IDENTIFICATION OF MUTIARA STRAIN OF AFRICAN CATFISH RESISTANT TO Aeromonas hydrophilla INFECTION USING MHC-I GENE AS THE MARKER
In the previous study, MHC-I gene has been identified as a candidate marker for resistance to Aeromonas hydropilla infection in Sangkuriang strain of African catfish. The results showed that the marker has high similarity to allele 9 and 17 of MHC-I gene. However, this marker has high polymorphism and was not effective when used for selective breeding program. The aim of this study was to evaluate MHC-I marker related to resistance against A. hydrophilla infection in Mutiara strain of African catfish strains resistant and susceptible to bacteria Aeromonas hydrophila with single and specific band of DNA, so this marker has been effective when applied in the aquaculture.
In this study, we used Bacteria pathogen, A. Hydrophila (Installation of Research and Development of Fish Diseases, Depok, Indonesia). The nucleotide coding sequences of MHC I (GenBank Accession numbers EU714302-EU714322) was aligned using a Pick primer NCBI to design 15 specific primer set for 22 allele. MHC I gene detection performed in live/survived/resistant and dead juvenile of African catfish post challenge test using PCR method. The PCR products were sequenced in 1 st Base Sequencing INT-Singapore. The data of sequencing were aligned by Bioedit software version 7.1.9. Furthermore, it was analyzed by BLAST (https://www.ncbi.nlm.nih.gov/). The MHC-I gene expression used PCR and β-actin as internal control. We detected candidate MHC-I marker from 454 broodstock (201 male fish and 253 female fish) and mated 60 female and 60 male fish, both having (P) and 3 female and 3 male fish no MHC-I (N) to progeny test.
The result of this study showed LD50 of A. hydrophila bacteria was 108 CFU mL-1. Among the 15 primer sets tested, a set primer is African Catfish grouped in two categories. First category is resistant/survival fish and it has been specific band about 1000 bp. Second, non-resistant (dead), it has not been PCR product (Figure 1). A set of primers and allele is MHC I (Clga-UAA) mRNA Clga-UAA 07 allele.
The results alignment of PCR product nucleotide at position 1000 bp sequence with GenBank: EU714308.1 showed similarities 99%, Identity 181/183(99%) and Gaps 1/183 (0%) (Figure 2).
Nucleotide sequence PCR product of about 1000 bp identical size as much as 99% with MHC I (Clga-UAA) mRNA Clga-UAA allele 07 of African catfish. Gene MHC I (Clga-UAA) mRNA Clga-UAA07 allele expressed in survived/resistant fish post challenge test with A. hydrophila. The expected size of its gene was 285 bp (Figure3).
The percentage of male broodstock of African catfish carried MHC-I gene about 97.01% (195/201) and 97.52% (247/253) for female fish and about 97.36% total broodstock carrying MHC-I gene. The results of the progeny test, both having MHC-I gene were 90%-100% carrying MHC-I gene. Otherwise, the progeny test from crossed male and female broodstock of African catfish that did not carry the MHC-I gene was all offspring negative MHC-I gene.