Epigenetic Map And Genetic Map Basis Of Complex Important Traits In Cassava (Manihot Esculenta Crantz) Population

The majority of studies on many species are based on the complex traits.Even though an increasing number of studies are investigating the heritable variation in the model plants,such as Arabidopsis thaliana and soybean,the heritable variations in cytosine methylation in non-model crops have not been investigated.In this study,we established in a cassava population with a highly heterozygous and large genome,and measured eight complex traits.Cassava（Manihot esculenta Crantz）,a starchy root crop,is a staple food and animal feed and serves as an important source of bioethanol.As a tropical root crop,cassava is sensitive to cold.Thus,chilling injury is the most important factor limiting cassava′s geographic distribution and productivity.The cold tolerance of cassava is very important for protection of the storage roots and propagation stems.Cold tolerance,high quality and a robust supply of cassava are urgently in demand.SC124exhibits a high yield and cold tolerance but lower proportion of starch.In contrast,KU50has many elite economic traits,such as a high proportion of starch and the ability to produce high yields,but is intolerant of cold temperatures.In this study,we established a cold-tolerance cassava mapping population that descended from a cross between the two non-inbred lines,with KU50 as the female parent and SC124 as the male parent.We then analyzed the methylation and genome structure in population,identified the QTL and QTL^epi and detected excellent candidate genes to provide theoretical basis for cassava molecular breeding with cold resistance and high yield and quality.1.The construction of high-density genetic map and comparative genomic map in cassava.Using a simple and rapid method for genome-wide SNP and methylation site discovery sequencing approach to construct the sequencing libraries.After silico enzyme digestion,lab enzyme digestion,AFSM-Sanger sequencing,AFSM-Ion Torrent sequencing and AFSM-Illumina Hiseq 2500 sequencing preliminary experiments,then AFSM-Illumina Hiseq 2500 sequencing was performed in 186 Cassava KS genetic population.In this study,573,557 SNP and indel markers were identified using this approach.Among the 573,557 SNPs and indels,10,627 were distributed in the CDS region,22,439 in the gene region and 8,709 in the promoter region.A high density KS genetic linkage map was produced for the KS cassava mapping population and 4648 out of 69,141 filtered AFSM markers were assigned to 18 linkage groups.The KS genetic linkage map spanned a total of 2190.34 cM,with the density 0.47 cM/Marker.Then38,380 AFSM markers were located on cassava genomic map.We also constructed a genomic map and a comparative genomic map across AM560,KU50 and W14 genome in cassava using only AFSM markers.2.The analysis of genome-wide DNA methylation,DNA methylation genetic patterns and DMRs with significantly differentially expressed genes,and the construction of epigenetic map in cassava.DNA methylation plays an important role in regulating gene expression and silencing transposons.In this study,55,657 fully methylation and 17,555 hemi-methylation sites were detected and the epigenetic map was constructed in cassava.Besides,genome-wide distribution and classification of CCGG hemi-methylation and full-methylation were clarified.The total full-methylation density was higher than the hemi-methylation,especial enriched in gene-body,which is consistented with the previous studies.Methylation sites were grouped in cassava population,according to the methylation genetic patterns among the parents and their offspring.Classes D2-4 contained the most methylated sites,methylated sites exhibiting differing methylation patterns in the F1 progeny and methylated sites exhibiting disparate methylation patterns between the parents.To assess the function of the differentially methylated regions（DMRs）in cassava,and to clarify the relationship between them and differentially expressed genes,we also constructed RNA-seq libraries and performed deep sequencing on the transcriptome of the parents′roots and leaves belonging to the KS population.DMRs with significantly differentially expressed genes（DMR-DEGs）tended to exhibit enrichment of cellular components,including small nucleolar ribonucleoprotein complex and intracellular membrane-bounded organelle.Enriched molecular function for DMR-DEGs included translation factor activity,nucleic acid binding,ATPase activity.For biological processes,organ morphogenesis,cell differentiation,response to light stimulus and cellular response to extracellular stimulus.3.Identification of QTLs^epi,QTLs and candidate genes that were significantly associated with cold tolerance or storage root yield and quality,based on the complex important traits from cassava KS population,including eight cold resistance,yield and quality traits.We detected 77 much candidate QTLs^epi for cold stress in 186 cassava KS population and identified CT-QTL^epi candidate genes such as GH family 2β-mannosidase,MAPKK4,Photosystem II protein D1 and POD.We also constructed AFSM QTL map and identified 260 much candidate QTL genes for cold stress,especially including CDPK,CBL4,CaLB and IQD10 in Ca²⁺dependent CDPK transduction pathways,and other cold stress signal response CT-QTLs genes,such as NCED3,ARF 2,OPR3,WRKY,MYB55,MYB70,MYB40,bHLH and ZFP6.The measurements of storage root yield,number of storage root,storage root dry weigh rate and fresh root starch content rate were also performed and calculated.Besides,we detected 103 much candidate QTLs^epi for storage root quality and yield in population and identified QY-QTL^epi candidate genes such as in photorespiration and photosynthesis（PSAT,TOC33 and Rubisco large subunit N-methyltransferase）and the storage of photosynthetic product related（NADH-GLT1,RNA methyltransferase,Snrnp auxiliary factor,small subunit,RING-H2-type finger and CPN20）.Further more,301 much candidate QTL genes for storage root quality and yield were detected in this study,especially including sucrose synthesis（SPS and Susy）,starch synthesis（Ructokinase and NINV）and starch degradation（β-AMY）.