| Apple(Malus pumila Mill.)is a rosaceae Juss apple genus(Malus Mill.)plant,is China’s largest deciduous fruit tree,planting area and yield are more than 50% of the world’s total,ranking first in the world.Apple varieties in production by grafting on the rootstock to improve resistance,China’s research and cultivation of apple rootstock started late,rootstock can choose less varieties,at present many apple-producing areas in China,especially Xinjiang is subject to serious environmental constraints such as salinity and alkali drought,so the breeding of resistant rootstock is of great significance to the development of the apple industry.In this study,the hybrid population was constructed with ’Qing anvil No.1’ as the mother and Xinjiang wild apple as the father,and the hybrid offspring were identified for their phenotype after salt stress,and then the transcriptome sequencing technology combined with BSR technology was used to screen the genes related to the salt tolerance of apple rootstock,which provided a basis and reference for molecular means to assist the breeding of new rootstocks.The findings are as follows:(1)In this experiment,’Xinjiang Wild Apple’(♂)was used as the male parent,and ’Qingzhen No.1’(♀)was used as the female parent for cross-pollination,and 150 seeds were obtained after the fruit matured.Because of the strong apomictic phenomenon of ’Qingzhen No.1’,there may be apomictic progeny and’Xinjiang Wild Apple’(♂)×’ in the 150 seeds that are consistent with the genotype of the female parent Qingzhen No.1 The hybrid progeny produced by Qingzhen No.1’(♀).After 150 seeds were sown,the hybrid progeny among them were screened using CAPS marker technology.A total of 30 pairs of CAPS primers were designed.Through the marker detection of the parental DNA,6 pairs of CAPS markers that could screen the hybrid progeny in the progeny were screened.Using the 6 pairs of CAPS markers screened out,a total of 150 progeny were screened out of ’Qing anvil No.1’(♀)× Xinjiang wild apple(♂)75 hybrid offspring.(2)The 75 hybrid offspring of ’Qing anvil No.1’(♀)× Xinjiang wild apple(♂)were subjected to salt stress with 0.4% concentration of Na Cl monosalline,and after 40 days of stress,the hybrid offspring group had significant phenotypic separation of salt stress,and according to the different salt damage phenotypes produced by hybrid offspring,the degree of salinity damage suffered by each single plant in the hybrid group was evaluated and graded,and a total of 6 grades were divided,from grade 0(extremely salt tolerance)to grade 5(extremely weak salt tolerance)The number of single plants accounted for 11%,43%,8%,and 15% of the total,respectively.、11%、13%。According to the degree of salt damage of individuals in the hybrid group,the salt damage index of the hybrid group can be calculated,and the calculation results show that the group can be divided into moderate salt tolerant groups.(3)18 salt-tolerant individuals and 18 salt-intolerant individuals were selected from the population of hybrid progeny after stress to construct a mixed pool,and 778 significantly differentially expressed genes were obtained by transcriptome sequencing,and the differential genes were found by GO functional annotation.Most of them are enriched in biological processes and molecular functions,and the remaining few are enriched in cellular components,among which the main enrichment function is stress response,which is of great significance in salt stress.The differential genes were annotated with KEGG,and the main enriched pathways were metabolic pathways and secondary metabolite generation pathways,which were significantly enriched under abiotic stress.Through further analysis of differential gene expression and annotation function,some genes that may be related to salt tolerance were screened out.Among them,MD07G1173900,MD15G1283200,MD15G1303500,MD13G1237300,MD16G1267200 and other genes have matching homologous genes in Arabidopsis thaliana.Various studies have shown that they may be related to salt tolerance,and the remaining genes need further experiments to verify.(4)By using BSR technology,the data obtained from transcriptome sequencing was compared with the reference genome to compare the distribution of SNPs in the genome,and 8 QTL intervals were located on the six chromosomes of Chr05,Chr07,Chr10,Chr11,Chr13 and Chr16,and there were 58979 SNPs and42 significantly differently expressed genes.28 of the 42 differential genes can be matched to homologous genes in the Arabidopsis database and functionally annotated.The remaining 14 failed to match homologous genes in the Arabidopsis database and could not be functionally annotated.Further analysis of28 genes with annotated functions found that the homologous genes of MD10G1338600,MD11G1303100,MD11G1287900,MD16G1010900 in Arabidopsis thaliana have been reported to be salt tolerance related genes,and the remaining genes need to be further tested. |
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