The Study On Genomics Of Genetic Origination And Stress Tolerance In Malus Prunifolia

Apple(Malus × domestica Borkh.),as one of the most representative temperate fruits,not only has a high nutritional value,but also has important cultural attributes and economic value.China ranks first in the world in terms of apple planting area and yield,but the country still faces problems such as a single main planting variety and a lack of good dwarf rootstocks.The high-quality development of China’s apple industry requires excellent dwarf rootstocks,and the current apple dwarf rootstocks generally exhibit weak resistance.It is important to accelerate the genetic improvement and breeding of highly resistant dwarf rootstocks for the long-term development of the apple industry.In fact,wild apple rootstocks are abundant in China,especially Malus prunifolia ‘Fupingqiuzi’,which has excellent characteristics such as vigorous growth,great grafting affinity and multiple stress resistance.This study aims to analyses the genome of ‘Fupingqiuzi’,understand its genetic origin,explore the source of its resistance genes,to seek its molecular mechanism of resistance as a rootstock,and provide genetic resources and theoretical basis for the selection and breeding of resistant dwarf rootstocks.For this purpose,we used multiple sequencing technologies to sequence and assemble the genome sequence of M.prunifolia ‘Fupingqiuzi’,then analyzed the origin of resistance genes in ‘Fupingqiuzi’ by comparative genomics.Next,we demonstrate the evolutionary history of the hybrid origin of M.prunifolia using data from population resequencing.Finally,we revealed the mechanism of stress resistance that M.prunifolia ‘Fupingqiuzi’(rootstock)enhanced scion(M.× domestica ‘Golden Delicious’)through a multi-omics analysis of homo-and hetero-grafting.The main findings of this study are as follows:a)High-quality genome assembly and annotation of Malus prunifolia ‘Fupingqiuzi’genome.We used a hybrid assembly strategy combining Illumina sequencing,Pac Bio sequencing and Hi-C sequencing to assemble the ‘Fupingqiuzi’ genome.The size of its assembly was approximately 712 Mb,containing 44,997 protein-coding genes with 95.1%BUSCO completeness.b)The origin of resistance genes in Liriodendron fupingii is related to gene family expansion and species-specific gene formation.Based on comparative genomics and functional enrichment analyses,we found that genes from expanded gene families and species-specific genes are closely related to resistance to biotic and abiotic stresses in Malus prunifolia‘Fupingqiuzi’,which provides important evidence for the formation of resistance genes in‘Fupingqiuzi’.c)The genetic origin of M.prunifolia accessions is a genetic mixture from M.sieversii and Malus Section Baccatus.The population genomics analysis revealed that the rowan family is closely related to the M.sieversii and M.baccata.Further,there is gene flows in M.prunifolia accessions from both M.sieversii and Malus Section Baccatus.In addition,the demographic history analysis confirmed that M.prunifolia accessions is of hybrid origin with M.sieversii and Malus Section Baccatus,with a speciation time of 30,000-50,000 years ago.d)‘Fupingqiuzi’ rootstock enhanced the resistance of the scion(‘Golden Delicious’)by altering the DNA methylation pattern and transcriptional expression level of the scion(‘Golden Delicious’).We performed abiotic and biotic stresses such as drought,heat,freezing and disease infestation on homo-grafts(‘Golden Delicious’ grafted onto ‘Golden Delicious’ and ‘Fupingqiuzi’ grafted onto ‘Fupingqiuzi’)and hetero-rootstock(‘Golden Delicious’ grafted onto ‘Fupingqiuzi’),then found that hetero-grafted scion(‘Golden Delicious’)was more tolerance than homo-grafted scion(‘Golden Delicious’).By conducting DNA methylation and transcriptome analyses of homo-and hetero-grafts under control and drought conditions,we found that compared to ‘Golden Delicious’ as rootstock,‘Fupingqiuzi’ as rootstock caused changes in the overall DNA methylation pattern of the scion(‘Golden Delicious’)and induced differences in the transcriptional expression of genes related to stress adaptation and flowering.In particular,the DNA methylation level of the Md LECRK3 gene was significantly reduced and its transcript level was significantly increased in hetero-grafted scions ‘Golden Delicious’ under drought conditions.Moreover,the transient expression of Md LECRK3 in tobacco enhanced the drought tolerance of tobacco leaves.In addition,under drought conditions,a total of 3102 drought-responsive differentially expressed genes(1217 from M.sieversii and 1147 from M.baccata)were identified between homo-and hetero-grafted ‘Golden Delicious’(scion),which are consistent with the hybrid origin of M.prunifolia ‘Fupingqiuzi’,suggesting that both M.sieversii and M.baccata contribute to the formation of resistance in ‘Fupingqiuzi’.In this study,we have assembled and annotated a high-quality genome of M.prunifolia‘Fupingqiuzi’,laying the foundation for the functional genomics study of M.prunifolia;The hybrid origin of M.prunifolia provides new insights into the evolution of Chinese apples;The important genes associated with graft-mediated resistance were identified,which offers a theoretical basis and genetic resources for genetic breeding and improvement of dwarf resistant rootstocks.