Development of EST-SSRs by an efficient FIASCO-based strategy: A case study in rare minnow (Gobiocyrpis rarus)

Type I markers are useful for comparative mapping and other genetic analyses, but relatively difficult to develop. In the present study a microsatellite (SSR)-enriched cDNA library was constructed for the first time using the fast isolation by AFLP of sequences containing repeats (FIASCO) method in a small fish, Chinese rare minnow (Gobiocypris rarus). A total of 97.4% of the expressed sequence tags (ESTs) contained targeted CA-repeats, in which 29 unique EST-SSRs were identified. Ten out of the 28 loci for which primer pairs were designed were polymorphic with alleles ranging from three to seven (mean 4.50). Some of these EST-SSRs can be amplified in other species. These results proved that cDNA-FIASCO is an efficient way to isolate novel EST-SSRs in a fish.

DEVELOPMENT OF MICROSATELLITE LOCI FOR PINUS KORAIENSIS (PINACEAE)

Premise of the study: Microsatellite markers were developed for Pinus koraiensis to characterize its genetic diversity and understand its population structure. Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 20 primer sets were developed in Chinese populations of P. koraiensis. Three of the markers showed polymorphism with two alleles per locus when assessed in a sample of two populations of P. koraiensis from the Changbai Mountain in the Jilin Province of China. Five and three loci were successfully amplified in P. taiwanensis and P. massoniana, respectively. The amplification size of these loci matches those in P. koraiensis. Conclusions: These markers may be useful for further investigation of population genetics of P. koraiensis.

胭脂鱼微卫星富集文库的构建及其应用前景展望

以湖北武汉和四川宜宾人工增殖放流的胭脂鱼子一代酒精浸泡鳍条样本为材料,提取完整基因组DNA,选用人工合成的生物素标记(AAAG)7探针,采用FIASCO(fast isolation by AFLP of sequences containing repeats)磁珠富集法构建胭脂鱼(Myxocyprinus asiaticus)微卫星富集文库。以MseI-N引物组和设计合成的微卫星核心序列引物(AAAG)5,用双引物PCR法筛选含有微卫星的阳性克隆,从54个阳性克隆中共获得22个微卫星序列,其中完美型(

长江江豚微卫星DNA分离的初步研究

为了开发物种特异性微卫星标记,本文采用一种改良的快速微卫星分离法(FIASCO)从长江江豚(Neophocaena phocaenoides asiaeorientalis)的基因组中筛选得到72条微卫星DNA序列。根据重复单元的排列特点,完美型、非完美型及复合型序列所占的比例分别为58.3%、22.2%和19.5%。选择其中30条序列设计PCR扩增引物,并用12个随机选择的长江江豚样品进行多态性筛选。初步结果表明其中14对引物的扩增产物稳定并且具有多态性;在每个座位上获得2-13个等位基因,平均等位基因

Development and characterization of polymorphic microsatellite loci in the endangered Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis)

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species widely distributed in the shallow coastal waters of the Indo-Pacific Oceans. The population size of the Yangtze subspecies (N. p. asiaeorientalis) has sharply decreased in the last two decades and access to objective data on its population structure and genetic diversity would be of great assistance for their proper management. Here we report on the isolation of nine polymorphic microsatellite using the "Fast Isolation by AFLP of Sequences Containing repeats" (FIASCO) protocol. Polymorphism was assessed using 30 porpoise individuals randomly sampled in the Yangtze River. The number of alleles per locus varies from 2 to 9, with an average value of 5.56, whereas the ranges of observed and expected heterozygosi ties were 0.300-0.633 (mean 0.496) and 0.473-0.804 (mean 0.659), respectively.

Sixteen polymorphic microsatellites in bighead carp (Aristichthys nobilis) and cross-amplification in silver carp (Hypophthalmichthys molitrix)

A (GT)(n) enriched partial genomic library of bighead carp (Aristichthys nobilis) was constructed by employing the (fast isolation by AFLP of sequences containing repeats) FIASCO protocol. Sixteen loci exhibited polymorphism with two to seven alleles/locus (mean 3.263) in a test population and the observed heterozygosity ranging from 0.100 to 0.690 (mean 0.392). Eleven of the 16 bighead carp microsatellites were found to be also polymorphic in silver carp. These polymorphic loci should provide sufficient level of genetic diversity to evaluate population structure of bighead carp.

Isolation and characterization of polymorphic microsatellites in a sex-reversal fish, rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a fish of economic importance in China and some Asian countries. From a (GT)(n)-enriched genomic library, 30 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Thirteen loci exhibited polymorphism with two to 13 alleles (mean 7.9 alleles/locus) in a test population and observed heterozygosity ranged from 0.3125 to 0.9688 (mean 0.7140). These loci should provide sufficient level of genetic variation to study the fine-scale population structure and reproductive ecology of the species.

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish, brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)(n)-enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure of C. heterodon.

Isolation and characterization of microsatellites in Chinese sturgeon, Acipenser sinensis

From (GATA)(n) and (AAAG)(n) enriched genomic libraries for the Chinese sturgeon (Acipenser sinensis), 50 primer pairs were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Forty-six primer pairs exhibited highly polymorphic with two to 11 alleles per locus, while the rest four displayed monomorphic. These markers yielded 246 alleles in a survey of eight specimens of wild A. sinensis. Average observed heterozygosity ranged from 0.13 to 1.00. These loci should provide sufficient levels of genetic diversity to allow parentage analysis for artificial stocking management and delineation of fine-scale population structure.