Discovery Of Genome-wide SNP Markers And Assessment Of Genetic Diversity For Four Chinese Indigenous Rabbit Breeds

As a result of a short history and single center of domestication,the overall genetic variation of modern domestic rabbits(Oryctolagus cuniculus)is relatively small.Therefore,to conserve their genetic diversity is particularly important.China has a long history of raising rabbits and the abundant genetic resources.However,due to the poor economic performance of Chinese indigenous rabbit breeds and the wide use of Western cultivated rabbit breeds,the population of many Chinese indigenous rabbit breeds has decreased dramatically and their genetic resources faces the risk of loss.With the rapid development and wide utilization of high-throughput sequencing technology,discovery of molecular marker and assessment of genetic diversity at the genome-wide level have become the prevailing technologies and common research strategies.In this study,104 unrelated individuals were randomly collected from three indigenous rabbit breeds(including Sichuan White,Fujian Yellow and Fujian Black)and one indigenous cultivated rabbit breed(Tianfu Black).We firstly employed the restriction site-associated DNA sequencing(RAD-seq)technology to develop and screen the genome-wide SNP molecular markers of those individuals,and the high-density SNP map was further constructed.Based on SNP molecular markers,we ultimately evaluated the intra-breed genetic diversity for every one of the four rabbit breeds and analyzed the inter-breed genetic distance of four rabbit breeds.The main research results are as follows:1.Discovery of genome-wide SNP.In this study,Eco RI was first selected to digest genomic DNA.Subsequently,DNA fragments were used to construct the sequencing libraries according to the standard procedure.Finally,the Illumina Hi Seq2000 platform was employed to sequence the constructed libraries and generate150 bp paired-end reads.Based on 295 Gb of raw data,we obtained a total of 260 Gb clean data after the strict quality filtering.After mapping clean reads to the reference rabbit genome,we ultimately obtained 7,955,814 raw SNPs.To avoid potential biases,we strictly selected a high-confidence set of 113,973 SNPs for the further analysis according to the three filtering criteria,including(i)coverage depth of reads≥3 for every sample,(ii)calling rate of 100%(i.e.,no any missing in the samples),and(iii)biallelic SNPs.2.Construction of SNP map.We analyzed the distribution regions and density of SNP loci across the 22 chromosomes.The results indicated that those SNP loci distributed on all of the 21 autosomes,and the distribution density was relatively even.On the contrary,the distribution on the X chromosome was the least,and the distribution density was uneven.Additionally,minor allele frequency(MAF)mainly concentrated around 0.35,and SNPs with MAF> 0.05 accounted for 98.91% of the total.After SNPs annotation,the ratio of transition vs.transversion was 2.23,and33.54% of SNPs were distributed in the gene region.3.Assessment of intra-breed population genetic diversity.The analysis found that Fujian Yellow rabbits had the highest genetic diversity,which the mean values of expected heterozygosity,observed heterozygosity,polymorphism information content and nucleotide diversity were 0.2857,0.3418,0.2281 and 0.2902,respectively;whereas Fujian Balck rabbits had the lowest genetic diversity,which the four indexes were 0.2511,0.3072,0.2000 and 0.2678,respectively.Genetic diversity of Sichuan White rabbits was between Fujian Yellow rabbits and Fujian Black rabbits,which the four indexes were 0.2644,0.3158,0.2109 and 0.2689,respectively.The mean Wright’s inbreeding coefficient ranged from-0.1109(FY)to-0.0390(TB).4.Analyses of inter-breed genetic distance.The results of pairwise Fst,phylogenetic tree,principal component analysis and population structure revealed that the individuals of all the four Chinese indigenous rabbit breeds could cluster together and be distinguishable from each other.However,compared with the other three indigenous rabbit breeds,Tianfu Black rabbits had the relatively higher intra-population genetic differentiation.In conclusion,to three indigenous rabbit breeds and one indigenous cultivated rabbit breed,we developed genome-wide SNP markers,constructed SNP map and evaluated genetic diversity.The results provided important reference data for the conservation and development of genetic resources of Chinese indigenous rabbits.