Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

Development of microsatellite markers in Cordia bifurcata (Boraginaceae) and cross-species amplification in Cordia inermis and Cordia pringlei

We developed 16 microsatellite markers in Cordia bifurcata, a Central and South American shrub. The markers show low polymorphism in C. bifurcata, a species suspected of self-fertilization or apomixis. Of four polymorphic loci, three had only two alleles. However, current research indicates that these markers hold value for interpopulational comparisons of C. bifurcata and for analyses of congeners. In Cordia inermis, a dioecious or subdioecious shrub, seven of the markers produced interpretable amplification products of which five showed polymorphism. In Cordia pringlei, a distylous shrub, nine of the markers produced interpretable amplification products of which six showed polymorphism.     

Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR

Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.     

泰国Khao Yai国家公园豹猫和云豹的生态和保护

于1997年10月到1999年10月在泰国KhaoYai国家公园对两种同域分布的物种（即豹猫和云豹）进行了研究。使用带诱饵的陷阱捕捉了这些动物,进行麻醉,确定了性别、年龄,并戴上了无线电项圈,对6只雌性豹猫和4只雄性豹猫进行了1—18个月的无线电追踪。豹猫在干旱季节的利用区域大于雨季,雄性利用的区域大于雌性。在夜间和晨昏时节,豹猫活动增加,但是并没有节律性活动。所有豹猫个体在旱季和雨季的活动都相似,但雄性个体的白天活动多于雌性。豹猫的行走距离有性别差异但是没有季节差异;其食物以鼠类为主。分别对1只雌性云豹和1只雄性云豹进行了17个月和7个月的无线电追踪,雌性个体的活动面积为39．4km^2,而雄性个体活动面积为42．2km^2,核心区都是2．9km2。云豹对半绿林的使用大于其它类型的植被,在晨昏和夜间的活动增加,其活动无节律。     

Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.     

The systematic status of the Iriomote cat (Prionailurus iriomotensis Imaizumi 1967) and the subspecies of the leopard cat (Prionailurus bengalensis Kerr 1792)

The taxonomic status of the Iriomote cat ( Prionailurus iriomotensis Imaizumi 1967) has been the subject of considerable discussion. Wozencraft's (1993) decision that it is merely a subspecies of Prionailurus bengalensis has been adopted by IUCN, which may affect the chances of conserving this unique animal. This paper reports on an investigation into the relationship between the Iriomote cat and other species, using skull measurements to calculate skull configurations, from which species and subspecies may be compared with each other. The results prove that the Iriomote cat cannot be subsumed as a subspecies of Prionailurus bengalensis but is a species of its own. It is closely related to the genus Prionailurus but also has affinities with the genera Pardofelis and Profelis . It appears to be a very ancient species, a 'missing link', nearer to the common root of the cat tribe than any other extant species.     

Twelve new polymorphic microsatellite markers from the loggerhead sea turtle (Caretta caretta) and cross-species amplification on other marine turtle species

We describe 12 new polymorphic dinucleotide microsatellite loci and multiplex Polymerase Chain Reaction conditions from the loggerhead sea turtle Caretta caretta. Levels of polymorphism were assessed in 50 individuals from the nesting population of the Cape Verde Islands. Number of alleles ranged from 3 to 13 (average of 7.33) and the values of observed heterozygosities from 0.32 to 0.80 (average of 0.61). Cross-species amplification on three other marine turtles, Chelonia mydas, Eretmochelys imbricata and Dermochelys coriacea, revealed polymorphism and variability at eight, eleven and three loci, respectively.     

Genetic diversity and population structure of the endangered medicinal plant Phellodendron amurense in China revealed by SSR markers

Phellodendron amurense is an endangered tree with important medicinal and economic value in China. In this study, eight nuclear SSR primer pairs were employed to assess the genetic diversity and structure of 22 natural populations, including 516 individuals. A total of 66 alleles were detected with an average of 8.3 alleles per locus ranging from 3 to 17. The expected heterozygosity (He) of each SSR locus varied from 0.347 to 0.877 (average 0.627). Analysis of molecular variance (AMOVA) revealed that the main variation component existed within populations (95.11%) rather than among populations (4.89%). The Wilcoxon's sign-rank tests did not show any recent bottleneck effect in any population. A Mantel test displayed a significant correlation between the geographic distances and genetic distances for all populations (r = 0.566, P = 0.0001), indicating conformity to the isolation by distance model. Bayesian clustering and UPGMA supported grouping the populations into two groups. The present genetic structure of P. amurense may be explained by geographical isolation. The lack of genetic structure and genetic diversity decreased with increasing latitude within the Northeast China group may be due to postglacial northward expansion from a single refugium. Proper conservation measures are proposed for this species.     

Characterization and cross‐species amplification of microsatellite markers in cherimoya (Annona cherimola Mill., Annonaceae)

Fifteen polymorphic microsatellites were developed using a CT/AG‐enriched genomic library of cherimoya cv. Fino de Jete and screened on 23 genotypes from different geographical areas. This is the first set of microsatellites developed in Annonaceae. Fourteen of the microsatellites detected a single locus and only one detected two loci. The number of alleles in the single locus microsatellites ranged from two to six and the observed and expected heterozygosity ranged from 0.17 to 0.61 and from 0.12 to 0.76, respectively. Most of the simple sequence repeats were transferable to other species in the family.     

Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species

? Premise of the study: Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. ? Methods and Results: Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. ? Conclusions: These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.     

Ecology driving genetic variation: a comparative phylogeography of jungle cat (Felis chaus) and leopard cat (Prionailurus bengalensis) in India

Background

Comparative phylogeography links historical population processes to current/ecological processes through congruent/incongruent patterns of genetic variation among species/lineages. Despite high biodiversity, India lacks a phylogeographic paradigm due to limited comparative studies. We compared the phylogenetic patterns of Indian populations of jungle cat (Felis chaus) and leopard cat (Prionailurus bengalensis). Given similarities in their distribution within India, evolutionary histories, body size and habits, congruent patterns of genetic variation were expected.

Methodology/Principal Findings

We collected scats from various biogeographic zones in India and analyzed mtDNA from 55 jungle cats (460 bp NADH5, 141 bp cytochrome b) and 40 leopard cats (362 bp NADH5, 202 bp cytochrome b). Jungle cats revealed high genetic variation, relatively low population structure and demographic expansion around the mid-Pleistocene. In contrast, leopard cats revealed lower genetic variation and high population structure with a F _ST of 0.86 between North and South Indian populations. Niche-model analyses using two approaches (BIOCLIM and MaxEnt) support absence of leopard cats from Central India, indicating a climate associated barrier. We hypothesize that high summer temperatures limit leopard cat distribution and that a rise in temperature in the peninsular region of India during the LGM caused the split in leopard cat population in India.

Conclusions/Significance

Our results indicate that ecological variables describing a species range can predict genetic patterns. Our study has also resolved the confusion over the distribution of the leopard cat in India. The reciprocally monophyletic island population in the South mandates conservation attention.     

A set of chloroplast microsatellite primers for Eucalyptus (Myrtaceae)

We present a set of 35 chloroplast microsatellite primers for Eucalyptus. Ten of the microsatellites displayed intraspecific polymorphism, and identified nine haplotypes among 16 Eucalyptus globulus individuals. Primer conservation was high, with a polymerase chain reaction (PCR) success rate of 98% when tested on four other Eucalyptus species and seven additional myrtaceous genera. Chloroplast microsatellites have applications in phylogeographic studies, fingerprinting and progeny analysis.     

Conservation of rare species with island-like distributions: A case study of Lasthenia conjugens (Asteraceae) using population genetic structure and the distribution of rare markers

Californian vernal pools, a patchy, island-like habitat, are endangered as a result of habitat destruction. Conservation of the remaining vernal pool habitat is essential for the persistence of several endangered species. We present the first study examining DNA-level genetic diversity within and among populations of a vernal pool plant species. We investigated genetic variation across eight populations of the US federally endangered vernal pool endemic Lasthenia conjugens (Asteraceae) using intersimple sequence repeat (ISSR) markers. Genetic diversity within the species was high (Nei's gene diversity estimate was 0.37), with moderate differentiation among populations (Bayesian F _ST analog of 0.124). Using an amova analysis, we found that the majority of the genetic variation (84%) was distributed within populations. There is a significant relationship between geographical distance and pairwise genetic differentiation as measured by the Bayesian estimate θ^B. The alternative hypotheses of historic geological processes within the Central Valley and contemporary gene flow are discussed as explanations of the data. Because of the vulnerability of the populations, we calculated a probability of loss for rare alleles (fragments) in the populations. Calculations show that sampling only one of the eight populations for ex-situ conservation or restoration will capture approximately 54% of the sampled rare fragments. We believe that one of the sampled populations has become extinct since it was sampled. When removing this population from the above-mentioned calculations, sampling one population will capture only 41.3% of the sampled rare fragments. We recommend sampling strategies for future conservation and restoration efforts of L. conjugens.     

Microsatellite DNA variation in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and cross-species amplification in the Acipenseridae

Overharvest and habitat alteration have led toa collapse of most commercial Atlantic sturgeon(Acipenser oxyrinchus oxyrinchus)fisheries while pushing the species to rarityor extirpation in most of its historical range. A biologically sound conservation program forthis species requires knowledge of its geneticdiversity and of the evolutionary relationshipsamong geographic populations. To address theseresearch needs, six microsatellite loci wereisolated from A. o. oxyrinchus. Pedigreeanalysis suggested that all are inherited in acodominant Mendelian pattern. The six lociwere tested in ten additional sturgeon speciesfrom three genera and three apparent ploidylevels (4n, 8n, 16n). Approximately 70% ofsuccessful locus-species amplifications werepolymorphic. Polysomy was observed most oftenin 8n and 16n species. Genetic diversity andpopulation structure of A. o. oxyrinchuswere assayed using three polymorphic Aoxmarkers and four markers developed from lakesturgeon (A. fulvescens). A. o.oxyrinchus were sampled from the AltamahaRiver, Georgia, USA north to the St. LawrenceRiver, Quebec, Canada. Gulf sturgeon, A.o. desotoi, were sampled from the SuwanneeRiver, Florida, USA, to assess differentiationbetween the subspecies. Seventy-seven alleleswere observed to segregate into uniquemultilocus genotypes for each of the 392individuals assayed. Mean diversity wasgreatest in the Chesapeake Bay (9.7 alleles perlocus) and Delaware River (7.4 alleles perlocus) collections, and lowest in the St.Lawrence River (4.6 alleles per locus). Meanheterozygosity across seven loci ranged from44.3% (St. Lawrence River) to 62.6% (Altamaha River). Significant allelic heterogeneity wasobserved in 82% of pairwise comparisons aswell as a global test (p < 0.0001) for A.o. oxyrinchus collections. Genetic distancesuggests the presence of at least sixsubpopulations in A. o. oxyrinchus: St.Lawrence River, St. John River, Hudson River,Delaware River, Albemarle Sound, and AltamahaRiver. Genetic and geographic distances werepositively correlated (r = 0.57, p < 0.03) amongA. o. oxyrinchus, suggesting isolation bydistance and philopatry. Hierarchical genediversity analysis indicated significantgenetic population structure at every level. Maximum likelihood assignment tests correctlyassigned individual fish to collection with ahigh rate of success (mean = 87.5%); this andother lines of evidence indicated that theChesapeake Bay collection represents a mixedpopulation of sub-adult sturgeon from northernand southern Atlantic coast populations. Population structure was correlated with thatsuggested by earlier mitochondrial (mt) DNAanalyses. Significant diversity was observedbetween two Canadian populations from whichonly a single mtDNA haplotype has beenreported.     

Population genetic characterization of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) using novel microsatellite markers

The dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) has long been considered an endangered insect in South Korea; the detection of recent population increases leaves its endangered status uncertain. Population genetic analysis subsequent to development of molecular markers is essential for establishing proper conservation strategies. In this study, we developed ten microsatellite markers specifically for C. tripartitus. Sixty-eight individuals of C. tripartitus collected from six South Korean localities were genotyped to validate these markers and preliminarily assess population genetic characteristics. Per-locus observed number of alleles, observed heterozygosity (H_O), and expected heterozygosity (H_E) ranged from 5–12, 0.499–0.958, and 0.54–0.743, respectively. All populations showed higher H_O than H_E, negative values of inbreeding coefficient, and, overall, no sign of recent population bottlenecks (excluding one population, Seosan). This suggests that C. tripartitus did not suffer from genetic drift and inbreeding, which are typically severe in small, isolated populations. Nevertheless, detection of only one of the two gene pools in some populations and resultant genetic subdivision into two population groups may suggest that the population size is not enough to cover both gene pools. Thus, a more extended period of protection may be required to ensure higher genetic diversity of widespread populations and achieve the long-term conservation goal.     

韩国豹猫与其它豹猫及西表猫之间的遗传分化:基于IRBP、12S rRNA 和Cyt b 序列的分析

利用韩国豹猫的IRBP、12S rRNA 和细胞色素b (Cyt b)序列,并将其与GenBank 中的豹猫及西表猫的上述基因进行比较,以此来重新分析韩国豹猫和其它豹猫及西表猫之间的遗传分化情况。首先,我们从18 只韩国豹猫的IRBP、12S rRNA 和Cyt b 序列分析中分别检测到6 个等位基因、1 个单倍型和3 个单倍型,推测曾经发生 在豹猫种群的瓶颈事件可能是本文发现的不同分子标记之间存在基因型丢失差异的原因。基于Cyt b 序列分析,证实东亚的豹猫形成一个单独的进化枝,本文得到的Cyt b 序列和之前发布结果支持一个分类理论,即认为东亚的豹猫就是东北森林猫。我们发现在IRBP、12S rRNA 基因上,韩国豹猫和西表猫存在相同的序列,支持将西表猫和豹猫视为同一物种。     

Genetic diversity of the endangered Chinese endemic herb Saruma henryi Oliv. (Aristolochiaceae) and its implications for conservation

Saruma henryi Oliv., the only representative of the monotypic genus Saruma Oliv. (Aristolochiaceae), is an endangered perennial herb endemic to China. It is a phylogenetically, ecologically, and medicinally important species. In the present study, inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 14 populations. A total of 16 selected primers yielded 175 bright and discernible bands, with an average of 10.94 per primer. POPGENE analysis showed that the genetic diversity was quite low at the population level (h = 0.0447–0.1243; I = 0.0642–0.1853; PPB = 10.29–36.57%), but pretty high at the species level (h = 0.2603; I = 0.3857; PPB = 73.71%). The hierarchical analysis of molecular variance (AMOVA) revealed a high level of genetic differentiation among populations (67.18% of total variance components, P < 0.001), in line with the gene differentiation coefficient (G _ST = 0.6903) and the limited among-population gene flow (N _m = 0.2243). Both Principal Coordinates Analysis (PCoA) and UPGMA cluster analysis supported the grouping of all 14 populations into three geographic groups, among which there occurred a moderate level of genetic differentiation (33.18% of total variance components, P < 0.001) as shown by AMOVA analysis. In addition, Mantel test revealed a significant correlation between genetic and geographic distances among populations (r = 0.7792, P = 0.001), indicating the role of geographic isolation in shaping its present population genetic structure. The present levels and patterns of genetic diversity of S. henryi were assumed to result largely from its breeding system, geographic isolation, clonal growth, its unique biological traits and evolutionary history. The high genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered herb.