A range wide geographic pattern of genetic diversity and population structure of Castanea mollissima populations inferred from nuclear and chloroplast microsatellites

Understanding geographical pattern of genetic diversity and population structure is of great importance for formulating conservation and utilization strategies. In this study, we investigated the genetic diversity and population structure of 28 natural populations of Castanea mollissima in China using eight nuclear and six chloroplast microsatellite makers (nSSRs and cpSSRs). Populations from central China harbored the highest genetic diversity at both nSSR and cpSSR markers (nSSR: H _E?=?0.705; cpSSR: H?=?0.461). The standardized measure of genetic differentiation estimated as G′ _ST was 0.447 for nSSR and 0.803 for cpSSR, respectively. The G′_ST-based pollen to seed flow ratio is 3.043, indicating that pollen flow is not extensive among C. mollissima populations. No obvious population genetic structure by geographical locations was found by STRUCTURE analysis based on nSSR data, and similarly, no signal of phylogeographic structure was detected for cpSSR analysis. Five boundaries defining zones of maximum genetic differences within the network of the C. mollissima populations were found, and the locations of those barriers were consistent with those of four mountains, i.e., Daloushan Mountain, Dabashan Mountain, Wushan Mountain, and Qingliangfeng Mountain, indicating that those mountains might act as genetic barriers obstructing the genetic exchange among natural C. mollissima populations. These results provide valuable baseline data for conservation and utilization of this species.     

Genetic diversity and differentiation of the endangered Japanese endemic tree Magnolia stellata using nuclear and chloroplast microsatellite markers

Genetic diversity and differentiation were analyzed in 11 populations of Magnolia stellata (Sieb. and Zucc.) Maxim. (Magnoliaceae) in the Tokai district, Japan. Variation at four nuclear microsatellite (nSSR) loci was examined, three chloroplast microsatellite (cpSSR) markers were developed and 13 haplotypes identified. The 11 populations were divided into three groups (A, B and C). Each population within the group was separated less than 40 km. Group B harbored the highest gene diversity (H) and allelic richness (Ar) for nSSR (H=0.74 and Ar=8.02). Group C had the highest diversity of chloroplast haplotypes (H=0.79 and Ar=6.8): 2.5 times more haplotypes than the other groups. Each population contributed differently to the total diversity, with respect to nSSR and cpSSR. AMOVA revealed that 58% of haplotypic and 15% of nSSR variation was partitioned among populations within groups. A Mantel test revealed significant correlations between population pairwise geographic ln(distance) and F_ST/(1−F_ST) for both nSSR (r=0.479; P=0.001) and cpSSR (r=0.230; P=0.040). Dendrograms of populations for nSSR, based on Nei’s genetic distance, were constructed using UPGMA and the neighbor-joining method. These results suggest that populations in group C have diverged from the other populations, while those in group B are similar to each other. For group B, fragmentation between populations should be avoided in order to maintain gene flow. For group C, the uniqueness of each population should be given the highest priority when planning genetic conservation measures for the species.     

Development, characterization, genetic diversity and cross-species/genera transferability of ILP markers in rubber tree (Hevea brasiliensis)

To construct a linkage map enriched with tapping panel dryness (TPD)-related markers, we firstly utilized rubber tree ESTs associated with TPD to develop intron length polymorphism (ILP) markers. In this study, 52 new ILP markers were further developed. Together with the ILP markers previously reported, 102 ILP markers developed from TPD-related ESTs were analyzed within 39 Hevea germplasm in detail. The PCR success rate and polymorphism rate of ILP markers was 97.06 and 61.62 %, respectively. The results based on PCR amplification and sequence analyses provided the evidences on cross-species/genera transferability of rubber tree ILP markers. The average polymorphic information content (PIC) values of 39 Hevea germplasm were about 0.1719, indicating that the genetic base of Hevea germplasm selected in this study was very narrow. Among 39 Hevea germplasm, the PIC value of wild rubber tree accessions was the highest, followed by Hevea species and cultivated rubber tree clones. Based on the similarity coefficient of ILP markers, 39 Hevea germplasm were divided into three groups including cultivated clones, wild accessions and Hevea species, suggesting that the classification was generally related to the characterization of Hevea germplasm. The ILP markers developed in this study further enriches the number of molecular marker in rubber tree, and the ILP markers will have a wide application in DNA fingerprinting, genetic diversity, marker-assisted selection and genetic mapping, etc. Moreover, the ILP markers transferred cross-euphorbiaceae family might be utilized in cassava, castor bean and physic nut.     

Consequences of multiple mating‐system shifts for population and range‐wide genetic structure in a coastal dune plant

Evolutionary transitions from outcrossing to selfing can strongly affect the genetic diversity and structure of species at multiple spatial scales. We investigated the genetic consequences of mating‐system shifts in the North American, Pacific coast dune endemic plant Camissoniopsis cheiranthifolia (Onagraceae) by assaying variation at 13 nuclear (n) and six chloroplast (cp) microsatellite (SSR) loci for 38 populations across the species range. As predicted from the expected reduction in effective population size (N_e) caused by selfing, small‐flowered, predominantly selfing (SF) populations had much lower nSSR diversity (but not cpSSR) than large‐flowered, predominantly outcrossing (LF) populations. The reduction in nSSR diversity was greater than expected from the effects of selfing on N_e alone, but could not be accounted for by indirect effects of selfing on population density. Although selfing should reduce gene flow, SF populations were not more genetically differentiated than LF populations. We detected five clusters of nSSR genotypes and three groups of cpSSR haplotypes across the species range consisting of parapatric groups of populations that usually (but not always) differed in mating system, suggesting that selfing may often initiate ecogeographic isolation. However, lineage‐wide genetic variation was not lower for selfing clusters, failing to support the hypothesis that selection for reproductive assurance spurred the evolution of selfing in this species. Within three populations where LF and SF plants coexist, we detected genetic differentiation among diverged floral phenotypes suggesting that reproductive isolation (probably postzygotic) may help maintain the striking mating‐system differentiation observed across the range of this species.     

Geographic patterns of genetic variation in native pecans

A structured collection of 80 seedling pecan trees [Carya illinoinensis (Wangenh.) K. Koch], representing 19 putatively native pecan populations across the species range, was evaluated at three plastid and 14 nuclear microsatellite (simple sequence repeat, SSR) loci. Data were analyzed using a priori population designations and also within a Bayesian framework, in which individuals were assigned to clusters regardless of population of origin. Population genetic analyses using a priori populations, clusters based on chloroplast microsatellite data (cpSSR), and clusters based on nuclear microsatellite data (nSSR) yielded consistent results. For all groupings, cpSSR variation exhibited more geographic structure than the nSSR data. Furthermore, cpSSR microsatellite diversity decreased with increasing latitude, but this pattern was not observed with the nuclear data. Contrasting patterns in plastid and nuclear genetic diversity demonstrate unique aspects of postglacial recolonization reflected in the movement of seeds versus pollen. These data suggest that plastid SSRs are useful tools for identifying population structure in pecan and hold promise for ongoing efforts to identify and conserve representative germplasm in ex situ collections.     

Development of Chloroplast Microsatellite Markers and Analysis of Chloroplast Diversity in Chinese Jujube (Ziziphus jujuba Mill.) and Wild Jujube (Ziziphus acidojujuba Mill.)

Ziziphus is an important genus within the family Rhamnaceae. This genus includes several important fruit tree species that are widely planted in China and India, such as the Chinese jujube (Ziziphus jujuba Mill.), the wild jujube (Z. acidojujuba), and the Indian jujube (Z. mauritiana). However, information about their domestication based on the chlorotype diversity of Chinese jujube population is lacking. In this study, chloroplast microsatellite (cpSSR) markers were developed and used to investigate the genetic relationships between and domestication of jujube cultivars and wild jujube populations. Primer sets flanking each of the 46 cpSSR loci in non-coding regions of the chloroplast genome sequence of Z. jujuba Mill. cv. ‘Junzao’ were designed. In total, 10 markers showed polymorphisms from 15 samples (9 jujube cultivars and 6 wild jujube individuals), of which 8 loci were due to variations in the number of mononucleotide (A/T) repeats and 2 were due to indels. Six cpSSR markers were used in further analyses of 81 additional samples (63 jujube cultivars, 17 wild jujube samples, and 1 Indian jujube). Using these cpSSR markers, the number of alleles per locus ranged from two to four. In general, the Shannon Index (I) for each cpSSR ranged from 0.159 to 0.1747, and the diversity indices (h) and uh were 0.061 to 0.435 and 0.062 to 0.439, respectively. Seven chlorotypes were found; the Indian jujube showed distinct chlorotypes, and both the Chinese and wild jujube had four chlorotypes and shared two chlorotypes. A dominant chlorotype (G) accounted for 53 of 72 jujube cultivars and 13 of 23 wild jujube individuals. All chlorotypes were highly localized along the Yellow River, from the mid- to the lower reaches, suggesting a wide origin of jujube. These cpSSR markers can be applied to population and evolution studies of Chinese jujube and wild jujube.     

Genetic structure of gall oak (<Emphasis Type="Italic">Quercus infectoria</Emphasis>) characterized by nuclear and chloroplast SSR markers

Quercus infectoria, commonly known as gall oak, is a small shrub found in Iran. Unfortunately, it is subjected to genetic erosion, and so, its conservation and evaluation are desirable. Thus, in the current research, 16 microsatellite primer pairs (seven nuclear simple sequence repeats (nSSRs) and nine chloroplast simple sequence repeats (cpSSRs)) were used in an attempt to assess the genetic diversity of 121 individuals of Q. infectoria belonging to 11 populations from three provinces in northern Zagros forests of Iran. In total, 69 alleles of nSSR and 18 alleles of cpSSR were detected among the individuals. The results of the overall analysis of molecular variance based on nSSRs indicated that 89.00% of the variation was due to differences within populations and 11.00% occurred among populations, while according to cpSSRs, 94.00% of the variation resided among populations, and only 6.00% could be attributed to variation within populations. A higher genetic differentiation of Q. infectoria populations was found according to cpSSR data in comparison to nSSR data. Cophenetic correlation coefficient values were statistically insignificant between nSSR and cpSSR data. The unweighted pair group method with arithmetic mean and Bayesian cluster analyses grouped the studied individuals into two main clusters based on both nSSR and cpSSR data. nSSR data could not completely clustered individuals next each other according to their geographical collection area. Information detailed by nSSR loci revealed that north-Zagros gall oak preserves average levels of genetic diversity at the species level, high level of within-population genetic diversity, and moderate level of genetic variation among populations. The present results provide valuable data for in situ or ex situ conservation and utilization of the studied germplasm.     

Isolation and characterization of chloroplast microsatellite markers in four mangrove species,Aegiceras corniculatum,Avicennia marina,Acanthus ilicifolius and Lumnitzera racemosa

One, three, seven, and six of polymorphic chloroplast microsatellite (cpSSR) markers were developed from four mangrove species, Acanthus ilicifolius, Aegiceras corniculatum, Avicennia marina, and Lumnitzera racemosa, respectively. Characterization of 229, 509, 369, and 216 individuals of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa, collected from different natural mangrove populations (A. ilicifolius, 6; A. corniculatum, 14; A. marina, 10; L. racemosa, 6) in the southern coastline of China showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.005 and 0.675. Combining the polymorphic cpSSR loci of each species, 3, 5, 11, and 4 of cpSSR haplotypes were separately detected in populations of A. ilicifolius, A. corniculatum, A. marina, and L. racemosa in the southern coastline of China. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of these four species.     

Spatial and temporal population genetic variation and structure of Nothotsuga longibracteata (Pinaceae), a relic conifer species endemic to subtropical China

Nothotsuga longibracteata, a relic and endangered conifer species endemic to subtropical China, was studied for examining the spatial-temporal population genetic variation and structure to understand the historical biogeographical processes underlying the present geographical distribution. Ten populations were sampled over the entire natural range of the species for spatial analysis, while three key populations with large population sizes and varied age structure were selected for temporal analyses using both nuclear microsatellites (nSSR) and chloroplast microsatellites (cpSSR). A recent bottleneck was detected in the natural populations of N. longibracteata. The spatial genetic analysis showed significant population genetic differentiation across its total geographical range. Notwithstanding, the temporal genetic analysis revealed that the level of genetic diversity between different age class subpopulations remained constant over time. Eleven refugia of the Last Glacial Maximum were identified, which deserve particular attention for conservation management.     

Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Habitat fragmentation,genetic diversity,and inbreeding depression in a threatened grassland legume: is genetic rescue necessary?

Kincaid’s lupine (Lupinus oreganus), a threatened perennial legume of western Oregon grasslands, is composed of small, fragmented populations that have consistently low natural seed set, suggesting they may have accumulated high enough levels of genetic load to be candidates for genetic rescue. We used simple sequence repeat (SSR) loci, both nuclear DNA and chloroplast DNA, to screen populations throughout the species’ range for evidence of severe inbreeding and recent genetic bottlenecks due to habitat fragmentation. After genotyping about 40% of the known populations, only one of 24 populations had strong statistical evidence for a recent genetic bottleneck (H _e > H _eq). Both mean nSSR fixation coefficients and genetic diversity did not statistically differ between very small, small, medium, and large lupine population size classes. Within population chloroplast DNA haplotype number was high for an animal pollinated species, ≈4.2 haplotypes/population, and within population haplotype diversity was also relatively evenly distributed. Within population patterns of nSSR and cpSSR genetic diversity suggest that genetic diversity has not been lost over the last century of habitat fragmentation. With genet lifespan thought to exceed 100 years, overlap of several to many generations, and substantial reductions in seed set from inbreeding depression that shifts cohort composition towards those generated by outcrossing events, Kincaid’s lupine is likely maintain the currently high levels of within population genetic diversity. The case of Kincaid’s lupine provides an example of how the assumptions of severe inbreeding depression with small population size and habitat fragmentation can be inaccurate.     

Chloroplast microsatellite markers for the mangrove tree species Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa, and cross-amplification in other mangrove species

Chloroplast microsatellite (cpSSR) markers were developed for three ecologically and economically important tree species in the mangrove family, Rhizophoraceae: Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa. Noncoding regions of chloroplast DNA (cpDNA) from each species were separately amplified using universal chloroplast primers. Six, two, and three polymorphic cpSSR loci in B. gymnorrhiza, K. candel, and R. stylosa, respectively, were developed from amplified noncoding cpDNA regions. Characterization of 216, 156, and 253 individuals of B. gymnorrhiza, K. candel, and R. stylosa, respectively, collected from different natural mangrove populations (B. gymnorrhiza, 9; K. candel, 7; R. stylosa, 9) on Iriomote Island in Japan showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.027 and 0.480. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of the three species. Several of these markers may also be useful in similar studies of other mangrove species.     

Development of ten microsatellite loci for <Emphasis Type="Italic">Gentiana crassicaulis</Emphasis> (Gentianaceae)

Genetiana crassicaulis is one of famous Chinese medicinal plant. The over-collection for its root has caused its dramatic reduction. In order to devise adequate conservation and management strategies for this species, it is important to characterize its genetic diversity and understand its population structure. Here, 10 polymorphic microsatellite markers have been developed. AC/TG microsatellite was enriched by combining biotin capture method. Polymorphism of each locus was assessed in 30 individuals from six populations. The number of alleles ranged from 2 to 9 and the expected heterozygosity ranged from 0.32 to 0.78.     

Isolation and characterization of microsatellites in a medicinal plant,Phyllanthus emblica (Euphorbiaceae)

• Premise of the study: Microsatellite markers were isolated and characterized in a medicinal plant, Phyllanthus emblica, to study population genetics for designing an effective in situ and ex situ conservation of genetic resources of the species. • Methods and Results: Six microsatellite markers were developed using an enrichment and magnetic separation protocol. They were characterized in two natural populations of P. emblica. Out of the six microsatellites, five showed polymorphism, with the number of alleles ranging from four to seven. Observed and expected heterozygosities ranged from 0.360 to 0.760 and 0.499 to 0.806, respectively. • Conclusions: The five polymorphic microsatellite markers will be useful for studying the genetic structure, reproductive biology, and for identification of clones and provenances of this important medicinal plant.     

Population differentiation and gene flow within a metapopulation of a threatened tree, Magnolia stellata (Magnoliaceae)

We examined genetic differentiation among eight local populations of a metapopulation of Magnolia stellata using 10 nuclear and three chloroplast microsatellite (nSSR and cpSSR) markers and evaluated the influence of historical gene flow on population differentiation. The coefficient of genetic differentiation among populations for nSSR (F(ST) = 0.053) was less than half that for cpSSR (0.137). An isolation-by-distance pattern was detected for nSSRs, but not cpSSRs. These results suggest that pollen flow, as well as seed dispersal, has significantly reduced genetic differentiation among populations. We also examined patterns of contemporary pollen flow by paternity analysis of seeds from nine seed parents in one of the populations using the nSSR markers and found it to be greatly restricted by the distance between parents. Although most pollen flow occurred within the population, pollen flow from outside the population accounted for 2.5% of the total. When historical and contemporary pollen flows among populations were compared, the levels of pollen flow seem to have declined recently. We conclude that to conserve M. stellata, it is important to preserve the whole population by maintaining its metapopulation structure and the gene flow among its populations.     

Molecular evolution and functional characterisation of haplotypes of an important rubber biosynthesis gene in Hevea brasiliensis

Hydroxy‐methylglutaryl coenzyme‐A synthase (HMGS) is a rate‐limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non‐coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three‐dimensional structural studies highlighting the structural positioning of non‐synonymous SNPs from different HMGS haplotypes revealed that the ligand‐binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.     

Characterization of chloroplast microsatellite loci from whole chloroplast genome of Camellia taliensis and their utilization for evaluating genetic diversity of Camellia reticulata (Theaceae)

This study characterized chloroplast microsatellite markers for Camellia reticulata, a famous ornamental and edible economic tree species only distributed in Southwestern China. Thirty-two chloroplast microsatellite markers were originally annotated in the whole chloroplast genome of Camellia taliensis, ten polymorphic microsatellite markers were tested in 96 individuals from four natural populations of C. reticulata. Alleles numbered from two to four, and average value of Shannon's Information index, Nei's gene diversity, total genetic diversity, genetic diversity within populations, gene differentiation coefficient and gene flow index were 0.408, 0.225, 0.217, 0.102, 0.530 and 0.443, respectively. Our results showed high genetic differentiation and limited gene flow among the studied populations, which may be explained by recent fragmentation of the remnant populations due to human destruction and disturbance of natural habitats of the species. The identified cpSSR markers will be useful for the future studies on population genetics, conservation biology and phylogeography of C. reticulata.     

Morphological and molecular diversity among populations of Quercus brantii Lindl. in western forest of Iran

Abstract

This study represents a preliminary step toward understanding the genetic structure of Persian oak in Iran. The genetic variability of Quercus brantii in Western forest of Iran was evaluated by amplified fragment length polymorphism (AFLP), chloroplast microsatellite and leaf morphology. Fifty-five trees from eight regions were sampled from across the range of Chaharmahal va Bakhtiari province of Iran. Twenty morphological traits were analyzed through clustering and ordination method. At morphological level, the applied statistics suggest that macromorphological traits significantly differentiate between populations. The overall sample shows a proportion of AFLP polymorphic markers of 92.1%, denoting a high level of variability. Based on AFLP data, differences among populations within geographic regions account for 11.6% of the total variation and only 0.57% is attributed to variation among regions. Based on chloroplast microsatellite (cpSSR), 34% of total variation was found among populations, suggesting a high within-population haplotype diversity. The dendrogram obtained from cpSSR showed a general pattern quite different from the pattern obtained from morphological analysis and AFLP markers.     

Phylogenetic relationships within Hevea brasiliensis as deduced from a polymorphic mitochondrial DNA region

We have cloned a 4.5-kb mtDNA fragment showing a high RFLP polymorphism between various Hevea genotypes. Subcloning and sequencing of a 1.4-kb segment of this clone allowed us to design PCR amplification primers to isolate homologous mtDNA segments of about 0.9 kb from 23 representative genotypes of Hevea. Complete sequences from 4 genotypes showed between 6.7% and 20.2% of nucleotide diversity, suggesting the presence of a hypervariable, or hotspot, region. A sequence of 345 nucleotides within this region was determined for the 23 genotypes. The phylogenetic relationships inferred from the sequence comparison are in general agreement with the results obtained from mtDNA RFLP analysis, indicating that this polymorphic mtDNA region is a useful molecular marker for phylogenetic analysis within Hevea.