The Application Of Molecular Markers In The Selective Breeding Of Marsupenaeus Japonicas

The Marsupenaeus japonicas is one of the most commercial important prawn inChina as well as its selective breeding work. In the present study, microsatellitetechnology was used for genetic analysis among different breeding groups and aestimating method for genetic parameters based on SSR was constructed which wouldlaid a solid foundation for the marker assisted selective work in M. japonicas. Theheat resistance trait of breeding group of M. japonicas was tested and its relationshipamong growth trait and inbreeding were analysed and SLAF-sequencing was used toscreen heat resistance trait associtated SNPs for assisting breeding M. japonicas withheat resistance ability.The genetic structure for two generations of cultured M. japonicas wasinvestigated by8pairs of microsatellite primers. In this study, a total of108alleleswere tested, and the length of alleles ranged from196bp to528bp; the number ofalleles per locus ranged from6to23with an average of13.5. For the parents andoffspring populations, the average numbers of alleles were8.5and11.5, respectively;the observed heterozygosities were0.7211and0.6319, respectively; the averageexpected heterozygosities were0.7748and0.7635, respectively; the averagepolymorphism information contents were0.7315and0.7286, respectively. Thepedigree structure for the parents and progeny populations was inferred by maximumlikelihood method according to the microsatellite marker information. The parentpopulation was divided into6groups, and the genetic distance among groups rangedfrom0.2773to2.356with an average1.5097. The offspring population was dividedinto17groups, and the genetic distance among groups ranged from0to2.5929withan average of1.1132. The genetic distance between parents and offspring sub-groupsranged from0to3.0892with an average of1.2380. An evolutionary tree for the23sub-groups populations was drawn by unweighted pair-group method with arithmeticmeans (UPGMA) according to the Nei’s genetic distance. Eighteen sub-groupsaggregated into the main stretch after forming three sub-stretches. However, five sub-groups (O15, O12, O13, P21and P18) were obviously away from the mainstretch. In this study, the heterozygosity and polymorphism of the offspringpopulation were lower than their parent. The result showed that loss of heterozygosityof parents has been partly lost during the genetic inbreeding.286cultured kuruma shrimps were chosen randomly from a pond, and their heatresistance and eight morphological traits were tested. Growth traits were used asindependent variables and heat resistance was used as the dependent variable incorrelation and path analysis.150individuals were chosen randomly for genetictyping by eight microsatellites, then the microsatellite inbreeding coefficient for eachshrimp was worked out by their genotype, and the corresponding of microsatelliteinbreeding coefficient with growth trait and heat resistance was calculated after that. Itwas found that the correlation coefficient between all the eight morphological traitsand upper temperature tolerance (UTT) were statically significant (p<0.01). The pathcoefficient (Pi) of body length, carapace width,6th abdominal segment length, widthbetween2th and3th abdominal segment, height between2th and3th abdominalsegment were statically significant, and these five traits were reserved for constructinga multiple regression equation with UTT. Most of the150individuals were detectedwith inbreeding, we found that the correlation between inbreeding coefficient andtraits were not statically significant.Fifteen male shrimps and thirty female shrimps were chosen from well-grownPenaeus japonicus preparatory parents to be a mating group then they mated in anatural way. At last, we harvested twelve male shrimps and twenty fertilized femaleshrimps, and six families were successfully constructed in the end. Genome DNA wasextracted using the muscle tissues of every parent and the whole bodies of mixedoffspring of every family. Twelve pairs of microsatellite primers were used to do PCRamplification for each DNA sample, and the parental analysis was done in use of themicrosatellite parting consequence. In the end, five families found the true fatherwhile two families shared a same father, and one family lost the father. This articletested the feasibility of finding out the family’s father in an early growth period inbreeding works for penaeus japonicus.12microsatellite loci were chosen for studying the genetic relations among15Marsupenaeus japonicus culturing groups from15families in need of the futurebreeding work.241alleles were tested from the12loci, each locus has5-30allelesand20.08on average; average observed and expected heterozigosity value is0.804and0.899, respectively; average polymorphism information content is0.889. Average total inbreeding coefficient (FIT) is0.1081, average inbreeding coefficient within-families (FIS) is-0.2338. Average genetic differentiation coefficient on all loci (FST)is0.2771and the average gene flow coefficient (Nm) is0.6521. A UPGMAdendrogram constructed according the Nei’s genetic distance among the15groupsrevealed group B4and C7has the closest while group C10has the furthest geneticrelation with other groups. Every individual was accurately divided into its ownfamily by family reconstruction.8of the12loci were found holding null allele. Thestudy reveals that the groups in current research have a relatively high geneticdiversity, and the genetic differentiation among groups is also high, while the geneflow is little. Groups with close genetic relationship like B4and C6should not bemated for avoiding inbreeding in the future mating project. The12SSR loci could beused in genetic analysis, but loci with a high null allele frequency like No.76shouldbe careful used in the future. Pedigree and molecular relatedeness were respectivelyused for estimating genetic parameters for10months old G2generationMarsupaneaus japonicas, which would be reference valuable for the future work.We used bulked segregant analysis as a method for rapidly identifying SNPmarkers linked to high-temperature tolerance traits in M. japonicas. Two bulked DNAsamples are generated from a high-temperature tolerance experience, one contains30shrimps dead in the initial time and the other contains28shrimps dead in the last time.SLAF sequencing technology based on paired-end sequencing was used for SNPscreening. At last, we got4537440bp sequenced sequences and50416high-qualitySLAFs, with an average sequencing depth of64.48x.4697SLAFs contain putativeSNPs, accounting for9.13%, no indels were found. In the SLAFs containing SNPs,1151were dominant in one of the two bulks, among these if one genotype is morethan five times opposite the other in one locus, then we consider this locus may berelated to the high-temperature tolerance traits,46and42markers were found fromthe non-high-temperature tolerance bulk and the high-temperature tolerance bulkrespectively. Each SLAF contains one to four putative SNPs, among putative SNPsmay be related to high-temperature tolerance, the number of transition type is1.39times of the transversion type.