Genetic Structure of Wild Rice Oryza Glumaepatula Populations in Three Brazilian Biomes Using Microsatellite Markers

The existence of Oryza glumaepatula is threatened by devastation and, thus, the implementation of conservation strategies is extremely relevant. This study aimed to characterize the genetic variability and estimate population parameters of 30 O. glumaepatula populations from three Brazilian biomes using 10 microsatellite markers. The levels of allelic variability for the SSR loci presented a mean of 10.3 alleles per locus and a value of 0.10 for the average allelic frequency value. The expected total heterozygosity (H_e) ranged from 0.63 to 0.86. For the 30 populations tested, the mean observed (H_o) and expected heterozygosities (H_e) were 0.03 and 0.11within population, respectively, indicating an excess of homozygotes resulting from the preferentially self-pollinating reproduction habit. The estimated fixation index ( _IS ) was 0.79 that differed significantly from zero, indicating high inbreeding within each O. glumaepatula population. The total inbreeding of the species (_IT ) was 0.98 and the genetic diversity indexes among populations, _ST and _ST, were 0.85 and 0.90, respectively, indicating high genetic variability among them. Thus, especially for populations located in regions threatened with devastation, it is urgent that in situ preservation conditions should be created or that collections be made for ex situ preservation to prevent loss of the species genetic variability.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

濒危羊踯躅子代幼苗遗传多样性的SSR分析

利用本课题组此前开发的20对多态性较高的羊踯躅SSR引物,分析来自3个省份的4个羊踯躅野生幼苗自然居群(共64个株系)的遗传多样性和遗传结构,以期为羊踯躅保护提供科学依据。结果显示:(1)20对SSR引物共扩增出314个等位基因,每个SSR位点的平均等位基因数为15.700,多态信息含量PIC和有效等位基因数N_e的均值分别为0.850和4.457。(2)羊踯躅幼苗自然居群的基因多样性指数H和Shannon多样性指数I的均值分别为0.717和1.557,其中江西金溪(JX)居群的遗传多样性最丰富,浙江磐安(PA)的最低。(3)基于无限等位基因模型(IAM)分析发现,羊踯躅幼苗种群的基因流N_m和遗传分化系数F_(st)分别为1.372和0.155;AMOVA分析表明,羊踯躅幼苗种群的86.0%变异发生于居群内,仅有14%变异发生在居群间。(4)遗传距离法聚类NJ分析和Structure分析结果基本一致,分别聚为3大类和4大类。综合比较分析发现,羊踯躅幼苗种群中的各个遗传多样性相关的指标参数均低于成年自然居群,表明生境片段化已对羊踯躅的生存构成了极大的威胁。     

Assessing the genetic diversity of Panicum coloratum var. makarikariense using agro‐morphological traits and microsatellite‐based markers

Panicum coloratum var. makarikariense is a perennial C₄ grass native to South Africa with relatively good forage production under limited‐resource conditions. Genetic characterisation and breeding efforts have been scant, thus limiting its use in cattle raising systems. The goal of the present study was to assess the genetic diversity of a collection of P. coloratum var. makarikariense using agro‐morphological traits and molecular markers, in comparison with one accession of var. coloratum and one population of Panicum bergii. Agro‐morphological variability between and within accessions of var. makarikariense in a common garden setting was observed, showing that there is still opportunity for selection. Some accessions performed better than the commercialised material in relation to potential forage production. A total of 117 ISSR bands and 48 SSR alleles allowed the detection of genetic variability between and within accessions. The presence of accession‐specific bands suggested distinctness and limited gene flow. The genetic variability encountered in the commercialised material suggested that it is a stabilised population which has not undergone a strong selection process. Low correlation between agro‐morphologic and molecular variability was observed indicating that both approaches provide complementary information. Both morphological and molecular markers reveal genetic differentiation between varieties and species. This study provides a set of new SSR markers available for diversity assessment and valuable information that can be applied directly in collection management for breeding and conservation programmes.     

Genetic characterization of <Emphasis Type="Italic">Brycon hilarii</Emphasis> (Characiformes) populations within the Pantanal: Aspects of their conservation within a globally important neotropical wetland

Brycon hilarii, a characid species endemic to the Upper Paraguay hydrographic basin, is important to regional artisanal and sports fisheries. To develop effective strategies for conservation of this species in the face of potential environmental changes in the Pantanal region, we characterized genetic structuring within and among six B. hilarii collections based on variation at five microsatellite DNA markers. Within-population genetic variability was high, with 75 different alleles; mean average allelic richness per locus per sample location ranged from 6.06 to 7.99. Nei’s gene diversity (hs) varied among drainages from 0.66 (±0.2) to 0.69 (±0.2), with an average across the four genetically identified populations of 0.68 (±0.02). Analyses of Jost’s D _EST and F _ST-like indices, AMOVA, and Structure-based clustering analyses indicated that B. hilarii populations exhibit a low level of genetic structure, with some indications that the Taquari River population is somewhat distinct from others. Results of K-means analysis suggested little or no structuring, with weakly differentiated populations above and below the confluence of the Paraguay and Taquari rivers. Because B. hilarii populations in the Pantanal are linked by high levels of gene flow, habitat alterations that would interfere with gene flow may jeopardize the long-term persistance of the species.     

Is the genetic diversity of small scattered forest tree populations at the southern limits of their range more prone to stochastic events? A wild cherry case study by microsatellite-based markers

Wild cherry (Prunus avium L.) is a widespread, partially asexual, noble hardwood European species characterized by a scattered distribution, small population sizes, and human exploitation for its valuable wood. These characteristics, especially at the southern limits of the species natural distribution where additional varying stresses may occur, render P. avium populations prone to potential stochastic, genetic, and demographic events. In this study, we used dominant inter simple sequence repeat (ISSR) and codominant simple sequence repeat (SSR) markers to infer the genetic structure of P. avium. Five populations from northern Greece were evaluated based on 46 ISSR and 11 SSR loci. Populations presented a relatively high level of genetic variation, with a mean genetic diversity of H _e?=?0.166 and H _e?=?0.740 regarding ISSR and SSR analysis, respectively. We observed moderate population differentiation for ISSR (G _ST?=?0.113) and SSR (F _ST?=?0.097) markers. AMOVA also detected significant differentiation among populations for ISSRs (?? _ST?=?0.338) and SRRs (?? _ST?=?0.162). According to linkage disequilibrium analysis, estimates of effective population size were generally sufficient for maintaining extant genetic variability and evolutionary potential. A possible bottleneck was detected for only one population. In general, it appears that despite the particular characteristics of the P. avium populations studied, genetic stochasticity events were not apparent. The studied populations, located at the rear edge of the species European distribution, reveal a wealth of genetic variation that is very valuable for the genetic conservation of local adaptive gene complexes, especially under contemporary climatic change scenarios.     

Isolation and characterization of new microsatellite markers from the Japanese scallop (<Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>)

Twenty polymorphic microsatellite loci were isolated from the Japanese scallop (Patinopecten yessoensis) using a method of hybridizing twice. The genetic diversity was analyzed by these microsatellite markers in field specimens from Zhangzidao Island in Liaoning Province, China. Observed heterozygosity (H _o), expected heterozygosity (H _e), number of effective alleles (N _e), and polymorphism information contents (PIC) were calculated. The heterozygote deficiency or excess was detected by the fixation index (Fis). The Hardy–Weinberg equilibrium was tested by possibilities (P _HWE) using chi-square test. Results indicate that the average of allele numbers was 6.0. The mean values of the parameters H _o, H _e, N _e, and PIC were 0.7921, 0.7076, 4.4347, and 0.6684, respectively. Only 4 of 20 microsatellite markers showed heterozygote deficiency including loci FJ262370, FJ262381, FJ262384, and FJ262400. The P _HWE value indicates the wild population deviated somewhat from the Hardy–Weinberg equilibrium. These newly isolated markers increase the available molecular resources of the Japanese scallop.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Past bottlenecks and current population fragmentation of endangered huemul deer (<Emphasis Type="Italic">Hippocamelus bisulcus</Emphasis>): implications for preservation of genetic diversity

Small populations in fragmented habitats can lose genetic variation through drift and inbreeding. The huemul (Hippocamelus bisulcus) is an endangered deer endemic to the southern Andes of Chile and Argentina. Huemul numbers have declined by 99% and its distribution by 50% since European settlement. The total population is estimated at less than 2,000 individuals and is highly fragmented. At one isolated population in Chilean Patagonia we sampled 56 individuals between 2005 and 2007 and genotyped them at 14 microsatellite loci. Despite low genetic variability (average 2.071 alleles/locus and average H _O of 0.341), a low inbreeding coefficient (F _IS) of 0.009 suggests nearly random mating. Population genetic bottleneck tests suggest both historical and contemporary reductions in population size. Simulations indicated that the population must be maintained at 75% of the current size of 120 individuals to maintain 90% of its current genetic diversity over the next 100 years. Potential management strategies to maintain genetic variability and limit future inbreeding include the conservation and establishment of habitat corridors to facilitate gene flow and the enlargement of protected areas to increase effective population size.     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.     

Analysis of molecular genetic diversity and population structure in Amaranthus germplasm using SSR markers

We assessed the molecular genetic diversity and population structure of Amaranthus species accessions using 11 simple sequence repeat markers. A total of 122 alleles were detected, and the number of alleles per marker (N_A) ranged from 6 to 21 with an average of 11.1 alleles. The frequency of major alleles per locus ranged from 0.148 to 0.695, with an average value of 0.496 per marker. The overall polymorphic information content values were 0.436–0.898, with an average value of 0.657. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged from 0.056 to 0.876 and from 0.480 to 0.907, with average values of 0.287 and 0.698, respectively. The average H_O (0.240) was lower than the H_E and gene flow (Nm), and showed substantial genetic variability among all populations of amaranth accessions. The sample groupings did not strictly follow the geographic affiliations of the accessions. A similar pattern was obtained using model-based structure analysis without grouping by species type. Knowledge of the genetic diversity and population structure of amaranth can be used to select representative genotypes and manage Amaranthus germplasm breeding programs.     

A genetic linkage map of microsatellite,gene-specific and morphological markers in diploid <Emphasis Type="Italic">Fragaria</Emphasis>

Knowledge of the genetic diversity of a species is important for the choice of crossing parents in line and hybrid breeding. Our objective was to investigate European winter triticale using simple sequence repeat (SSR) markers and the coancestry coefficient (f) with regard to genetic diversity and grouping of germplasm. Three to five primer pairs for each of the 42 chromosomes were selected to analyse 128 European winter triticale varieties and breeding lines. SSR analysis resulted in the identification of 657 alleles with an average of 6.8 alleles per primer pair. The average polymorphism information content (PIC) for polymorphic markers was 0.54. Correlation between f and genetic similarity (GS) estimates based on Rogers Distance was low (r_f×GS(ABDR)=0.33). The analysis of molecular variance (AMOVA) revealed that 84.7% of the total variation was found within breeding companies, and 15.3% among them. In conclusion, SSR markers from wheat and rye provide a powerful tool for assessing genetic diversity in triticale. Even though no distinct groups within the European winter triticale pool could be detected by principal co-ordinate analysis, this study provides basic information about the genetic relationships for breeding purposes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by H.C. Becker     

Microsatellite variation in China’s Hainan Eld’s deer (<Emphasis Type="Italic">Cervus eldi hainanus</Emphasis>) and implications for their conservation

Hainan Eld’s deer (Cervus eldi hainanus) experienced a dramatic decline in the late 1960s through early 1970s and by 1976 only 26 deer remained in Datian of Hainan Island, China. Since then, conservation efforts have successfully rescued this deer from extinction. We employed 10 microsatellite DNA loci to index genetic variation in the one source (Datian) and two introduced populations (Bangxi and Ganshiling) and suggest implications for the conservation of the species. A total of 40 alleles at 10 loci were examined from 198 deer blood samples. The source population harbored all 40 alleles, while the Bangxi and Ganshiling translocated populations contained 24 and 26 alleles, respectively. The genetic variability was low (H _e ≈ 0.33) for each of the three populations. No significant difference in genetic variability between the three populations was detected (P > 0.05); yet significant differentiation was found among the three populations. Our results suggest that founder effects and genetic drift have affected the two translocated populations. For conservation we recommend the three populations be managed as a meta-population. When establishing future reintroductions, the founder population should have a size larger than the original 26 founders in Datian population or be composed of a cohort of over 20 same-age individuals with 1:1 sex ratio. Genetic monitoring for both the source and translocated populations should be continuously conducted in order to assess the effectiveness of deer conservation in the future.     

Genetic diversity of an endangered plant, <Emphasis Type="Italic">Cypripedium macranthos</Emphasis> var. <Emphasis Type="Italic">rebunense</Emphasis> (Orchidaceae): background genetic research for future conservation

Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, n _a and n _e were 1.85 and 1.28 respectively, and H _o and H _e were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future.     

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (H_E) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.     

A Study of Conservation Genetics in Cupressus chengiana,an Endangered Endemic of China,Using ISSR Markers

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (H _e) was 0.3120, effective number of alleles (A _e) was 1.5236, and Shannon’s information index (I) was 0.4740. At the population level, PPB = 48%, A _e=1.2774, H _e=0.1631, and I=0.2452. Genetic differentiation (G _st) detected by Nei’s genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (N _m ) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r=0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.     

Development of robust genomic simple sequence repeat markers for estimation of genetic diversity within and among bulb onion (Allium cepa L.) populations

Improved understanding of genetic diversity in onion and shallot (Allium cepa L.) is required to inform breeding and genetic resource conservation, and to enable development of association genetics and seed quality assurance methods. To develop quantitative estimates of diversity we estimated within- and among-population heterozygosity in a set of onion populations using genomic simple sequence repeat (SSR) markers developed by genomic skim sequencing. Primer sets (166) designed to flank SSR motifs identified were evaluated in a diverse set of lines, with 80 (48?%) being polymorphic. The 20 most robust single copy markers were scored in 12 individuals from 24 populations representing short-day to long-day adapted material from diverse environments. The average genetic diversity estimate (H _e) per population was 0.3 (SD 0.08) and the average per marker was 0.49 (SD 0.2). The onion populations assessed in this survey were distinct with moderate to large population differentiation but also had high within-population variation (F _st?=?0.26). There was evidence of inbreeding (F _is?=?0.22) with observed heterozygosities lower than the expected. This marker resource will be applicable for DNA fingerprinting, measuring levels of inbreeding in breeding lines, assessing population structure for association mapping and expanding linkage maps that are principally based on expressed sequence tag-based markers. A Galaxy workflow was developed to facilitate bulk SSR marker design from next-generation sequence data. This study provides one of the first quantitative views of population genetic variation in onion and a practical toolset for further genetics.     

Development of thirty new polymorphic microsatellite primers for <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Four new microsatellite primer pairs were developed in tree peony (Paeonia suffruticosa) based on the database mining and other twenty-six primer pairs by fast isolation by AFLP of sequences containing repeats (FIASCO) method. The polymorphism of each locus was further evaluated in 40 individuals of one population plus 5 tree peony related species. The number of alleles per locus ranged from 3 to 7 and the expected (H_e) and observed (H_o) heterozygosity at each locus ranged from 0.42 to 0.78 and 0.28 to 0.59, respectively. These microsatellite markers will be useful for investigating genetic diversity and studies of population genetic structure of tree peony.