation exhibit more genetic variation than samples from middle and low elevations.

A new set of microsatellite markers for the peach palm (Bactris gasipaes Kunth); characterization and across‐taxa utility within the tribe Cocoeae

AbstractA (GA)n microsatellite‐enriched library was constructed and a new set of 18 nuclear simple sequence repeat loci was isolated in Bactris gasipaes var. gasipaes. The loci were found to be highly variable in the target species and readily transferable to related Bactris species as well as to the Astrocaryum and Elaeis genera of the same Cocoeae tribe. These microsatellite resources are made available to study the genetic diversity and gene flow within the Bactris complex for a better understanding of the domestication process of the peach palm and for further Cocoeae genetics.

An extreme cytoplasmic bottleneck in the modern European cultivated potato (Solanum tuberosum) is not reflected in decreased levels of nuclear diversity

We have used the polymorphic chloroplast (cp) and nuclear simple sequence repeats (SSRs) to analyse levels of cytoplasmic and nuclear diversity in the gene pool of the European cultivated potato (Solanum tuberosum ssp. tuberosum). Primers designed from the complete chloroplast sequence of tobacco (Nicotiana tabacum) were used to amplify polymorphic products in a range of potato cultivars. Combining the data from seven polymorphic cpSSR loci gave 26 haplotypes, one of which (haplotype A) accounted for 151 out of the 178 individuals studied and corresponded to the T-type cytoplasm previously identified in cultivated potatoes using chloroplast restriction fragment length polymorphism analysis. Phylogenetic and diversity analyses of the relationships between cpSSR haplotypes confirmed much higher levels of cytoplasmic diversity outwith the T-type group. Diversity levels at eight nuclear SSR loci, however, were not significantly different between cytoplasmic groups, suggesting a severe maternal bottleneck in the evolution of the modern cultivated potato. These results highlight the importance in quantifying levels of cytoplasmic as well as nuclear diversity and confirm the need for a change in breeding practices to increase levels of non-T-type cytoplasm in the cultivated gene pool, thus helping reduce problems associated with pollen sterility. This may be facilitated by germplasm analysis using cpSSRs, which will allow efficient selection of diverse cytoplasm donors.