Identification of Anti-BmNPV molecular marker of silkworm NC99R based on BSA-Seq

【Objective】By using BSA-Seq sequencing analysis to determine the resistance SNP markers and resistance genes of the resistant parent NC99R of the nuclear polyhedrosis resistant variety Guican N2, lay a foundation for the identification of NC99R resistance genes and provide marker resources for silkworm nuclear polyhedrosis resistant molecular marker assisted breeding.【Method】A hybrid F₁, F₂, and BC1 genetic population were prepared using the disease resistant variety NC99R and the susceptible variety Furong as parents, and their genetic patterns were analyzed by feeding nuclear polyhedrosis virus(BmNPV). In the F₂ population of NC99R near allelic gene line NC99R-NIL hybridized with Furong, high and low concentration feeding groups with virus concentration differences of 4000 times were set up. Non-diseased individuals were selected from the high concentration feeding group to construct an extreme resistance material DNA mixing pool. Individuals with typical disease symptoms were selected from the low concentration feeding group to construct an extreme susceptibility material DNA mixing pool. Then the resistance DNA mixing pool, susceptible DNA mixed pool and two parental genomes were completed sequencing using second-generation sequencing, and locating resistance linked regions with population segregation analysis(BSA).【Result】The results showed that NC99R exhibited stable high resistance to BmNPV, and the genome carried anti BmNPV genes, which were jointly regulated by significant resistance major genes or closely linked genes. A total of 19276670 SNP loci and 4789615 InDel loci were obtained through whole genome sequencing analysis of extreme resistance DNA mixing pool, extreme susceptibility DNA mixing pool and parental DNA. Based on the LOESS regression fitting analysis of ΔSNP-index, two regions associated with resistance to nuclear polyhedrosis were identified, Chr. 25: 150-11069kb and Chr. 27: 8700-11009kb respectively. When the ΔSNP-index was set to≥0.8, a total of 3865 SNP markers significantly related to resistance were identified. Among them, 78 SNP markers were screened in the association interval of chromosome 25 and 2694 SNP markers were screened in the association region of chromosome 27. After screening for non-synonymous mutations at the amino acid level, the Zinc carboxypeptidase gene on chromosome 25 and the Facilitated trehalose transporter Tret1 gene on chromosome 27 were identified.【Conclusion】Using BSA-Seq, 3865 SNP marker resources related to resistance against nuclear polyhedrosis are found in silkworm NC99R, and two genes related to resistance against BmNPV are preliminarily identified(Zinc carboxypeptidase gene on chromosome 25 and Facilitated trehalose transporter Tret1 gene on chromosome 27), providing marker resources for molecular marker assisted breeding of silkworms resistant to nuclear polyhedrosis.