| Pea(Pisum sativum L.),which is an important legume crop for both food and feed,plays an important role in agricultural production and national economic development in China.However,bruchids(Bruchidae)are the most destructive insect pests of various legumes and can devastate pea in the field and during the storage,resulting in a yield loss of about 50%,which seriously limits the development of the pea production.Chemical pesticides are effectively used against bruchids for a long time,but are simultaneously associated with a number of drawbacks including high costs and concerns about environmental pollution and food safety.The use of bruchid-resistant cultivars is an effective and environment-friendly management option against storage pest.Therefore,the studies of screening bruchid resistant germplasm,identification of the resistance gene(s)and analysis of the resistance mechanism are of great significance for understanding the interaction between pea and bruchids and the breeding of bruchid resistant pea.So far,there are few reports on the resistance of peas to Callosobruchus chinensis and Callosobruchus maculatus.Therefore,the pea accessions resistance to C.chinensis and C.maculatus were screened in this study,and combined with the agronomic analysis of these accessions,the bruchid resistant accession PWY19 and bruchid susceptible accession PHM22 were screened out.Using the segregated F2 population constructed by crossing between PWY19 and PHM22,the QTL loci related to the resistance of pea to C.chinensis and C.maculatus were identified by molecular marker technology.In order to perform finemapping of the major QTL for the bruchid resistance,a larger F2 population of the cross between PWY19× PHM22 and newly developed InDel and SSR markers was used.The molecular mechanism of bruchid resistance was studied by sequence variation analysis,gene expression pattern analysis and subcellular localization analysis of the candidate gene.The results obtained were as follows:1.The method of artificial inoculation(‘Free-Choice’and ‘No Free-Choice’)was used for identifying the resistance of 70 pea germplasm resources to Callosobruchus chinensis and Callosobruchus maculatus.The results showed that the seed damage rate caused by both of C.chinensis and C.maculatus were different among the pea germplasm resources.The ‘Free-Choice’ method identified 28 and 46 materials with high resistance to C.chinensis and C.maculatus,accounting for 40.0% and 65.7% of the total identified materials,respectively.Based on the ‘No Free-Choice’,there were 6 and 7 pea accessions showed high resistance against C.chinensis and C.maculatus,accounting for 10.0% and 8.6% of the total identification materials,respectively.Through the analysis of the frequency distribution of the damage rate of seeds based on ‘Free-Choice’ and ‘No Free-Choice’,it was found that the damage rate of ‘Free-Choice’ was biased towards 0%-10%,suggesting the choice of infestation of C.chinensis and C.maculatus resulting in less damage to some resources.2.Through Identifying the resistance of pea germplasm resources to C.chinensis and C.maculatus based on ‘No Free-Choice’,11 resistant pea resources with the seed damage rate of less than 30% caused by the damage of the both of the two bruchid were screened out.Besides,there were 10 high-sensitivity resources with a seed damage rate of over 90%.The correlation analysis between the seed damage rate caused by C.chinensis and C.maculatus based on ‘No Free-Choice’ and the agronomic traits of peas showed that the resistance against C.chinensis and C.maculatus was related to pea growth habit,leaf color,flower color,pod type,pod width,plant height and number of main stem nodes.However,the resistance of bruchids was not related to seed shape and seed color,indicating that the resistance of pea seeds to bruchids might be related to the insect-resistant substances inside the seeds.3.Transcriptome sequencing and analysis of the developing seeds of PWY19 and PHM22 at different stages(10 d,20 d,and 30 d after flowering)showed that a total of 1035 differentially expressed genes(DEGs)were identified.GO annotation of DEGs showed that functional areas related to “defense response”,“pathogenesis” and “stress response” were enriched.KEGG enrichment analysis showed that “phytohormone Signal Transduction” and “MAPK Signaling Pathway” were enriched in the seeds at the stage of 20 d after flowering.Based on the transcriptome data,173 candidate InDel markers distributed throughout the chromosome were developed of which 97 InDel markers were polymorphic between PWY19 and PHM22,accounting for 56.07% of the total detected markers.The developed InDel markers were used for the gene mapping of bruchids resistance in this study.4.Seed damage rate caused by C.chinensis and C.maculatus was investigated in the F2 populations(F2Y and F2N),which derived from PWY19(resistant)and PHM22(susceptible).The genotypes of the F2 Y and F2 N populations were analyzed using SSR molecular markers.The genetic map of F2 Y and F2 N population were constructed by using 126 molecular markers with polymorphism between two parents.Genetic map constructed for the population F2 Y and F2 N consisted of 8 linkage groups with a total length of 1307.2 c M and 1145.4 c M,respectively.QTL analysis for the resistance to C.chinensis and C.maculatus in the F2 Y and F2 N was run using QTL Ici Mapping 4.2 software.In all cases,a major QTL was identified located on LG2 between markers 18339 and PSSR202109.In the F2 Y,the LOD values of the detected QTL against C.chinensis and C.maculatus were 66.76 and 46.32,respectively,and phenotypic variance explained(PVE)value of the QTL were 70.94% and 61.35%,respectively.In the F2 N,the LOD values of the detected QTL against C.chinensis and C.maculatus were 32.84 and 25.38,respectively,and PVE value of the QTL were 52.66% and 50.91%,respectively.Since the QTLs were consistently identified at the same interval with similar genetic effect for different bruchid species across different populations that grown in different environments,we considered these QTLs the same locus and designated this locus as qPsBr2.1.5.In order to perform fine-mapping of the major QTL qPsBr2.1,a larger F2 population(F2F)from the cross PWY19×PHM22 was used and recombinant plants were screened for the test of bruchid reisistance.With the use of newly developed markers,the qPsBr2.1 locus was narrowed down to the genomic region between markers PSSR2021082 and PSSR2021017,which were 1.07 Mb apart on the chromosome 2.PsXI(Psat2g026280)encodes a xylanase inhibitor was considered as candidate gene for the bruchid resistance.For analyzing sequence variation of PsXI between PWY19 and PHM22,de novo assembly of the genomic sequences of PWY19 and PHM22 was performed,Together with sanger sequencing,the results indicated that there is an unknown length insertion in the intronic region of PWY19 that results in sequence difference between PWY19 and PHM22 and a change in transcription sequence of PWY19.Full-length c DNA sequences of PsXI in PWY19 and PHM22 were obtained using RACE-PCR,and the results showed that,compared with PHM22,the open reading frame(ORF)of PsXI in PWY19 lacks the signal peptide and N-terminal structure.Relative expression levels analysis of PsXI in different tissues in PWY19 and PHM22 showed that the gene exhibited low level expression in the root,stem,leaf,and seed.In different tissues of seed,the expression level of PsXI in embryo of PWY19 was significantly higher than that of PHM22.The evaluation of the subcellular localization showed that the PsXI of PWY19 and PHM22 was targeted to different localizations.The PsXI-PWY19-GFP fusion was co-localized with plasma membrane marker and nuclear marker,whereas the PsXI-PHM22-GFP fusions were mainly localized to the plasma membrane in Nicotiana benthamiana foliar cells.Based on the changes of protein coding in the PsXI of PWY19,the difference of expression in the embryos of the PWY19 and the difference styles of sub-cellular location between PsXI-PWY19 and PsXI-PHM22,PsXI was expected to be the candidate gene that triggered the resistance of PWY19 to bruchids.Our results of bruchid resistant accessions screening and the mapping of QTLs for bruchids provided useful genetic resources for improving the bruchid resistance of pea.The results of fine mapping of the QTL qPsBr2.1 and the preliminary analysis of the resistance gene PsXI laid the foundation for elucidating resistance mechanism of resistance against C.chinensis and C.maculatus in pea. |
