Development And Preliminary Application Of Chromosome-specific BAC-FISH Probes In The Large Yellow Croaker

Large yellow croaker(Larimichthys crocea)is one of the most important marine cultured fish in China.In recent years,the research on genetics of large yellow croaker have advanced rapidly.However,the stusies on the chromosomal level still insufficient.Similar to other fishes,the chromosomes of large yellow croaker have the the characteristics of small size,single morphology,and continuous length.So it is difficult to identify chromosomes and fragments.Accurate identification of chromosomes has been the bottleneck of the cytogenetics research in large yellow croaker.In this study,we selected some BAC clones from the BAC library for end-sequencing.According to these end sequences,72 chromosome-specific BAC clones were screened out by bioinformatics method and verified by FISH.This study provides a cytogenetic tool for the chromosome identification of the large yellow croaker,and helps to integrate the genome sequence map and the cytogenetic maps of large yellow croaker.Meanwhile,the BAC-FISH was performed in Collichthys lucidus by using the chromosome-specific clones of large yellow croker.It provides a research tool for chromosome indentification of Collichthys lucidus.and also laid a basis for further cytogenetic studies in yellow croaker and other Sciaenids.The results were as shown:(1)Preliminary analysis of the end sequence of BAC cloneBased on the BAC library of large yellow croaker.500 BAC clones were selected in random and sequenced from the two ends of the inserts.After trimming and quality filtering.1100 BAC end sequences(BESs)including 1050 paired-ends BESs were obtained.The lengths of the BESs ranged from 100 to 1200 bp with an average length of 870 bp.The total base number was 960629 bp,with 41.51%GC,representing 0.13%of the large yellow croaker genome.Analysis of the BESs indicated that the repeated sequence accounts for 5.49%of the total number of bases and the primary repeats were DNA transposons.In addition,107 SSRs were predicted from 91 BESs,in which dinucleotide repeats were the most common type.(2)Screening and validation of chromosome-specific BAC clonesChromosome-specific BAC clones were screened by bioinformatics method.The BESs were aligned to the reference genome sequence of the large yellow croaker,of which 870(85.6%)and 146(14.4%)were located on chromosomes and scaffold,respetively.There were 435 BAC clones with pair-ends located on the same chromosome,with an average of 18 BAC clones per chromosome.Five BAC clones for each chromosome were selected,for they contained relative little repeatitive sequence which was known from checking the corresponding segment in the reference genome.Among them,3 BAC clones were further selected for FISH analysis according to their relative location on the chromosome.After being validated with FISH,2 chromosome specic BAC clone were gained for Chr.5,6,18,23,and 24,and 3 chromosome specic BAC clone were gained for the other chromosomes.The signal sites located on each chromosome are collinearity in consistent with the result of BAC end sequence alignment.(3)Development of chromosome recognition method for large yellow croakerMulticolor FISH was used to realize the recognition of all chromosomes of large yellow croaker.A panel of chromosomal specific BAC clones were prepared with different haptens to form a probe pool for FISH hybridization.According to the number of signal sites,color and chromosome morphology,the recognition of each chromosome was realized.(4)Cross-spceice application of the chromosome-specific BAC clones of large yellow croakerThe BAC end sequence of large yellow croaker was aligned and compared to the reference genome sequence of Collichthys lucidus.The results showed that a total of 219 BAC clones were aligned to the reference genome of C.lucidus.By FISH,Clone 101-J21 and 101-K12 that was chr.5 specific in large yellow croaker,were located on the same pair chromosomes in C.lucidus,suggesting that the chromosome-specific BAC clones of large yellow croaker developed in the present study can be partially used for C.lucidus.