SNP MARKERS ASSOCIATED WITH GROWTH TRAITS IN Clarias magur (HAMILTON, 1822) IDENTIFIED BY RNASeq

Clarias magur is a popular indigenous catfish species due to its taste and nutritional qualities. It can be cultured in limited space and can tolerate poor water quality due to its air breathing nature. Its growth is however much slower than the African catfish C. gariepinus that is now contaminating Indian waters. Genetic improvement for faster growth can improve the aquaculture prospects of this species. Single nucleotide polymorphisms (SNPs) explain 90% of the genetic differences between individuals and are most suitable for genetic evaluation and marker assisted selection. In an ongoing genetic selection programme for improved body weight at ICAR-CIFE, three stocks of magur from Andhra Pradesh, Assam and West Bengal were used to develop the base population by complete diallel crossing using single paired mating design. Five high and five low growth performing brooder pairs were selected with average body weights of 330 g and 240 g, respectively, and bred to produce the F1 generation. Tissues (liver, kidney, muscle, and brain) were collected from the selected parents and 10 specimens of their offspring. Equal quantity of each tissue from each specimen was pooled and total RNA was isolated for cDNA library preparation. Finally, cDNAs of each family were pooled in equimolar quantities and barcoded separately. The ten barcoded paired-end cDNA libraries were sequenced on Illumina MiSeq platform.  A total of 9 GB transcriptome data with 78.96 million high-quality reads was obtained. De Novo transcriptome assembly resulted in 52,237 contigs with an average length of 917 bp and N50 length of 1330 bp. Total 36642 contigs could be annotated to the known sequence databases with significant BLAST scores using Blast2GO software. The reads of each family were mapped to the assembled reference contigs for calling SNPs. A total of 25,231 different heterozygous SNPs could be identified using stringent parameters with minimum coverage greater than 10 and base quality score greater than 30. The estimated mean Ts : Tv ratio was 1.34:1. Total 10,125 SNPs were found to be unique to the high growth performing families. Of these, 52 SNPs present in 52 growth related genes were selected for genotyping using high resolution melting curve analysis (HRM) method to determine their association with growth performance of magur. Changes in amino acid classes and peptide stability were recorded for 13 non-synonymous SNPs while changes in codon preference were noted for 19 synonymous ones. Rest 20 SNPs are present in the UTR region of the genes, of which 5 SNPs are found to affect the transcription factor binding sites. Once validated, the growth associated SNPs will be useful in genetic selection.