Identification Of ZW Chromosomal DNA Fragments Using Whole-genome Resequencing Approach And BAC Library Screening Method In The Chinese Mitten Crab,Eriocheir Sinensis

The Chinese mitten crab,Eriocheir sinensis,a member of the Grapsidae family of decapod crustaceans,widely distributed in coastal lakes in north and south China.Due to the female crab’s ’crab roe’ has high edible and medicinal value,the study of its sex control has important application significance in the parthenogenetic reproduction of Chinese mitten crabs,but the study on its sex determination mechanism still needs to be further explored so far.It has been previously reported that the sex determination mechanism of the crab is WZ/ZZ system through genomic research and the establishment of genetic linkage map.Because of the large and small number of chromosomes in crustaceans such as crabs makes it impossible to identify sex chromosomes by karyotype analysis,and genome sequencing assembles diploid sequences into haploid sequence,making it even more impossible to distinguish sex chromosomes.The identification of putative DNA fragments on Z and W chromosome can be effectively solved using the whole-genome resequencing approache in Eriocheir sinensis,and then using chromosome candidate fragments as probes to screen BAC library not only provide sex-specific markers for the study of sex determination mechanism of the crab to distinguish its sex chromosome sequence,but also provide theoretical basis for the follow-up study of its complete sex chromosome and sex-related genes.In order to obtain candidate fragments on the Z and W chromosome in Eriocheir sinensis,our team previously used next-generation sequencing to establish genetic linkage map to isolate 70 sex-difference SNP(single nucleotide polymorphism)markers located on the No.48 sex-linked group(LG48),which mapped 16 different genome scaffolds.In this study,genomic DNA from two males and one female is sequenced independently and aligned to female reference sequences.The male to female ratio of the number of alignments to a reference sequence,a parameter called the chromosome quotient(CQ),is used to determine whether the scaffold is Z-linked or W-linked.Using the CQ method,we successfully identified Z-linked scaffolds(CQ=2)and W-linked scaffolds(CQ=0)in Eriocheir sinensis and compared with these in LG48;Screening 40 scaffolds(CQ=2)of Z chromosome DNA using quantitative PCR,in which males were 2 times the number of female copies of DNA.The results confirmed that five scaffolds with 2 times relationship were Z chromosome DNA,and 35 scaffolds presented a 1:1 relationship,indicating the reliability of theoretical Candidate Fragments of Z chromosome DNA(CQ=2).We design 80 pairs of specific primers based on W-linked scaffolds for PCR amplification in 8 pairs of individual genome DNA of mitten crabs,and obtained only one unique band(560bp)exclusive to the female genome,but no specific segment appeared in the male genome.To discover the Z chromosome derived fragment,we design specific primers S820F/R based on the downstream region of 560 bp W fragment located on scaffold47455,and then two bands(1156bp and 820bp)appeared in the female genome,while only one band(1156bp)appeared in the male genome.A further 20 pairs of Xinghua population of crabs were selected for verification and similar results were reached,which confirmed that the sex determination mechanism of the mitten crab was female heterogametic type(WZ)and male homozygous type(ZZ).Afterwards,cloning sequencing found that the DNA fragment(1156bp)common to both males and females was of the same sequence,while the female-specific 820 bp band of DNA fragments compared with a 1156 bp fragment missing about 300 bp and 70 base mutation points.Furthermore,we confirmed the reliability of Z chromosome derived fragment using quantitative PCR,which can be concluded that the 1156bp-band is from Z sex chromosomes,the 820bp-band is from W sex chromosomes,respectively named Z and W chromosome derived fragment.These two sets of fragments provide a molecular marker for sex determination of the mitten crab,but for more genetic information anchored to sex chromosome,we ulteriorly screened BAC library using the Z and W fragments as the probes.401 level one pools and 520 level two pools were prepared,using specific primers of S820F/R and the pooled BAC plasmids as templates for D screening on the pools by PCR.In the end,we successfully obtained a total 7 positive clones of the Z and W putative fragments.Herein,a W-derived(P282E18)BAC clone was selected to sequence by Pac Bio method randomly,which a 45388 bp W chromosome DNA sequence was obtained,and then collinear alignment of homologous regions with the reference sequence of W chromosome in the genome were preliminarily analyzed,and the similarity reached 95%;6 functional genes were predicted from the W BAC insert,which opened a new chapter for the further exploration of the sex chromosome genetic information in Eriocheir sinensis.