The Draft Genome Of Chinese Bayberry(M.rubra)and Genetic Analysis Of Sex Determination Between Male And Female Plants

Chinese bayberry（Morella rubra Sieb.and Zucc.）（syn.Myrica rubra）is one of theimportant subtropical fruit crops native to the South of China and Asian countries.It is used to be a neglected fruit,now large scale commercial production and distribution in China promoted researches in recent years.In genetics and genomics,previous researches were mainly in the development of molecular makers from genome survey and ESTs and applied in cultivar（female）diversity.Here we reported the new SSR makers from genome,whole genome sequencing project,and genetic and expression studies on the male and female diversification.The main results are as follows:1)In this study,107 novel simple sequence repeat（SSR）molecular markers,a powerful tool for genetic diversity studies,cultivar identification and linkage map construction,were developed and characterized from whole genome shotgun sequences.In total,828 alleles across 45 accessions were detected,with an average of eight alleles per locus.The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08.The polymorphic information content（PIC）varied from 0.13 to 0.89,with an average of 0.63.Moreover,these markers could also be amplified in their related species M.cerifera and M.adenophora.Seventy-eight SSR markers can be used to produce a genetic map of a cross between ’Biqi’ and ’Dongkui’.A neighbor-joining（NJ）tree was constructed to assess the genetic relationships among accessions,and the elite accessions ’Y2010-70’,’Y2012-140’（new variety ’Xia Zhi Hong’）and’Y2012-145’ were characterized as potential new genotypes for cultivation.We also identified the accession ’Y2012-145’ was the most homozygous accession among 43 bayberry accessions,which is suitable for whole genome sequencing.2)In this study,we sequenced M.rubra accession ’Y2012-145’ using a whole genome strategy on the Illumina HiSeq2000 and PacBio system,and assembled the genome with SOAP denovo and HABOT softwares.The genome size was estimated as 322.7 Mb.The genome assembly consists of 962 scaffolds and 2,939 contigs（>2 kb）,with the N50 sizes of 635.18 kb and 192.56 kb for scaffolds and contigs,respectively.The GC content of the assembled genome was 36.9%.The genome contains 110.6 Mb of repetitive sequence（35.4%of the assembled genome）,among these repetitive sequences,95.6%of assembled genome are transposable elements（TEs）.Using RNA-seq data we have generated from root,stem,leaf,bud and fruits,integrated with ab initio gene predictions and homologous sequence searching,a total of 29414 protein-encoding genes were predicted,the function of 26316 genes were noted.There are 13432 gene families in Chinese bayberry,among them 962 are unique.A total of 50 MADS-box gene families were identified in bayberry genome,14 of them are highly expression in flower.3)We constructed a RAD library using genomic DNA from ’Biqi’ x ’Dongkui’ F1 population.Sequencing of 101 F1 accessions and two parents were performed on Illumina HiSeq 4000,and 8461 polymorphic SNP loci were produced.After filtering the distorted segregation data,a total of 4441 SNPs and 63 SSRs were used for constructing linkage map.We firstly constructed a high-density linkage map of Chinese bayberry,overall,1132 SNP and 38 SSRs markers were mapped to 8 linkage groups corresponding to 8 chromosomes.The map spans 563.01 cM with an average marker distance of 0.48 cM.4)Using 84 polymorphic SSRs,we genotyped 213 M.rubra individuals（99 male individuals,113 female varieties and 1 monoecious）and compared the difference in genetic diversity between the female and the male populations.Neighbour-joining cluster analysis separated M.rubra from three related species,and the male from female populations within M.rubra.By structure analysis,178 M.rubra accessions were assigned to two subpopulations:Male dominated（98）and Female dominated（80）.Nest structure analysis show that Male dominated can subdivided into 4 subpop,and Female dominated can divided into 2 subpop:’Biqi’ female series and ’Fenhong’ female series.The well-known cultivars ’Biqi’and’Dongkui’,and the landraces ’Fenhong’ are derived from three different gene pools.Female population had a slightly higher values of genetic diversity parameters（such as number of alleles and heterozygosity）than the male population,but not significantly different.The SSR loci ZJU062 and ZJU130 showed an empirical Fst value of 0.455 and 0.333,respectively,which are significantly above the 95%confidence level,indicating that they are outlier loci related to sex separation.Those two marker were mapped on scaffold₁004 and scaffold₉1 which located on chromosome LG5 and LG2,respectively.This research provides genetic information on male and female Chinese bayberry and will be a good reference for genome localization of sex determining regions and breeding programs.5)’BiQi’ male and female flower,and monoecious flower RNA libraries were used for high-throughput sequencing,and produced 12104480,12242575 and 11911476 clean reads,respectively.We identified 194 known miRNAs belongs to 32 miRNA families and 33 novel miRNAs.Five known miRNA（miR156,miR157,miR166,miR167 and miR168）families were highly expressed in three libraries.And a total of 46 unique miRNAs were differently expressed in three libraries.We also identified 58 targets for 14 known miRNA families and 7 for 4 unique novel miRNAs by using degradome sequencing.Among the 65 identified miRNA targets,45 targets were annotated by GO,and 11 miRNA targets were categorized into 7 biological process based KEGG analysis.