Localizing Genome-wide Linkage Groups And Sex-determination Region Using BAC Clones And Fluorescence In Situ Hybridization (Fish) In The Large Yellow Croaker

Large yellow croaker(Larimichthys crocea)is one of the most important marine cultured fish in China.Despite the published whole genome sequences,the cytogenetic information is still largely unknown.In this study,we constructed the Hind-III BAC library of the male large yellow croaker,screened specific BAC clones of 24 linkage groups from BAC library and made a preliminary evaluation and validation.This study can provide abundant probes for improving chromosome analysis and lay the foundation to construct cytogenetic map of large yellow croaker.Meanwhile,screenning the BAC clones of male sex determination candidate genes of Dmrt1 can contribute for further study of molecular mechanism of sex determination in large yellow croaker.The main results were as follows:1?Constructing and evaluating the large yellow croaker BAC libraryWe constructed a Hind-III BAC library using DNA from brain of male fish.The BAC library consists of 41,472 clones with 1.25% empty,an average insert size of 133 kb,and 8.1 haploid genome equivalents.2?Screening and preliminary analysis of specific BAC clones of linkage groupsA three-step PCR screening system of BAC library was constructed using an orderly mixing of the BAC clones,we obtained 72 positive clones of linkage groups.The 72 BAC clones were then sequenced and obtained 141 high-quality BAC end sequences,the average length of which was 625 bp.The total base number was 88,195 bp,with 41.45% GC,which was comparable to the reference genome GC content(41.71%).Forty-one gene annotations were detected in 40 out of 151 BAC end sequences.Thirteen microsatellite makers(2~6 base)were discovered in 12 genome sequences using the Tandem Repeats Finder(TRF)software.High-quality BAC end sequences were aligned to the genome and genetic linkage group of large yellow croaker.Results showed that 85(60.3%)of the BAC end sequences were aligned to 1 locus of prospective linkage groups;22(15.6%)of the BAC end sequences were aligned to 2 loci of prospective linkage groups;12(8.5%)of the BAC end sequences were aligned to scaffold of unknow linkage group;18(12.8%)of the BAC end sequences were aligned to unexpected linkage groups;and 7(5.0%)of the BAC end sequences were aligned to several loci in the genome of large yellow croaker.The FISH results showed that LG9(18O3,20F6,30C22)BAC clones FISH signals were detected 2 pairs,1 pair and 10 pairs respectively,which indicated the three clones were no synteny.The LG10(20N3,8C9,29H16)BAC clones FISH signals were detected 1 pair,2 pairs and 1 pair repectively,which also indicated the three clones were no synteny.And the LG19(75P10,77I4,60P17)BAC clones FISH signals were detected 1 pair,8 pairs and 9 pairs respectively,which indicated the three clones were unable to estimate synteny.3?Screening and preliminary analysis of Dmrt1 BAC clonesFive Dmrt1 positive BAC clones(8G22,30N15,49L4,71K22 and 78O2)were screened out from the BAC library using the three-step PCR screening method.The reuslts of PCR product sequences analysis indicated that 5 clones were in XY genotype coinciding to the rate of above 94.50% in the reference genome.Sex-linked markers detection results showed that the clone 30N15 was from Y chromosome and the clone 8G22,49L4,71K22 and 78O2 were from X chromosome.We sequenced 5 BAC clones and obtained 10 high-quality BAC end sequences,with an average length of these sequences of 783 bp.The total base number was 7,826 bp,with the GC percent of 39.50%,which was less than that of the reference genome(41.71%).The alignment of the genome of large yellow croaker and genetic linkage group showed that 5 BAC end sequences were aligned to expected linkage groups,and 5 BAC end sequences were aligned to unexpected linkage groups.And 6 and 2 BAC end sequences were aligned to 1 and 2 locus,repectively.In addition,2 BAC end sequences were aligned to several loci in the genome of large yellow croaker.BAC-FISH results suggested that 5 Dmrt1 gene positive clone were located at the proximal centromere region of chromosome,which was associated with the loci of 18 S rDNA.The morphology of chromosomes and Dmrt1 positive signals were not obviously differed from male to female individuals.