Sequencing Of A Wild Apple (Malus Baccata) Genome And RNA-sequencing Transcriptome Profiling Of Apple Floral Induction

Malus baccata is one of four wild apple species that can hybridize with the cultivated apple species(Malus domestica).It is widely used in high-latitude apple-producing areas as a rootstock and breeding resource because of its disease resistance,and cold tolerance.A lack of a reference genome has limited the application of M.baccata for apple breeding.Floral induction is an important stage in the apple tree life cycle.In‘Nagafu No.2',which was derived from a‘Fuji'bud sport,flower bud formation is associated with serious problems.Moreover,the molecular regulatory mechanisms underlying apple floral induction remain unknown.To characterize these mechanisms,we compared the RNA-sequencing-based transcriptome profiles of buds during floral induction in profusely flowering‘Qinguan'and weakly flowering‘Nagafu No.2'apple varieties.1)For our de novo genome assembly,173.61 Gb(248-fold genome sequence coverage)of high-quality sequence was obtained and used for the de novo genome assembly.The contigs(excluding gaps)totaling 665Mb accounted for approximately 85.3%of the estimated M.baccata genome.The result statistics of our final assembly showed that the contig N50 and scaffold N50 values were 44.7 and 452.7 Kb,respectively.We estimated the completeness of the M.baccata genome assembly by attempting to align Malus EST and plant CEGMA(Core Eukaryotic Genes Mapping Approach)gene set.We observed that 84.84%of the EST and 93.20%of the plant BUSCO sequences searched were present in M.baccata scaffolds.2)To assemble pseudomolecules,we implemented the genotyping by restriction site-associated DNA sequencing method to construct a high-density M.baccata genetic map.We established a high-density genetic map with 3,065 bin markers using 390 F₁ progenies from a cross between M.baccata‘Shanjingzi'and M.domestica‘Danxia'.A total of 1,480 scaffolds were anchored to the high-density genetic map by these markers,accounting for 72.57%of the assembly(521.75 of 718.98 Mb).We identified 17 chromosome pseudomolecules and determined the sequence orientation of 53.13%of the anchored scaffolds(382.02 Mb)based on genetic distances.The collinearity relationship between the two genomic chromosomes was constructed by using the genetic linkage map bin-labeled SNP flanking sequence at the location of the cultivated apple genome.It was found that the 17 chromosomes had good collinearity,but the homologous chromosomes of chr05 and chr13 were found.The location has a chromosomal rearrangement.3)Transposable elements(TEs)contributed 58.56%(413.09 Mb)of the M.baccata genome sequence.The long terminal repeat(LTR)retrotransposons were the most abundant transposable elements.Among the LTR retrotransposons,Gypsy and Copia constituted 29.18%and 16.00%of the M.baccata genome sequence,respectively.The abundance of LINE/RTE content(8.03%)in the M.baccata and M.domestica genomes is much greater than that of other Rosaceae plant species suggesting a unique evolutionary event occurred in the Malus genome.We identified 46,114 high-confidence protein-coding gene models.We identified 2,345 TF-encoding genes in 58 families in the M.baccata genome.M.baccata genome was observed to carry more M-type MADS,MYB?related,and C2H2 TFs than the M.domestica genome.We compared the M.baccata gene families with those of four other Rosaceae species and ancestral species.M.baccata and M.domestica diverged from each other approximately 6.9–11.9 million years ago.There are 598 gene families were specific to M.baccata.We determined that 1,049and 1,715 gene families had expanded and contracted,respectively,in the M.baccata genome relative to its most recent common ancestor.The gene families related to the isoflavonoid biosynthesis pathway and tyrosine metabolism pathway expanded in M.baccata,but contracted in M.domestica.4)In M.baccata,119 coiled coil-NBS-LRR(CNL),111 toll/interleukin receptor(TIR)-NBS-LRR(TNL),126 NBS-LRR,54 NBS,23 CC-NBS,and 29 TIR-NBS genes were identified.The M.baccata genome contained more RPK-type R genes and a greater proportion of NBS-type R gene clusters than the M.domestica genome.Furthermore,the proportion of COR genes carrying the DREB motif was greater in M.baccata than in M.domestica.The additional MYC motif in the promoter of the M.baccata CBF gene was due to the insertion of a transposable element.M.baccata and M.domestica CA genes(associated with cold acclimation)were annotated with 44 and 4 unique GO terms,respectively.5)Genes differentially expressed between the buds of the‘Qinguan'and‘Nagafu No.2'apple were mainly related to carbohydrate,fatty acid,and lipid pathways.Additionally,the steady up-regulated expression of genes related to the fatty acid and lipid pathways and the down-regulated expression of starch synthesis-related genes in the carbon metabolic pathway of‘Qinguan'relative to‘Nagafu No.2'were observed to contribute to the higher flowering rate of‘Qinguan'.Additionally,global gene expression profiling revealed that genes related to cytokinin,indole-3-acetic acid,and gibberellin synthesis,signalling,and responses were significantly differentially expressed between the two varieties.The up-regulated expression of genes involved in abscisic acid and salicylic acid biosynthesis via shikimate pathways as well as jasmonic acid production through fatty acid pathways in‘Qinguan'buds were also revealed to contribute to the floral induction and relatively high flowering rate of this variety.