The Screening And Mapping Of BmNPV Resistant Genes In The Silkworm,Bombyx Mori

The silkworm is the model insect of Lepidoptera, is also an important economic insect. The silk with very high economic value is the main income for the farmers in many areas of the developing countries such as China and India. However, the development of sericulture industry is being hampered by the silkworm diseases. According to incomplete statistics, every year about three percent loss of the total output is caused by silkworm diseases which seriously restricted the stable development of sericulture industry. Virus disease is the most harmful one of all diseases, in which the harm of blood type pus disease caused by Bm NPV(Bombyx mori nuclear polyhedrosis virus) is the greatest accounted for about 70% of silkworm disease and loss. The measure for this disease is still in the stage to prevent. Therefore, it is an important task to search for the resistance genes of Bombyx mori nuclear and group match of resistant varieties in terms of production and safety.This aticle selects inbred strain resistant strain AN that mating formation of silkworm varieties resistanting to Bm NPV Hua-kang No.2 F1 hybrids mated with moth batch, resistant parent field BN beyond silkworm varieties resistanting to Bm NPV Yesanyuan N, medium resistant silkworm varieties breed(P50) and usceptible strain C108 as research material. The resistance of different silkworm varieties to Bm NPV is studied firstly. Then, major resistance gene linkage analysis populations and the mapping populations are constructed and finely mapped by genetic breeding technology, SSR molecular marker technology and HRM analysis technology; minor resistance gene population are respectively constructed by low resistance strains AN, P50 and C108 and preliminary linkage analysis is made by SSR molecular marker technology and HRM analysis technology. The main experimental contents and results are as follows:1. Finely mapping the major resistance gene of the Bombyx mori strain AN to Bm NPV.According to the enetic law of resistance major gene and the tolerance of the silkworm to Bm NPV, resistant strain AN is selected as the female parent(P1), susceptible strain C108 is selected as male parent(P2) and backcross, then group the two to F1 population. Female individual of F1 backcross male individual of C108 and group to BC1 F. Feed these groups to molted second-instar and feed mulberry leaves with 1*108 /m L Bm NPV. Collect the survival individuals as BC1 F linkage analysis population. HRM analysis technology is used to genotype analyze for the mapped gene population. Male individual of F1 backcross male individual of C108 and group to BC1 M. Feed these groups to molted second-instar and feed mulberry leaves with 1*108 /m L Bm NPV. Collect the survival individuals as BC1 F mapping analysis population. And so on, group BC2 M and BC2 M population. Screening resistance gene linked polymorphic markers with P1, P2 and F1 as a template; BC1 F and BC2 F are used to linkage analysis for resistance major gene; BC1 M and BC2 M are used to mapping analysis.The identification results AN strain resistance to Bm NPV show when feed molted second-instar with Bm NPV virus, half lethal concentration(LC50) is 2.154*109 /m L, half lethal dose(LD50) is 1.436 *107 / head silkworm.Comprare half lethal concentration(LC50) of F1, BC1, BC2 feed with Bm NPV, the results show that resistant strain AN has high resistance to Bm NPV which controlled by a Chang autosomal located in dominant major gene and uninfluence by sex chromosomes.Linkage analysis showed that the resistance major gene of the strain AN resistant to Bm NPV was in chromosome 27. Through localization analysis for more than 600 individual genomic in the BC2 M mapping population, draw a total genetic distance 29.1c M ans lines Bm NPV resistance gene genetic linkage map. The SSR markers s2800-9(nscaf2800: 1311765~1311847) and s2801-35(nscaf2800: 117840~118038) were located on both sides of the gene and and the genetic distances between the main genes were 5.2c M and 2.9c M respectively.Because of a gap in the silkworm genome sequence between the two markers, it is impossible to realize the further fine mapping.2. Mapping analysis for the resistant major gene of Bombyx mori strain Ye BN to Bm NPV.Resistant strain Ye BN is selected as the female parent(P1), susceptible strain C108 is selected as male parent(P2) and backcross, then group the two to F1 population. Female individual of F1 backcross male individual of C108 and group to BC1 F. Feed these groups to molted second-instar and feed mulberry leaves with 1*108 /m L Bm NPV. Collect the survival individuals as BC1 F linkage analysis population. HRM analysis technology is used to genotype analyze for the mapped gene population. Male individual of F1 backcross male individual of C108 and group to BC1 M. Feed these groups to molted second-instar and feed mulberry leaves with 1*108 /m L Bm NPV. Collect the survival individuals as BC1 F mapping analysis population. And so on, group BC2 M and BC2 M population. Screening resistance gene linked polymorphic markers with P1, P2 and F1 as a template; BC1 F and BC2 F are used to linkage analysis for resistance major gene; BC1 M and BC2 M are used to mapping analysis.The identification results Ye BN resistance to Bm NPV show when feed molted second-instar with Bm NPV virus, half lethal concentration(LC50) is 2.083*109 /m L, half lethal dose(LD50) is 2.5*107 / head silkworm.Linkage analysis showed that a resistance major gene of the strain Ye BN resistant to Bm NPV existed in chromosome 27 and also maybe in other chromosome.The result based on HRM analysis to BC2 M mapping analysis population in 4 moth batch and 6 polymorphic markers in chromosome 27 show that the strain Ye BN has a resistance major gene tolerancing mulberry leaves with 1*108 /m L Bm NPV in addition to that in the linkage group 27.3. Linkage analysis of minor resistance gene.(1) Resistance strain of ANThe resistance genes of silkworm resistant strain AN to Bm NPV contain many minor genes in addition to the major resistance genes. Resistant strain AN is selected as the female parent(P1), susceptible strain C108 is selected as male parent(P2) and backcross parent, then we can get the BC2 F group. Mulberry leaves soaked in a concentration of 1*107/ml Bm NPV polyhedrin suspension liquid, fed mulberry leaves to the newly molted second-instar larve to identify the resistance of surviving individuals in chromosome 27.Choose the moth batch of no resistance genes in chromosome 27. Construct BC3 F and BC4 F groups for minor resistance genes linkage analysis. Linkage analysis shows that the minor resistance gene is in chromosome 6.(2) Resistance strain of p50Resistant strain p50 is selected as the female parent(P1), susceptible strain C108 is selected as male parent(P2) and backcross parent, hybrid for F1 group. Mulberry leaves soaked in a concentration of 1*107/ml Bm NPV polyhedrin suspension liquid, fed mulberry leaves to the newly molted second-instar larve, the individual collection and survival as the BC1 F group.Linkage analysis results that resistance genes exist widely in the silkworm genome, there may be multiple micro effect genes, resulting in variety p50 to Bm NPV has higher resistance.