Cuiyun Lu

SLAF-seq: An Efficient Method of Large-Scale De Novo SNP Discovery and Genotyping Using High-Throughput Sequencing

Large-scale genotyping plays an important role in genetic association studies. It has provided new opportunities for gene discovery, especially when combined with high-throughput sequencing technologies. Here, we report an efficient solution for large-scale genotyping. We call it specific-locus amplified fragment sequencing (SLAF-seq). SLAF-seq technology has several distinguishing characteristics: i) deep sequencing to ensure genotyping accuracy; ii) reduced representation strategy to reduce sequencing costs; iii) pre-designed reduced representation scheme to optimize marker efficiency; and iv) double barcode system for large populations. In this study, we tested the efficiency of SLAF-seq on rice and soybean data. Both sets of results showed strong consistency between predicted and practical SLAFs and considerable genotyping accuracy. We also report the highest density genetic map yet created for any organism without a reference genome sequence, common carp in this case, using SLAF-seq data. We detected 50,530 high-quality SLAFs with 13,291 SNPs genotyped in 211 individual carp. The genetic map contained 5,885 markers with 0.68 cM intervals on average. A comparative genomics study between common carp genetic map and zebrafish genome sequence map showed high-quality SLAF-seq genotyping results. SLAF-seq provides a high-resolution strategy for large-scale genotyping and can be generally applicable to various species and populations.

Genome evolution trend of common carp （Cyprinus carpio L.） as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication (FSGD) may have occurred before the radiation of the teleosts. Common carp (Cyprinus carpio L.) has been considered to be a tetraploid species, because of its chromosome numbers (2n=100) and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution (genome duplication and diploidization) of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs (HLJ021 and HLJ332) were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition (dynamic) and equilibrium (static) were proposed to explain the common carp genome evolution between genome duplication and diploidization.

A Set of Polymorphic Trinucleotide and Tetranucleotide Microsatellite Markers for Silver Crucian Carp (Carassius auratus gibelio) and Cross-Amplification in Crucian Carp

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2–19), observed heterozygosity (0.1765–0.9706), expected heterozygosity (0.2392–0.9421), and polymorphism information content (0.2186–0.9236).

Development of 10 tri‐ and tetranucleotide microsatellite loci for population studies in the common carp (Cyprinus carpio L.)

Ten tri‐ and tetranucleotide microsatellite DNA markers were isolated and characterized from common carp (Cyprinus carpio L.) to estimate genetic potential. These markers were tested in the samples from two closely related carp populations (Cyprinus carpio var. xingguonensis and Cyprinus carpio var. wananensis). The number of the alleles ranged from three to nine, and observed and expected hererozygosities varied from 0.207 to 1.000 and from 0.499 to 0.900 in each population, respectively. No evidence for linkage disequilibrium was found, indicating that these markers will be useful for population studies.

Complete mitochondrial genome of sterlet (Acipenser ruthenus)

Abstract The complete mitochondrial genome of sterlet (Acipenser ruthenus) was determined in this study. The mitogenome is 16,790 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (the control region and the putative origin of the light strand replication) with a typical vertebrate mitochondrial gene arrangement. The overall base composition of the heavy strand is 30.26% for A, 29.00% for C, 16.23% for G and 24.51% for T, with a slight AT bias of 54.77%.

Complete mitochondrial genome of Amur sturgeon (Acipenser schrenckii).

Abstract The complete mitochondrial genome of Amur sturgeon (Acipenser schrenckii) was determined in this study. The mitogenome is 16,684 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (the control region and the putative origin of the light strand replication) with a typical vertebrate mitochondrial gene arrangement. The overall base composition of the heavy strand is 30.07% for A, 29.36% for C, 16.44% for G and 24.13% for T, with a slight AT bias of 54.20%.

Mapping QTLs of caudal fin length in common carp (Cyprinus carpio L.)

Common carp (Cyprinus carpio L.) exhibits significant morphological variability in the shape and size of the caudal fin. In this study, we used 190 progeny from two F1 parents to identify and map quantitative trait loci (QTLs) that influence caudal fin length (CFL) and the ratio (RCS) between caudal fin length and standard length (SL) based on a microsatellite genetic map of common carp. A total of 15 QTLs were detected in seven different linkage groups. One significant and eight suggestive QTLs affecting CFL were identified on LG8, LG14, LG29, LG32 and LG44, which explained 8.0%–22.1% of the phenotypic variation; six suggestive QTLs affecting RCS were detected on LG8, LG32, LG46 and LG48, which explained 7.0%–15.4% of the phenotypic variation. The QTLs for caudal fin length detected in this study may serve as a starting point for identification of genes involved in caudal fin development in common carp.

Isolation and characterization of 57 novel polynucleotide microsatellites from yellow catfish (Pelteobagrus fulvidraco) genome for genetic analysis

Yellow catfish (Pelteobagrus fulvidraco) is one of the important economic freshwater fish in Eastern Asia. In recent years, the wild resources were decreased sharply due to overfishing and pollution. It is necessary to conserve the wild stocks for the sustainable use of the species. In this study, 200 microsatellite markers were isolated from yellow catfish genome using Roche 454 pyrosequencing method. Among these markers, 57 exhibited polymorphism in yellow catfish population collected from Songhua River in China. The number of alleles ranged from 2 to 13 and the number of effect alleles ranged from 1.112 to 8.980. The observed heterozygosity (Ho) and the expected heterozygosity (He) varied from 0.106 to 0.957 and from 0.102 to 0.898, respectively. Most loci conformed to Hardy–Weinberg equilibrium with the exclusion of five loci. These polymorphic loci will be valuable for population genetic structure and genetic conservation for the P. fulvidraco.

The complete mitochondrial genome of Endangered fish Huso dauricus (Acipenseriformes: Acipenseridae).

Abstract In this study, we sequenced and obtained the complete mitochondrial genome of the Kaluga (Huso dauricus) for the first time. The circular genome (16,691 bp in length) contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region. The overall base composition of the novel mitogenome is 30.39% for A, 24.18% for T, 29.27% for C, 16.15% for G. AT content (54.57%) is higher than the GC content.

Development and characterization of new microsatellite markers for Amur sturgeon (Acipenser schrenckii)

Abstract Twenty-five polymorphic microsatellite markers were developed in Amur sturgeon (Acipenser schrenckii. In a survey population, these markers yielded a total of 168 alleles with the number of alleles per locus ranged from 2 to 18. Average observed heterozygosity of these loci ranged from 0.27 to 1.00. These markers could enrich the available molecular markers for genome mapping and genetic diversity analysis of sturgeons. These loci not only should detect sufficient genetic diversity to allow kinship analysis for broodstock management and stock assessment for delineation of fine-scale population structure, but also will contribute greatly to resource conservation.