Construction Of SSR Linkage Maps For Larimichthys Crocea By Using Distant Hybirdization

Whole genome amplification technique, universal sequence tailed and fluorescence labeled microsatellite analysis method by the universal M13 primer were established for the distant hybridization fingerprints of Larimichthys crocea×Nibea albiflora in this study. Those methods were employed to construct the SSR genetic linkage maps by distant hybridized fry of Larimichthys crocea (♀)×Nibea albiflora (♂) and Larimichthys crocea (♂)×Nibea albiflora (♀) separately. Additonally, the genetic distance between centromere and 32 typeⅠmicrosatellites were analyzed by inducing hybrid triploid lines of Larimichthys crocea. The major results were presented as following.1. M13-tailed microsatellite analysis method was developed by the fluorescent labeling universal M13 primer, which facilated to collect the genotyping datas of microsatellites automatically and accurately. The PCR system included three primers: forward tailed primer by M13 universal sequence, reverse primer, and labeled M13 universal primer by fluorescence dye. The desired PCR result could be obtained when the proportion of forward primer and reverse primer was 1:10, and the amount of the forward primer and M13 primer equaled to reverse primer. In this study, the PCR reaction system, thermal cycling procedure, and multi-loading method for PCR products had been optimized for the developed microsatellites. Comparing with the common PCR and silver-staining methods, our approach had the advantages of high-throughput, low-cost, and being accurate analysis.2. Although the normal distant hybridized fry which processed the parental haploid genomes and normal morphology could be created by the distant hybridization between Larimichthys crocea and Nibea albiflora, they could not ingest exogenous food and growed normally. The quality of their gomomic DNA, thus, was unsufficient for constructing of genetic linkage maps. Therefore, whole genome amplification was developed in this study for the amplifying genomic DNA of distant hybridized fry. The endonuclease TaqⅠwas used to digeste the genomic DNA, then the oligounclotide adaptor was ligated to the DNA fragments, and finally the PCR amplification was performed for the ligation products by the primer from the adaptor sequence and the high fidelity DNA polymerase. Genomic DNA could be multiplied about 2.5×107 folds, and the most amplified DNA fragments fall the range of 500 bp-1500 bp. Desried PCR amplification could be attained when the amplified gemomic DNA was used as PCR template. Whole genome amplification provided an aviliable solution for the unsufficient quality of genomic DNA when using fingerprints to construct genetic linkage maps.3. It was very difficulty to construct high density genetic maps by families of large yellow croaker alone, because there were a few genetic variances among parents, resulting in the lacks of genetic linkage information for many microsatellites. We, thus, attempted to use the distant hybridized fry of Larimichthys crocea and Nibea albiflora to construct the genetic maps for large yellow croaker, with the expecting to increase the markers'dencity in genetic maps. 144 and 96 F1 progenies from two reciprocal hybrid lines of Larimichthys crocea and Nibea albiflora were used. The major resources of microsatellites were those that lack linkage information in families of Larimichthys crocea, and those that had been mapped on the genetic maps of Larimichthys crocea with regarding as anchor markers. 221 microsatellites were tested, and there were 81 and 61 microsatellites of processing linkage information in reciprocal crossing families, while the female map and male map were consisted of 43 and 30 loci, with assembling into 15 and 11 linkage groups separately. The total length of two maps were 529.17 cM and 327.56 cM, with the expected length of 1138.96 cM and 614.48 cM, respectively. The coverage rates of two maps, hence, were 46.4% and 53.3% separately. Additionly, specific amplified products could be obtained by 32 microsatellites in Nibea albiflora, and 14 markers were polymorphic.4. When distant hybridization between Larimichthys crocea and Nibea albiflora, the releasing of secondary polar body was inhabited to found two hybrid triploid lines. The distance between centromere and 32 typeⅠmicrosatellites were analyzed by this triploid lines for Larimichthys crocea. The value of recombination rate for 21 markers was larger than 2/3, inllustrating there were long distance between centromere and those microsatellites. The recombination rate value between LYC1737 and centromere was 0.979, presenting that LYC1737 was possible at the terminal region of chromosome; while the value was 0 for LYC3211, displaying that LYC3211 was likely to be very close to the centromere.