Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.     

Development of 10 microsatellite loci for <Emphasis Type="Italic">Rheum tanguticum</Emphasis> (Polygonaceae)

Rhubarb is an important Traditional Chinese Medicine. However, the wild resource has been declining. In order to design appropriate conservation methods for the official species across their natural distributions, it is important to characterize their genetic diversity. Here, we describe the development of 10 new microsatellite loci for AC/TG/CCA in Rheum tanguticum. The microsatellites were enriched using the combined biotin capture method. The polymorphism of each locus was further assessed in 12 individuals from four geographically distinct populations of this species. The number of alleles ranged from three to seven and the expected heterozygosity ranged from 0.53 to 0.73. All markers have been checked in the other three species in the genus and two of them together comprise the official medicinal rhubarb resource with R. tanguticum. These microsatellite markers could provide a useful tool for genetic and conservation studies of the rhubarb species.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Ardisia crenata</Emphasis> (Myrsinaceae)

Ardisia crenata, an evergreen shrub native to East Asia, has been a serious invasive plant to the southeastern USA. Here 13 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of A. crenata. The average allele number of these microsatellites was four per locus, ranging from two to seven. The ranges of observed and expected heterozygosity were 0.000–1.000 and 0.239–0.789, respectively. These microsatellite markers will be useful for investigating population genetics and reproductive ecology of A. crenata.     

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.     

Development and characterization of microsatellite markers for an endangered shrub, <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> (Leguminosae) and cross-species amplification in <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis>

We developed 11 polymorphic microsatellite markers for an endangered shrub Ammopiptanthus mongolicus using the Fast Isolation by AFLP of Sequences Containing repeats protocol. Polymorphism of each locus was assessed in 24 individuals from five natural populations of A. mongolicus. The average allele number of these markers was 4.2, ranging from 2 to 7. The observed (H _O) and expected (H _E) heterozygosities were 0.044–0.956 and 0.044–0.827, respectively. Further assessment in the congeneric species A. nanus revealed that nine of eleven loci were successfully amplified, but only two loci showed polymorphism. These markers provide powerful tools for the conservation genetics studies of A. mongolicus.     

Isolation and characterization of nine microsatellite loci for the takin (<Emphasis Type="Italic">Budorcas taxicolor</Emphasis>)

We report on the isolation and characterization of nine microsatellite markers in the takin (Budorcas taxicolor) from genomic DNA-enriched libraries. Twenty-eight microsatellites were screened from the libraries, and nine of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 28 individuals ranged from two to seven, and the expected and observed heterozygosity was 0.105–0.758 and 0.071–0.821, respectively. Four loci (TK01, TK02, TK04 and TK08) of nine deviated from Hardy-Weinberg expectation and no significant linkage association was found among all these loci. These microsatellite markers provide useful tool for population genetic studies of the takin.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Characterization of novel microsatellite loci in rare minnow (<Emphasis Type="Italic">Gobiocypris rarus</Emphasis>) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2–7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.     

Development of microsatellite markers in a mangrove tree species Aegiceras corniculatum (Myrsinaceae)

Aegiceras corniculatum is an ecologically important mangrove tree species. We isolated 18 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with polymorphism of two to eight alleles per locus. The observed and expected heterozygosities ranged from 0.050 to 0.550, and from 0.097 to 0.736, respectively. These markers will contribute to research on the conservation, genetic diversity and mating patterns of A. corniculatum.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

Polymorphic microsatellite loci for the cardinal fish (<Emphasis Type="Italic">Apogon imberbis</Emphasis>)

Eight polymorphic microsatellite loci were isolated and characterized for the Cardinal fish (Apogon imberbis), a coastal-reef fish endemic to the Mediterranean Sea. Characterization of 30 Cardinal fish individuals form the western Mediterranean showed moderate to high allelic diversity ranging from 6 to 19 alleles per locus. Two loci showed significant departures from Hardy-Weinberg equilibrium presumably due to null alleles. No evidence of linkage disequilibrium was found for any locus pairwise comparasions. This microsatellite set could be useful for any basic population genetic studies of this species.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Cryptocarya chinensis</Emphasis> in lower subtropical China

We report on the isolation and characterization of eight microsatellite markers from repetitive DNA enriched libraries for Cryptocarya chinensis from lower subtropical China. These are the first microsatellite loci reported for Cryptocarya species. The number of alleles ranged from three to nine. Observed and expected heterozygosities ranged from 0.0518 to 0.9910 and 0.5241 to 0.7935 for polymorphic loci, respectively. These markers will allow analyses of the baseline genetic variability and population structure of C. chinensis to enrich our scientific understanding of forest fragmentation on genetic health of this species and provide strategies for effective conservation and management in this area.     

Development of microsatellite loci for the black-footed limpet, <Emphasis Type="Italic">Patella depressa</Emphasis>, and cross-amplification in two other <Emphasis Type="Italic">Patella</Emphasis> species

Limpets (Patella spp.) are suitable organisms to investigate the effects of climate change in marine systems. They are widespread over NE Atlantic intertidal rocky shores and have been extensively studied in terms of population dynamics and ecology. Within genus Patella, microsatellites have only been developed for Patella caerulea and cross-species tests are unknown. In this work, we describe 11 primer pairs for Patella depressa and the results of cross-species testing on Patella candei and Patella rustica.     

Characterization of 21 microsatellite marker loci in the ring-tailed lemur (<Emphasis Type="Italic">Lemur catta</Emphasis>)

Ring-tailed lemur (Lemur catta) is the only species in the Genus Lemur, distributed in the deciduous and spiny forests of southwestern Madagascar. This species is listed as endangered due to the loss and fragmentation of its natural habitat, a consequence of deforestation. Twenty-one nuclear microsatellite loci were isolated from a genomic DNA derived from a free-ranging ring-tailed lemur population from the Tsimanampetsotsa National Park, Madagascar. We report various parameter estimates and measures to establish the utility of this marker suite as screened among individuals this single forest fragment.     

Eight new polymorphic microsatellite loci from the eastern phoebe (<Emphasis Type="Italic">Sayornis phoebe</Emphasis>)

We describe eight new polymorphic microsatellite loci (5 dinucleotide and three trinucleotide) for the eastern phoebe (Sayornis phoebe) to complement five previously published loci. The number of alleles per locus ranged from 3 to 15 and observed heterozygosities ranged from 0.33 to 0.91. Preliminary screening revealed that loci were polymorphic in other Tyrannidae: Empidonax virescens (n = 10), Tyrannus tyrannus (n = 10), Tyrannus vociferans (n = 5), and Tyrannus melancholicus (n = 28).     

Twelve polymorphic microsatellite loci for <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> (Poaceae)

Microsatellites are often highly variable and abundant in most complex genomes, therefore are widely used in population genetic studies. In this study, twelve polymorphic microsatellite loci were isolated and characterized for the Achnatherum inebrians, a plant abundant in grasslands of Northwest China. Characterization of 24 A. inebrians individuals form four geographically distant populations (Gansu, Qinghai, Xinjiang and Inner Mongolia provinces) showed moderate to high allelic diversity ranging from 3 to 13 alleles per locus, and the expected heterozygosity ranging from 0.41 to 0.67. No evidence of linkage disequilibrium was found for any locus pairwise comparisons. The markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow of this species. Na Chen and Yan-Zhuo Yang contributed equally to this work.     

Fine mapping of the <Emphasis Type="Italic">qCTS4</Emphasis> locus associated with seedling cold tolerance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Rice seedlings are sensitive to low temperatures (≤15°C) and under prolonged or repeated exposure, yellowing and stunting are commonly observed. Damage to seedlings results in poor stand establishment and delayed maturation, which can cause significant reductions in yield. In general, japonica rice varieties exhibit more cold tolerance than indica varieties. Earlier genetic analysis of the California rice variety M202 revealed several quantitative trait loci (QTL) that contribute to its tolerance to low temperatures in comparison to the indica rice variety IR50. Among these QTL, qCTS4 is associated with tolerance to yellowing and stunting of rice seedlings and accounts for 40% of the phenotypic variation. Here we report on the fine mapping of qCTS4 to a 128 kb region of chromosome 4, which is highly suppressed for recombination in our mapping populations. Our results provide the necessary foundation for identifying the gene(s) underlying qCTS4 and the markers developed here may be used to introgress this region into indica varieties to improve seedling tolerance to low temperatures. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Isolation of microsatellite loci from the endemic and endangered Adriatic sturgeon (<Emphasis Type="Italic">Acipenser naccarii</Emphasis>)

With the aim of elaborating a breeding plan on a captive stock of the highly endangered Adriatic sturgeon (Acipenser naccarii), a total of 10 polymorphic microsatellite loci were isolated from an enriched library. The results of cross amplification of additional 8 loci previously isolated from A. oxyrinchus, A. fulvescens and Scaphyrinchus platorhynchus are also reported. Given the tetraploid condition of the species the genetic variability was estimated basing on the number of alleles per individuals and the average band sharing.