Genetic structure of refugial populations of the temperate plant Shortia rotundifolia (Diapensiaceae) on a subtropical island

Continental island systems harbour relict biota and populations that might have migrated during glacial periods due to the formation of landbridges. Here we analysed the genetic structure of relict populations of the temperate plant Shortia rotundifolia on the subtropical island of Iriomotejima, Japan. This plant, which inhabits riparian environments, is designated “near threatened”. Only five extant populations have been found on the island. Our analyses of 10 nuclear microsatellite loci detected genetic diversity of H _E = 0.488 and H _O = 0.358 for all populations of S. rotundifolia on the island. A high inbreeding coefficient for all populations together (F _IS = 0.316) and each population separately (F _IS = 0.258–0.497) might be attributable to crossing among closely related descendants within a population, an idea that is supported by the relatedness coefficient. These results and an examination of the populations’ demographic histories suggest that the extant populations on Iriomotejima have not experienced a recent population bottleneck. The five extant populations were genetically differentiated (F _ST = 0.283; P < 0.001), suggesting low seed dispersal by gravity and/or low pollen flow via pollinators in the riparian environment. In addition, population differentiation was not related to genetic distance, implying that at one time, ancestral populations might have been distributed over a wider area of the island. However, population fragmentation and range contraction might have occurred at random during the postglacial period.     

Isolation and characterization of polymorphic microsatellite loci in Candidia barbata (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Candidia barbata is an endemic cyprinid fish in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, a total of 14 polymorphic microsatellite loci from C. barbata were isolated and characterized using an optimized protocol to construct a microsatellite-enriched genomic library. The analysis of variability was performed in 30 specimens of Taiwan. The mean number of alleles across loci was 4.92 ± 1.44. The levels of expected and observed heterozygosities varied from 0.1266 to 0.5079, and from 0.0667 to 0.9667, respectively. Frequencies of null alleles of the 14 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population.     

Development and characterization of microsatellite loci in the endangered Tricyrtis ishiiana (Convallariaceae), a local endemic plant in Japan

Nine microsatellite loci were isolated and characterized for the endangered and tertiary relict perennial herb, Tricyrtis ishiiana. The number of alleles ranged from 2 to 33. The expected (H _E) and observed (H _O) heterozygosities were 0.207–0.944 and 0.215–0.813, respectively, from 96 individuals on one population. Five loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in this species may provide a useful tool for population genetics to establish conservation strategy.     

Microsatellite variation, population structure, and bottlenecks in the threatened copperbelly water snake

Habitat loss and isolation is pervasive in the Midwest U.S. Wetlands are experiencing particularly dramatic declines, yet there is a paucity of information on the genetic impacts of these losses to obligate wetland vertebrates. We quantified the genetic variation of extant populations of a shallow wetland specialist and evaluated potential reductions in population size (i.e. bottlenecks) using seven polymorphic microsatellite DNA markers. We analyzed 228 copperbelly water snakes (Nerodia erythrogaster neglecta), representing populations from three states. Moderate genetic differentiation exists among all three regions (F _ST = 0.12, P < 0.001), with evidence for low levels of differentiation within the federally protected Ohio region (F _ST = 0.025, P = 0.007), and moderate to strong differentiation within the Indiana region (F _ST = 0.23, P < 0.001). Furthermore, Bayesian clustering (i.e. STRUCTURE) supports the separation of the Indiana sites, both from each other and from all other sampling sites. However, it does not support the separation of the Ohio sites from the Kentucky sites. Differentiation among sampling sites did not appear to be related to geographic distance, but rather depended on the quality of terrestrial corridors used for dispersal. Mode shifts in allele frequencies and excess heterozygosity tests were negative, while M-ratio tests were nearly all positive, indicating the likelihood of historical rather than contemporary population bottlenecks. However, potential subspecific intergradation in the Kentucky region may have artificially lowered the M-ratio, and we suggest caution when using the M-ratio approach if intergradation is suspected. Our results have conservation implications for wetland management and management of the copperbelly populations, and emphasizes the importance of protecting wetland complexes.     

Isolation and characterization of polymorphic microsatellite DNA makers for Euphrasia nankotaizanensis (Orobanchaceae) and cross amplification in another Euphrasia L.

Euphrasia nankotaizanensis is an endangered flowering plant distributed restrictedly on rocky slope of high mountain peaks in central and northern Taiwan. In order to undertake a conservation program, especially given impacts of the global warming, it is essential to evaluate its genetic diversity and population structure. We described nine novel microsatellite primer pairs in E. nankotaizanensis and also examined its relative E. transmorrisonensis. The number of alleles per locus ranged from 7 to 29. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.83 to 0.98 and 0.00 to 0.95, respectively. Eight of the nine microsatellite loci displayed significant departures from Hardy–Weinberg expectations, likely due to the loss of habitats and the small population size. Significant linkage disequilibrium (LD) was detected in most loci. Cross-species amplification of microsatellites took place at five loci. These primers may provide a tool for understanding the demography and population structure of Euphrasia species in Taiwan. Kuo-Hsiung Wang, Ming-Jou Wu, and Tzen-Yuh Chiang contributed equally to this work.     

Isolation and characterization of 13 microsatellite loci in Rhinolophus pusillus (least horseshoe bat) with cross-amplification in five related species

We used the enriched genomic library method to isolate and characterize dinucleotide microsatellite loci in the least horseshoe bat, Rhinolophus pusillus. Seventeen loci were obtained and tested on 31 individuals sampled from Guangxi Province in southern China. Thirteen of these markers were polymorphic with expected heterozygosity ranging from 0.821 to 0.909. A total of 164 alleles were detected and the number of alleles per locus ranged from 9 to 16 (mean 12.6). These polymorphic markers will be used to assess population structure in R. pusillus. In addition, successful cross-amplification in five congeneric bat species suggests most of these markers will also be useful for studying related species.     

Development of 19 polymorphic microsatellite loci for the intermediate horseshoe bat, Rhinolophus affinis (Rhinolophidae, Chiroptera)

The intermediate horseshoe bat (Rhinolophus affinis) is a widespread species in Southeast Asia. We developed 19 novel microsatellite loci from an enriched genomic library of the bat, and tested their polymorphism using a single population from Guangdong province, southern China. The number of alleles ranged from 3 to 15 per locus with the expected and observed heterozygosity ranging from 0.397 to 0.920 and 0.280–0.926, respectively. Three markers significantly deviated from the Hardy-Weinberg expectations after Bonferroni correction and no linkage disequilibrium was detected in any of loci. These microsatellite loci will be useful in studying the phylogeography of this species.     

柔嫩艾美尔球虫EST序列中SSR的获取及分析

对柔嫩艾美尔球虫EST—SSR进行生物信息学分析,共获取Eimeria tenella EST序列34074条,总长度为16．45Mb,小于12bpSSR的ESTs达7651条,从中获得SSR序列19576条、总长度为0．35Mb,EST—SSRs的频率是48．00％,平均相隔S40bp出现一个长度不小于12bp的SSR。在E．tenella的核苷酸重复基元中,2、3、4、5、6和7bp重复序列在基因组中出现的种类分别有11种472条、49种14710条、31种525条、13种25条、21种43条和15种400条,3碱基重复序列是最丰富的重复单元,占总数的75．14％。各种SSRs中富含G、C碱基的重复单元以GCA出现频率最多（28．63％）,次为AGC（17．59％）,GCT（8．76％）,TGC（7．62％）,CTG（7．15％）。     

Genome-wide association study identifies novel loci associated with resistance to bovine tuberculosis

Tuberculosis (TB) caused by Mycobacterium bovis is a re-emerging disease of livestock that is of major economic importance worldwide, as well as being a zoonotic risk. There is significant heritability for host resistance to bovine TB (bTB) in dairy cattle. To identify resistance loci for bTB, we undertook a genome-wide association study in female Holstein–Friesian cattle with 592 cases and 559 age-matched controls from case herds. Cases and controls were categorised into distinct phenotypes: skin test and lesion positive vs skin test negative on multiple occasions, respectively. These animals were genotyped with the Illumina BovineHD 700K BeadChip. Genome-wide rapid association using linear and logistic mixed models and regression (GRAMMAR), regional heritability mapping (RHM) and haplotype-sharing analysis identified two novel resistance loci that attained chromosome-wise significance, protein tyrosine phosphatase receptor T (PTPRT; P=4.8 × 10⁻⁷) and myosin IIIB (MYO3B; P=5.4 × 10⁻⁶). We estimated that 21% of the phenotypic variance in TB resistance could be explained by all of the informative single-nucleotide polymorphisms, of which the region encompassing the PTPRT gene accounted for 6.2% of the variance and a further 3.6% was associated with a putative copy number variant in MYO3B. The results from this study add to our understanding of variation in host control of infection and suggest that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control.