Development of 10 microsatellite loci for Rheum tanguticum (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.

Assessment of cattle genetic introgression into domestic yak populations using mitochondrial and microsatellite DNA markers

Hybridization between yak Poephagus grunniens and taurine Bos taurus or indicine B. indicus cattle has been widely practiced throughout the yak geographical range, and gene flow is expected to have occurred between these species. To assess the impact of cattle admixture on domestic yak, we examined 1076 domestic yak from 29 populations collected in China, Bhutan, Nepal, India, Pakistan, Kyrgyzstan, Mongolia and Russia using mitochondrial DNA and 17 autosomal microsatellite loci. A cattle diagnostic marker-based analysis reveals cattle-specific mtDNA and/or autosomal microsatellite allele introgression in 127 yak individuals from 22 populations. The mean level of cattle admixture across the populations, calculated using allelic information at 17 autosomal microsatellite loci, remains relatively low (mY(cattle) = 2.66 +/- 0.53% and Q(cattle) = 0.69 +/- 2.58%), although it varies a lot across populations as well as among individuals within population. Although the level of cattle admixture shows a clear geographical structure, with higher levels of admixture in the Qinghai-Tibetan Plateau and Mongolian and Russian regions, and lower levels in the Himalayan and Pamir Plateau region, our results indicate that the level of cattle admixture is not significantly correlated with the altitude across geographical regions as well as within geographical region. Although yak-cattle hybridization is primarily driven to produce F-1 hybrids, our results show that the subsequent gene flow between yak and cattle took place and has affected contemporary genetic make-up of domestic yak. To protect yak genetic integrity, hybridization between yak and cattle should be tightly controlled.

Identification of monozygotic twin chimpanzees by microsatellite analysis

Zygosity determination is important for epidemiological, biological, obstetric, and prognostic studies in both human and nonhuman primates. In this study, microsatellite loci were used to screen a pair of chimpanzee (Pan troglodytes) twins and their parents. The twins share identical alleles at all loci tested. The probability of dizygotic origin is estimated to be 2.9 x 10(-11). Even after excluding linkage of loci on the same chromosome, the probability is still low enough (3.7 x 10(-9)) to exclude dizygotic origin. MHC typing was also done on Patr-DRB and Patr-DQB loci and the twins share identical alleles at both loci, consistent with the microsatellite results. Together these results demonstrate a monozygotic origin for the chimp twins. Our results suggest that microsatellite analysis is a powerful method for zygosity determination, which can be screened reliably and efficiently. Am. J. Primatol. 52:101-106, 2000. (C) 2000 Wiley-Liss, Inc.

Microsatellite polymorphisms of Sichuan golden monkeys

Previous study using protein electrophoresis shows no polymorphism in 44 nuclear loci of Sichuan golden monkey (Rhinopithecus roxellana), which limits our understandings of its population genetic patterns in the nuclear genome. In order to obtain sufficient information, we scanned 14 microsatellite loci in a sample of 32 individuals from its three major habitats (Minshan, Qinling and Shennongiia). A considerable amount of polymorphisms were detected. The average heterozygosities in the local populations were all above 0.5. The differentiations among local populations were significant. There was evidence of geneflow among subpopullations, but geneflow between Qinling and Shennongjia local populations was the weakest. Minshan and Qinling populations might have gone through recent bottlenecks. The estimation of the ratio of the effective population sizes among local populations was close to that from census sizes. Comparisons to available mitochondria data suggested that R. roxellana's social structures played an important role in shaping its population genetic patterns. Our study showed that the polymorphism level of R. roxellana was no higher than other endangered species; therefore, measures should be taken to preserve genetic diversity of this species.

Isolation and characterization of microsatellite markers for the endangered Taxus yunnanensis

Eleven polymorphic microsatellite loci were characterized for the endangered conifer Taxus yunnanensis. Eight loci were isolated through SSR-anchored PCR, one locus was developed by cross-species amplification tests, while the last two loci were obtained

Microsatellite diversity and population genetic structure of redfin culter (Culter erythropterus) in fragmented lakes of the Yangtze River

Redfin culter (Culter erythropterus) is a small lethic freshwater fish and widely distributed in the adjacent lakes of the Yangtze River of China. Five microsatellite loci were applied to investigate the genetic variation and population structure of redfin culter from seven lakes in the middle-and-lower reaches of the Yangtze River. The gene diversity was high among the populations (H > 0.9), the average number of alleles among seven populations was low with a range from 2.00 to 3.87. The mean observed (H-O) and expected (H-E) heterozygosity ranged from 0.111 to 0.419 and from 0.162 to 0.750, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found in 50% of the total locus-population combination tests in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percentage of variance among and within these populations were 6.18 and 93.82, respectively. The Fst values (0.062, P < 0.001) among studied populations indicated that there were significant genetic differentiations among redfin culture populations from the scattered lakes with different connections to the Yangtze River. These results are useful for the evaluation and conservation of small freshwater fishes. The factors that may be involved in low intra-population polymorphism and the pattern of the population genetic structure of redfin culter from the Yangtze River were discussed.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Genetic differentiation in populations of the cestode Bothriocephalus acheilognathi (Cestoda, Pseudophyllidea) as revealed by eight microsatellite markers

The genetic structure of populations of the fish cestode, Bothriocephalus acheilognathi collected from Bailianhe Reservoir (BLH), Changshou (CSH) and Liangzi (LZH) Lakes was investigated by using 8 microsatellite loci. A total of 108 adult worms were genotyped at each of the 8 loci. For the 3 populations, the mean number of alleles per locus ranged from 2.38 to 5.5, and the mean expected heterozygosity ranged from 0.432 to 0.559. The average polymorphic information content (PIC) was from 0.384 to 0.492. The significant F-is values indicated non-random mating within LZH and BLH populations. On the other hand, when samples were further classified into subpopulations at the level of host fish species, no or little heterozygote deficiency was detected at most loci, showing that cross-fertilization, predominantly, but not exclusively, must have occurred within the subpopulations. Microsatellite markers also revealed an unexpected high level of genetic differentiation, as measured by R-st and N-m values or by deltau(2) genetic distance among subpopulations from different hosts. Factors influencing the population genetic structure and the parasite host specificity are discussed.

Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono-crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

The haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore-derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open-sea cultivation. Sporophytic offspring originated from mono-crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono-crossing lines (M1 and M2), two self-breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono-crossing lines had a higher degree of identity (95.6-100%) than self-breeding lines (87.7-98.4%) and the wild population (81.5-92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono-crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.

Nine microsatellite DNA primers for Hippophae rhamnoides ssp sinensis (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the and 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.

Isolation and characterization of microsatellite DNA primers in Juniperus przewalskii Kom (Cupressaceae)

Juniperus przewalskii (Cupressaceae) is a dominant tree species endemic to the northeast Qinghai-Tibetan Plateau. This species plays an important role in maintaining the arid ecosystem in this region. However, natural distributions of this species have been declined. In order to develop effective conservation methods, it is important to know the distribution of the genetic diversity within and among populations. In this study, we developed nine new microsatellite loci for this species. We used the combining biotin capture method to enrich AG/CT/AC/GGT microsatellites. The polymorphisms of each locus were further assessed in 12 individuals from four geographically distant populations. The number of alleles per locus varied from three to six and expected heterozygosity ranged from 0.58 to 0.70. These loci together provide a useful tool to investigate the genetic diversity of this species. In addition, all markers have been crossly checked in the other four congeneric species.

用6个微卫星座位研究BMY牛和婆罗门牛的遗传多样性和群体遗传结构

对6个牛微卫星座位的遗传变异及多态性分析,以期了解BMY牛和婆罗门牛的群体遗传结构与育成情况.6个微卫星座位的多态信息含量为在0.524～0.752间,BMY牛、婆罗门牛两个群体的平均期望杂合度和观察杂合度值接近,分别为0.737 6和0.739 6,0 641 2和0.653 7,进入横交固定第二世代的BMY牛期望杂合度值较高,这与我们的育种进展相符.红安格斯的期望杂合度较低(0.460 9)接近日本和牛0.471,暗示红安格斯牛的同质性较高.

NONINVASIVE GIANT PANDA PATERNITY EXCLUSION

Giant panda hair samples obtained by noninvasive methods served as a source of DNA for amplification of seven giant panda microsatellite loci utilizing the polymerase chain reaction. Thirteen giant pandas held in Chinese zoos were tested for identification of paternity. Some males listed as sires have been excluded as the biological father of captive-born giant pandas. Because of the death of some potential sires, paternity is still not assigned for some giant pandas, although there is a high likelihood that paternity assignment could be made if postmortem samples are available for genetic analysis. The DNA microsatellite variation assayed by the test we have developed provides a rapid, highly informative, and noninvasive method for paternity identification in giant pandas. (C) 1994 Wiley-Liss, Inc.

Gynogenesis and sex determination in large-scale loach Paramisgurnus dabryanus (Sauvage)

Gynogenesis was induced using heterologous sperms in large-scale loach, Paramisgurnus dabryanus (Sauvage), in which a ZW/ZZ sex determination was previously proposed. Three microsatellite loci were used to monitor exclusive maternal inheritance of gynogenetic progenies. The results showed that high percentages of meiogynogens were produced at 4 min post-fertilization and mitogynogens were produced at 18 min post-fertilization by heat shocks, while meiotic gynogenesis was induced by cold shocks within a wide period and high heterozygosity was even observed in gynogens produced at 24 min post-fertilization. The sex ratios of the F, progenies in three gynogenetic families were significantly deviated from 1: 1 expectation with a female bias in two families and a male bias in one family (P < 0.05), and the other four gynogenetic families showed approximate 1:1 sex ratios. Moreover, the self-mating between gynogenetic F, progenies and mating between gynogenetic F, progenies and normal individuals produced all-female progenies or identical proportions of females and males. The data of sex ratios generally confirmed that the sex determination in large-scale loach was determined by the putative ZW/ZZ system, and the possible reasons causing the biased sex ratios are discussed.