The Analysis Of WSSV Resistance Of Fenneropenaeus Chinensis "Huanghai No.2"

In China, farming industry of fleshy shrimp Fenneropenaeus chinensis has almost collapsed because of the outbreak of WSSV (white spot syndrome virus) disease since 1993. Thereafter, to produce the fast-growth and/or disease-resistance shrimp varieties became an important approach which would lead to the second bloom of shrimp farming in China. "Huanghai No.2" is the first F. chinensis new variety with multi-traits (better growth performance, higher survival rate and longer survival time after WSSV infection) for farming in China. It was approved by National Aquatic New Variety Certification Committee in 2008. The application of this new variety alleviated and controlled the outbreak of WSSV disease to some extent. The purpose of this thesis is to provide theoretical bases for studying the molecular mechanism of WSSV resistance, to improve the selected traits and expedite the selected breeding progress of F. chinensis. In this thesis, the breeding technology of "Huanghai No.2" is summarized and the genetic parameters and genetic gain of WSSV tolerance and of survival rate are evaluated. Besides, the comparison of commercial seedling and "Huanghai No.2" is also conducted to prove the superiority of selected traits. In addation, WSSV amplification mode in postinfected shrimp is discussed. Moreover, the characters of EST sequences that generated from the next generation high-throuput sequencing technology are analyzed in "Huanghai No.2". The main results are showed as follows:1. Breeding technology of new F. chinensis variety "Huanghai No.2"Two cultured populations of "Huanghai No.1" variety and "Jikang 98" strain and four wild populations of F. chinensis are selected as base population via unbalanced nested breeding design. Afterward 3-time standardized breeding procedure of all shrimp families is conducted, then to carry out serials procedures to evaluate genetic parameters comprehensive selection index via Best Linear Unbiased Prediction (BLUP) and Restricted Maximum Likelihood (REML) methods, and design mating strategy, et al., to achieve the selected breeding of "Huanghai No.2". It's revealed that the heritability of WSSV resistance is as low as 0.017-0.026, the genetic gain of WSSV resistance is 6.38%,9.74% and 6.78%, respectively, based on statistical analysis of data from 2007 to 2009. The comparison of commercial seedling and "Huanghai No.2" in 2008 shows that WSSV-resistance traits of "Huanghai No.2" is higher than commercial seedling of Changyi, Rizhao and Hebei with ratio of 12.25%,14.59% and 20.70%, respectively.2. Quantitative analysis of "Huanghai No.2" to its viral loads after artificially infected with WSSVIn order to explore the amounts of WSSV in F. chinensis after oral feeding WSSV, the viral loads are examined by TaqMan fluorescent quantitative PCR in"Huanghai No.2"shrimp at different stage. The WSSV levels are 8.8×104-1.5×105,4.4×105-1.2×106 and 8.5×105-4.6×106 copy/ng DNA in shrimps died at early, middle and late periods of postinfection, with average of 9.5×105 copy/ng DNA, whereas that is 2.2×105 copy/ng DNA in survived shrimps. However, only 18.2% of survivals have viral loads over 1.0×105 copy/ng DNA and 52.3% had less than 1.0×102 copy/ng DNA. Statistical analysis indicates that "Huanghai No.2" has strong anti-WSSV ability and can survive for long time after WSSV infection.3. WSSV amplification character in shrimp after artificially infected with WSSVIn order to investigate the dynamic change of WSSV amplification after virus invades shrimp, TaqMan fluorescent quantitative PCR is applied to examine the viral loads of living shrimp that sampled at different time. Based on statistical analysis of 8 times within 120 hr of postinfection, the amounts of WSSV in shrimp are ascending all along. The trend of WSSV amplification is similar to that of the ratio of dead shrimp per unit time to the remainders. Whileas when the state of WSSV disease become stable and there are no more shrimp die continuelly, the WSSV loads of more than 80% survived shrimp decrease to original state. Hereby three amplification modes of WSSV in shrimp of postinfection are deduced.4. The differences of EST sequences of "Huanghai No.2" after infected by WSSVA next generation high-throuput sequencing approach, Roche 454 GS FLX technology is applied to sequence the transcritptome of WSSV-sensitive (group S) and WSSV-resistant (group R) shrimp of "Huanghai No.2". A total of 451,637 high-quality reads and 95,583,417 bases are obtained averaging 212 bp per read. A total of 48,681 unigenes, including 18,560 contigs and 30,121 singletons are assembled with CAP3 software. Of these, a total of 71.23% sequences are new. There were 16,790 specific contigs in group R and 12,573 in group S based on comparison of BLASTN. Of these,968 contigs with annotation and 427 with GO in group R and 1156 with annotation and 465 with GO in group S if not considering singletons. A total of 71,724 putative SNPs are detected, in which there are 53,449 and 18,275 SNP in coding region and non-coding region, respectively. Different SNP types of same sense, mis-sense and nonsense is 17,329, 34,642 and 1,478, respectively, in coding region.