Genomic Analyses Provide New Insights Into Apple Evolution,Domestication And Genetic Diversity

Human selection has reshaped crop genomes.Here we report a comprehensive apple genome variation map generated through genome sequencing of 117 diverse accessions.A total of 117 Malus accessions from 24 species were selected for genome sequencing.We generated a total of 1,060 Gb high-quality cleaned sequences,identifing a final set of 7,218,060 single nucleotide polymorphisms(SNPs).Furthermore,we identified 431,597 small insertions and deletions(indels).We first examined the phylogeny among wild and cultivated apples using SNPs at fourfold degenerate sites(4D SNPs).The tree showed that accessions of M.domestica and M.sylvestris formed a subclade within a large mixed clade comprising M.sieversii as progenitors of cultivated apples.A principal component analysis(PCA)illustrated a similar pattern.We use a Bayesian clustering algorithm to estimate ancestry proportions for each accession.ΔK analysis revealed that five populations(K=5)represent the best model.M.sieversii accessions segregated into two different subpopulations reflecting their geographical distributions.We propose a comprehensive apple evolutionary map illustrating the initial domestication from M.sieversii in Kazakhstan,the hybridization between M.sylvestris and the ancient domesticated apples via the Silk Road westward,and the rise of orient hybrid species from crosses between M.baccata and M.sieversii in Kazakhstan along the Silk Road eastwardWe then evaluated the genetic diversity of different apple subpopulations.Linkage disequilibrium(LD)analyses for each group further supported a very weak bottleneck,as well as in nucleotide diversity study(Pi value).The rapid LD decay of domesticated apple suggested that a large set of markers densely is preferred for apple breeding.The identified selective sweeps are enriched with genes associated with fruit sugar content,firmness,color,hormone and secondary metabolism in both Dom_SieK and Dom_Syl contrasts,while genes related to fruit acidity were only enriched in Dom_Syl,highlighting the constant selection of sweet and firm fruits in the history of apple domestication.These candidate genes are indicative of different selective forces for improving different agronomic traits from the two different contributors during domestication.As fruit size is one of the most desirable traits for crop domestication and improvement,apple domestication started with a great advantage and much lower evolutionary pressure than other crops.We performed a GWAS in 66 domesticated and wild accessions and identified two loci associated with fruit weight,which we designated as gwa_w1 and gwa_w2,respectively.The QTL gwa_w1 co-localized with four selective regions in the Dom_Syl contrast harboring 42 genes and a small sweep in the Dom_SieK contrast harboring five genes.we identified two additional miR172 genes(miR172g and miR172h)that might have contributed to the increase of fruit size during Malus speciation prior to domestication.Taken together,we propose a two-step evolution model for fruit size enlargement in apple to characterize its unique evolution process.The finding helped explain why a weak selection in a highly heterozygous Apple can still yield favorable large fruits suggesting that apple fruit size has great potential in future breeding practices.Enhancement of fruit firmness during apple domestication.We first performed an association study for flesh firmness and detected a major QTL(named gwa_f1)on chromosome 6.The gwa_f1 region was under intensive human selection in both Dom_SieK and Dom_Syl contrasts,harboring genes encoding key cell wall modifying enzymes including three polygalacturonases,one pectinesterase,and one glucan endo-1,3-beta-glucosidase.Furthermore,several other selective regions were also found to contain genes closely related to fruit firmness.Enrichment of fruit flavor during apple domestication.During the domestication from M.sieversii in Kazakhstan,a region on chromosome 12 has undergone intensive selection and co-localizes with a sorbitol QTL.Interestingly,genes encoding four sorbitol transporters and two sugar transporters cluster within this sorbitol QTL under selection,and all four sorbitol transporter genes were differentially expressed during apple fruit development.We identified a region around 1.5 kb upstream of Ma1 coding sequence(CDS)with substantially reduced nucleotide diversity in M.domestica compared to M.sylvestris,and another region about 750 bp downstream of Ma1 CDS when compared to M.sieversii in Kazakhstan,indicating that the Ma1 might be under strong selection during apple domestication.This study outlines genetic bases of apple domestication and evolution,and provides valuable information for facilitating marker-assisted apple breeding and improvement.