Premium
Rapid detection of SNP s in candidate genes regulating the growth of orange‐spotted grouper, E pinephelus coioides ( H amilton, 1822), using semiconductor sequencing
Author(s) -
Guo Liang,
Xia Junhong,
Yang Sen,
Li Mingming,
Chen Xinghan,
Lin Haoran,
Meng Zining
Publication year - 2017
Publication title -
aquaculture research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 89
eISSN - 1365-2109
pISSN - 1355-557X
DOI - 10.1111/are.12897
Subject(s) - biology , ion semiconductor sequencing , grouper , genetics , sanger sequencing , single nucleotide polymorphism , epinephelus , population , start codon , stop codon , genotype , gene , dna sequencing , fishery , nucleotide , demography , sociology , fish <actinopterygii>
Orange‐spotted grouper ( E pinephelus coioides ) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms ( SNP s) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNP s were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.