IDENTIFICATION OF MAJALAYA COMMON CARP STRAINS RESISTANT TO KHV INFECTION USING CYCA-DAB 1 * 05 ALLELE AS THE MARKER

The Cyca-DAB1*05 allele of major histocompatibility complex class II genes is recently suggested to have a link with the European common carp strain resistant to koi herpesvirus (KHV). In this study, a set of specific primers for Cyca-DAB1*05 was designed and applied as a marker to identify broodstocks of majalaya common carp strain subsequently used as a candidate resistant to KHV infection. From a total of 23 broodstock subjected to PCR analysis, two female and male fish, both having (P) and no Cyca-DAB1*05 (N), were selected and then diallelly mated. Disease resistance of progenies from 10 crosses was determined by a survival analysis in pond rearing and a laboratory challenge-test using cohabitation method. The results have revealed that the average survivals of PxP progenies for pond rearing and KHV challenge test were 86% and 100% higher (P < 0.05) respectively compared to that of NxN fish. Survival rate of PxN/NxP progenies was significantly lower (P < 0.05) than that of PxP fish. Furthermore, PCR analysis showed that almost 91% progenies of PxP crosses seemed to have a KHV resistant gene marker. Thus, this study suggests that the marker is associated with the KHV resistance in majalaya common carp strain, and farming of PxP progenies can be useful to increase common carp production.

infection in common carp, such as the use of phytomedicine (e.g.garlic extract and powder) and DNA vaccine (Nuryati et al., 2010).However, the wide application of those substances still has to be developed to meet the practical and economical means.
Improvement of broodstock and seed quality can be achieved by selective breeding program.However, the process requires more than five generations in order to obtain significant increase of genetic improvement.Recently, rese arche rs have de ve lo pe d mo le cular marker-assisted selection (MAS) to accelerate the selection (Liu & Cordes, 2004) by avoiding unintended crosses (Martinez, 2007).The use of MAS is also potential as a fast track to increase genetic gain (Sonesson, 2007;Ibitoye & Idowu, 2010).
It is commonly found that several individuals have survived when KHV infected a majalaya common carp population.The survived fish may be genetically resistant to KHV as found in European common carp (Rakus et al., 2009).KHV-resistant European common carp is linked to Cyca-DAB1*05 allele of major histocomapatibility complex (MHC) class II (Rakus et al., ibid).Other linked molecular markers for KHV-resistant in European common carp have also been reported by Kongchum et al. (2010).Furthermore, the average survival of progenies from crosses between European common carp strains is higher than the purebred against KHV infection (Odegard et al., 2010).In this study, a specific primer set for Cyca-DAB1*05 as a marker was designed and then used in PCR amplification to select the broodstocks of majalaya common carp carrying the marker.The broodstock subsequently can be used for mass production of KHV-resistant progenies, and crossbreed with other strains.
The majalaya common carp strain used in the study was the survived fourth generation after KHV infection and maintained in ponds at the Main Center of Freshwater Aquaculture Development, Sukabumi, Indonesia.The DNA genome of caudal fin from a total of 23 microchip-tagged fishes (13 females and 13 males) was isolated using a DNA Isolation Kit (Qiagen) according to the manufacturer's instructions.PCR analysis was performed in 20 µL of 10x Ex Taq buffer, 200 µM of dNTPs, 0.125 U of Ex Taq polymerase (Takara, Shiga, Japan), 2 µL of DNA as template, and 1 pmol of each primer.A total of 35 cycles of denaturation for 30 s at 94 o C, annealing at 62 o C for 30 s, and extension at 72 o C for 30 s were performed.PCR was also performed for the -actin gene as an internal control using this primer set: 5'-GTGCCCATCTACGAGGGTTA-3' and 5'-TTTGATGTCACGCACGATTT-3'.PCR amplification for the internal control was performed as follows: 5 min.at 94 o C, followed by 30 cycles of 20 s at 94 o C, 15 s at 68 o C, 15 s at 72 o C and 3 min.at 72 o C. One microliter of the reaction was then electrophoretically separated using 0.7% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light.

Progenies Production and Rearing
Four diallel crosses of Cyca-DAB1*05carried fish (P x P) and non-carried fish (N x N), and two crosses for Cyca-DAB1*05-carried and non-carried fish (P x N) were performed to produce 10 families.Egg ovulation was induced by hormonal manipulation using ovaprim (Syndel Laboratories, Ltd.).Triplicate batches of fertilized eggs from each cross were incubated in 90-L glass aquaria at 28 ± 1 o C. The larvae were fed with Artemia nauplii three times a day.Five-day-old larvae were transferred to a hapa (size 2 m x 2 m x 1 m) settled in a concrete pond of 300 m 2 .Larval rearing density was 300 fishes per hapa.Fish were maintained for 60 days and fed with a commercial diet (protein content 28%) for three times daily.Thirty fishes per hapa were sampled every two weeks to measure the body weight.Survival rate was calculated at the end of fish rearing.
Ten fish samples from each cross were randomly selected after 45 days of rearing to determine the fish carrying Cyca-DAB1*05 allele.The DNA genome of caudal fin was isolated using a DNA Isolation Kit (Qiagen) according to the manufacturer's instructions.

KHV Challenge Test
Sixty-day-old KHV-free common carp (8-15 g of body weight) with a total of 30 fishes per family were adapted in 90-L aquaria at 21 o C-23 o C for three days before challenge test.The status of KHV-free was determined using PCR analysis as described by Gray et al. (2002).Challe nge te st was co nducted using cohabitation method by adding three KHVinfected fish into each aquarium.Fish were reared for 30 days at 21 o C-23 o C, fed with a commercial diet (28% protein) for three times a day.Water quality condition was maintained by allowing water exchange for up to 50% every two days.The fish mortality was recorded everyday.Samples of five live and dead fish from each cross were collected to determine the presence of Cyca-DAB1*05 allele by using PCR analysis as described previously.

Statistical Analisis
Statistical analyses were performed by using the SPSS 11.0 microcomputer software package (SPSS, Chicago, IL, USA).Differences between crosses were determined by one-way ANOVA followed by Duncan's test.The level of significance was set at P = 0.05.

DISCUSSION
Marker assisted selection (MAS) has been suggested to be a useful tool for shortening the time of selective breeding to obtain high quality broodstocks.In this study, PCR method was applied with a specific primer set for Cyca-DAB1*05 allele as the MAS to identify the majalaya common carp broodstock capable of producing progenies resistant to KHV.As a result, challenge-test survivals of PxP progenies showed about two times higher compared to NxN progenies.This clearly indicates that there is a link between the molecular marker and survival of the progenies.Furthermore, the survival rates of PxN/NxP and NxN progenies were constantly lower than that of PxP when they were challenged in pond condition and cohabitated with KHV-infected fish.However, the survival rate of NxP/PxN fish in KHV challenge test was lower than that of in pond rearing.This suggests that other immunogenetic factors may take part in increasing pond survival of NxP/PxN progenies.Variation of KHV resistance in terms of survival rate of progenies from different PxP cross was observed (Figure 2 and 4).In fact, about 91% progenies derived from those PxP carry the molecular marker.In addition, the results of PCR amplification of DNA from five dead fish after KHV challenge test showed that about 20% fish carried the marker.In this study, Cyca-DAB1*05 of MHC class II was used as the marker.In vertebrate including fish, immune response occupies the MHC class I and class II (Gillund et al., 2008).Thus, it is likely that MHC I may also be involved in immune regulation of common carp against KHV infection.Indeed, allele heterozigosity in Cyca-DAB1, e.g.*02/*05, *05/ *05, and *05/*06, has been reported in European common carp (Rakus et al., 2009).This heterozigosity affects resistance of fish against KHV infection.Similar pattern may also exist in majalaya common carp strain, and this remains to be investigated in the future.
A similar size of PCR product of Cyca-DAB1*05 in PxP fish was obtained in several individuals of NxN progeny (Figure 3).Because of high homology of nucleotide sequences among Cyca-DAB1, specific primer set for Cyca-DAB1*05 was designed based on one and two nucleotide polymorphism.Changes in one nucleotide at 3' region of Cyca-DAB1 can allow annealing of the primer.Thus, it is more likely that DNA band of PCR amplification product from NxN fish is the result of annealing primer to the mutated nucleotide region of Cyca-DAB1.
As a consequence of variations in the resistance of common carp to KHV infection, fish breeders are required to select the suitable PxP crosses producing higher KHV-resistant seed.The seed from those crosses can directly be extended for nursery and grow-out as a shortcut way to increase common carp production.The use of selective breeding method to allow Cyca-DAB1*05 to be passed on from broodstocks to their progeny may also increase resistance of common carp to KHV infection.There are at least 6 common carp strains in Indonesia (Aliah et al., 1999).The use of Cyca-DAB1*05 marker to identify other common carp strains is also interested to be performed to provide various strains that are resistant to KHV.In addition, the survival of progenies from crosses of European common carp strains is higher than their purebred against KHV infection (Odegard et al., 2010).The KHV resistance of Indonesian common carp may also be improved by cross breeding program.

CONCLUSION
The molecular marker of Cyca-DAB1*05 allele can be used to identify individual broodstock of majalaya common carp strain producing progenies resistant to KHV infection.The progenies have high survival rates when it is cultured in ponds.

Figure 1 .
Figure 1.The results of screening for the presence of Cyca-DAB1*05 allele by PCR analysis with DNA templates extracted from caudal fin of broodstock common carp.A: PCR product using Cyca-DAB1*05 specific primers.Lanes 1 to 10, PCR product of broodstock individuals.M is 2-log ladder DNA marker (BioLabs, Inc., New England).The amplified fragment is about 260-bp in size (marked by arrow head).B: PCR product with b-actin gene primers [200-bp in size]