Study On The Heredity Of BmNPV Resistance Of Silkworm,Bombyx Mori,Based On The 2b-RAD Technology

Silkworm is a Lepidoptera model insect and is also an important economic insect.In China and India and other developing countries,silk has a high economic value,is the main source of income for farmers in many areas.However,the occurrence of silkworm disease to the silkworm industry has brought great distress.According to incomplete statistics,the annual loss caused by silkworm disease is about three percent of the total output,seriously restricting the stable development of sericulture industry.Virus disease is the most serious disease in the production of sericulture,in which the Bombyx mori nuclear polyhedrosis virus?Bm NPV?caused the blood type pus disease the greatest harm,about 70% of silkworm loss,the silkworm disease current Still stay in the prevention phase.Therefore,from the production and safety aspects,the silkworm nuclear polyhedrosis virus resistance gene search and resistance varieties of the combination is an important work.In this study,we used the resistance of Bm NPV silkworm variety Wukang 2generation hybrids with moths to form inbred lines AN,anti-Bm NPV silkworms wild ternary N resistant parent BN and susceptible strain C108 as the study Material,first of all different varieties of silkworm Bm NPV resistance to the size of the study.On the basis of this,we constructed the resistant isolates and established the 2b-RAD analysis database.The main contents and results were as follows:1.Identification of Bm NPV resistance of silkworm AN and wild BN?LC50?was 2.154 × 109 cells / m L,and the half lethal dose?LD50?was 1.436 ×107 / head in the 2nd instar silkworm Bm NPV polyhedron.Wild BN in the feeding Bm NPV virus polyhedron concentration reached 1 × 108/ m L when the occurrence of silkworm death,wild BN half LD50 2.5 × 107 / head.The morbidity of the susceptible cultivar C108 was high when the virus was inoculated,and when the polyhedrin concentration reached 1 × 107,the total lethal concentration?LC50?was as low as1.239 × 105 cells / m L,LD50 is 8.266 × 102 / head.The gap between the resistance strain AN and wild BN and the susceptible strain C108 reached above 104.Therefore,the use of Bm NPV polyhedron suspension concentration of 1 × 108/ m L impregnated mulberry leaves on the 2nd instar larvae for 12 h,can be effectively selected AN and wild BN strains and Bm NPV resistance gene carrying individuals.2.2b-RAD analysis of Bm NPV resistance gene in silkworm AN and wild BNstrains1)2b-RAD marker analysisIn order to analyze the genetic mechanism of Bm NPV resistance,the susceptible cultivars C108 were used as backcrosses to construct two varieties of BC4 M [C108 ×???AN × C108?× C108?× C108?)And BC3 M [C108 ×??wild BN × C108?× C108?×C108)] genetic separation population.Two survivors were isolated and cultured for 2days.The survivors were treated with 1 × 108 cells / m L NPV polyhedron suspension for 12 hours.Construction of the 2b-RAD library and the Illumina sequencing of the genomic DNA of three parents and two resistant isolates.A total of 11919 SNPs markers were obtained.The polymorphism SNP markers were distinguishable between SNPs with different BN and C108 lines.SNP molecular markers were mainly distributed in intron?44.13%?,intergenic?21.92%?,downstream?12.12%?and upstream?11.09%?and exon?8.82%?regions.The SNP synonymous mutation,missense mutation and nonsense mutation were 82.03%,17.70% and 0.27%,respectively.There was no significant difference between the Progeny₁ and the Progeny₂.2)AN and wild BN strains were analyzed for the distribution of chromosomes and the contribution rate of disease resistanceThe SNP index representing the Bm NPV resistance association corresponding to each polymorphic locus was calculated using the following formula: SNP index =resistance line type SNP detection frequency /?susceptible strain type SNP detection frequency + resistance strain type SNP The frequency of each chromosome fold line and abscissa represents the contribution of each chromosome to Bm NPV resistance,and the chromaticity SNP index of the chromosomes is accounted for.The line area of? ? the chromosome SNP index is calculated by dividing the chromosome SNP index by 50 Kb.Genome SNP index The percentage of the total area of ? ? the polyline as a contribution to the resistance of each chromosome.The Bm NPV resistance-related markers of the whole genome Bm NPV resistance were analyzed,and the Bm NPV resistance-related markers of AN and wild BN varieties were located on multiple chromosomes.The first five of the AN strains were the Chromosomes5,10,27,23 and 4.The top 5 of the BN varieties were the chromosomes 4,27,15,18 and 6.Multiple SNP index high-value loci have a high correspondence with the position of the Bm NPV resistance-related genes.Suggesting that AN,wild BN strain Bm NPV high resistance and the resistance of the main gene and other resistancegenes associated with the work.3.Preliminary Analysis on the Difference of Bm NPV Resistance between Silkworm AN Series and Wild BN Line1?Based on the difference in the contribution rate of each chromosomeThe Bm NPV resistance of silkworm AN and wild BN lines was associated with chromosome 27,and the contribution rates of Bm NPV in the two lines were respectively located in the third and second positions.There are large differences between the contribution rate of the two strains in Chromosome 3,chromosome 7 and chromosome 8.In silkworm AN and wild BN lines,the contribution rates of Chr3 were 4.84% and 2.29%,ranked ninth and twenty-second in each lines;the contribution rates of Chr7 were 4.16% and 1.44%,ranked eleventh and twenty-eights in each lines;the contribution rates of Chr8were1.06% and 4.53%,ranked twenty-sixth and ninth in each lines.Indicating that silkworm AN and wild BN may have a greater genetic blood and resistance source mechanism.2)Based on the difference value of the high number of SNP-index MarkersIn the resistance analysis of the two cultivars,SNP markers with SNP-index of0.5 ± 0.2 were defined as high correlation markers of NPV resistance,and The difference of the high correlation markers of NPV resistance among whole genome NPV The results showed that Chr 27,Chr 12,Chr 16,Chr 23,and Chr 25 were located in the first six positions,and the NPV resistance of the six chromosomes was higher than that of the six chromosomes.The total proportion reached 54.82%,indicating that the six chromosomes?Chr27,Chr 3,Chr 12,Chr 16,Chr 23,Chr 25?play an important role in the Bm NPV resistance of AN and BN and there were different resistance genes or processes among these chromosomes Resistance of the two varieties.In the scaffold level of these chromosomes,it was found that the number of NPV resistance markers of Bm_scaf48,Bm_scaf112,Bm_scaf55,Bm_scaf6 and Bm_scaf50 was the highest in the top 5,and the study of differences genes or differences mechanisms of anti-Bm NPV can focuse on these scaffold.