Identification and characterisation of microsatellite DNA markers in order to recognise the WSSV susceptible populations of marine giant black tiger shrimp, Penaeus monodon

Chakrabarty et al. Veterinary Research (2015) 46:110 DOI 10.1186/s13567-015-0248-2 VETERINARY RESEARCH RESEARCH ARTICLE Open Access Identification and characterisation of microsatellite DNA markers in order to recognise the WSSV susceptible populations of marine giant black tiger shrimp, Penaeus monodon Usri Chakrabarty, Sourav Dutta, Ajoy Mallik, Debabrata Mondal and Nripendranath Mandal* Abstract White spot disease (WSD) which is caused by white spot syndrome virus (WSSV) creates severe epizootics in captured and cultured black tiger shrimp, resulting a huge loss in the economic output of the aquaculture industry worldwide. Performing selective breeding using DNA markers would prove to be a potential cost effective strategy for long term disease control in shrimps. In the present investigation, microsatellite DNA fingerprints were compared between naturally occurring WSSV resistant and susceptible populations of Penaeus monodon. After PCR with a set of shrimp specific primers three reproducible DNA fragments of varying sizes were found, among which 442 bp and 236 bp fragments were present in considerably higher frequencies in the WSSV susceptible shrimp population (p ≤ 0.0001). After WSSV challenge experiment the copy no. of WSSV was determined using real-time PCR, where it was found to be almost 4 × 103 fold higher in WSSV susceptible shrimps than in the resistant ones. Thus, these microsatellite DNA markers will be useful to distinguish between WSSV susceptible and resistant brood stocks of P. monodon. Sequencing studies revealed that these DNA markers were novel in P. monodon. Highest WSSV resistance using these DNA markers, was observed in the shrimp populations of Andaman Island and Chennai among the different coastal areas of India, suggesting these places as safe for specific pathogen resistant brood stock shrimp collection. This study will be a very effective platform towards understanding the molecular pathogenesis of WSD for generation of disease free shrimp aquaculture industry. Introduction Aquaculture industry enjoys an exponentially profitable market worldwide and deals with various valuable marine and fresh water invertebrate and vertebrate species. Marine black tiger shrimp, Penaeus monodon, is one of the most economically important native cultivated species of India due to its bigger size, higher nutritional value and huge demand as food delicacies across the globe. They contribute to 95% of the total production of the captured and cultured shrimps in India [1]. Proneness of the shrimp species towards many deadly viral diseases is an alarming factor in aquaculture industry [2]. Among them, the white spot disease (WSD) caused due to white spot syndrome virus (WSSV), a rod shaped * Correspondence: Division of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII-M, Kolkata 700054, India dsDNA virus (genus Whispovirus of the family Nimaviridae) of 305.12 Kb length is the deadliest one. WSSV has a very wide host range and is able to sustain for a considerably long period of time in the virion state increasing the chances of further infection [3-5]. Several preventive measures were tested and applied throughout the world against the WSSV infection, but with a very less success rate [6-9]. It is well known that, some resistance phenomena always lies in the repository of the nature itself. Nature always favoured some individuals for better existance and resistance as well as preventive capability against any kind of odds like natural and artificial disasters. So that is obvious that, some kind of disease resistance phenomena might be present in shrimps also and some special genomic fingerprints may be accountable for this resistance. © 2015 Chakrabarty et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chakrabarty et al. Veterinary Research (2015) 46:110 Several DNA fingerprinting methods are used in population genetic studies, genetic diversity analysis, classifying germplasm and selective breeding in animals and plants for disease resistance [10-14]. Microsatellite markers are vastly used for study due to their reproducibility, codominant expression type, even genomic distribution, small locus size and high polymorphism. This enriched knowledge about DNA fingerprints can be very useful in the isolation of resistant individuals from an economically important species and cultivating them selectively as per the suitable genomic content. Disease resistance has been a major field of interest in several species of shrimps since decades. Eight markers associated with infectious hypodermal and hematopoietic necrosis virus (IHHNV) resistance or susceptibility in mutant and wild type populations of Litopenaeus stylirostris have been discovered using RAPD technique [15]. Evidence of clear association of a microsatellite marker was found with taura syndrome virus (TSV) susceptibility/resistance in Litopenaeus vannamei [16]. There are more instances where, one more microsatellite loci (RS0622) was identified to be associated with WSSV resistance in Fenneropenaeus chinensis [17]. Recently, single nucleotide polymorphism (SNP) discovery and association analysis detected several SNP markers associated with resistance to TSV in L. vannamei [18]. Previously, one 71 bp microsatellite DNA marker has been developed from two populations of P. monodon designated as WSSV resistant and disease susceptible which were collected from ponds, highly infected with WSSV [19]. Later both of these two populations of P. monodon were challenged individually by injecting WSSV and finally mortality as well as WSSV propagation was measured by quantitative real-time PCR. It was observed ~103 fold higher WSSV propagation was occured in the disease susceptible population than the WSSV resistant population [20]. This study is mainly focused on the identification of more microsatellite DNA markers associated to WSSV resistance or susceptibility in shrimps. From the sequence analysis some very useful novel DNA markers are identified and practical applicability of these DNA markers are subsequently confirmed by WSSV challenge experiment. The possible safer places for WSSV resistant brood stock collection for Indian shrimp aquaculture are also suggested in this investigation. Materials and methods Sample collection P. monodon samples were collected from 20 highly disease affected ponds in West Bengal, India. The mature adult shrimp samples of 75 days (...truncated)