Fine Mapping Of The Major QTL And Identify Of The Causal Genes Controlling In Vitro Tuberization In Tetraploid Potatoes (Solanum Tuberosum L.)

Potato(Solanum tuberosum.L),with rich nutrition in tubers and a high edible value,is one of the most important food and economy crops in the world together with rice,wheat and corn.In this study,based on the results of QTL and BSA RNA-seq,we tried to develop more SNP markers to fine map the major QTL and identify the causal genes controlling in vitro tuberization.The main results are shown as follows.1.Based on the data of BSA RNA-seq,464 SNP loci were selected for population genotyping and fine mapping.According to test by the SNP dosage information model and the tetraploid mapping software,we successfully constructedthe genetic linkage map of chromosome V of the parents including all eight homologous chromosomes.This integration linkage map covered a total length of 42.07 cM and contained 193 SNP markerswith 0.22 cM of the average distance of the markers.2.Use the 2-year phenotype data of MTI populationfinished previously,we performed QTL mapping on the new integrated map.The results showed that 2-year phenotype data all mapped QTL on this integration linkage map.One QTL was detected by the phenotypic data of 2014 with a peak LOD of 14 cM and one-LOD confidence interval of 13-18 cM.The other one was identified by the phenotypic data of 2016 with a peak LOD located at 32 cM and one-LOD confidence interval of 30.2-35.1 cM.In combination with the corresponding physical positions of the above-mentioned genetic segments on the potato DM reference genome,we mapped the physical region of the segment from 47.3Mb to 47.8 Mb of which contained 56 coding genes.3.In this study,187 SNP markers(single,double,and double single,not including multiple allelic markers)on the integrated map were analyzed for associations between markers and phenotypes.The association analysis showed that a total of 39 markers were significantly associated with the two-year phenotypic data.Of these,19 markers were extreme significantly associated with the phenotype(P< 0.01),and the remaining 20 markers were significantly associated with the phenotype(P< 0.05).Based on the location information of these 19 extremely significant correlation markers on the potato reference genome,we obtained a candidate interval of 47.3 Mb-4.08 Mb,which contains 78 coding genes on the potato reference genome.Among them,the gene PGSC0003DMG400027130 contains five markers that are extreme significantly related to tuber formation.4.In this research,we detected the expression patterns of 11 differentially expressed genes located on chromosome V in the parental pool and the mixed pool,and tested 15 differentially expressed genes in the MT05 segment of the mixed pool in different phenotypic materials.The results indicated that 3 genes(PGSC0003DMT400013500,PGSC0003DMT40006030 and PGSC0003DMT400060472)both expressed at the 5/6 sampling point of the tuber formation materials and expressed at the second sampling point of the non-tuber formation materials.The detection results of the interference strains and control materials indicated that the above three genes may also be located in the PHYF and/or PHYB signal pathways governing photoperiodic tuberization.5.In this study,we also tested the expression levels of 93 genes in the QTL segment and 4 genes in the recombination segment in the parental phenotypic extreme materials.Seven candidate genes PGSC0003DMG400007238,PGSC0003DMG400007237,PGSC0003DMG400046174,PGSC0003DMG402030099,PGSC0003DMG400021827,PGSC0003DMG400011638 and PGSC0003DMG401027172 may related to potato tuber formation.Based on previous studies,this study developed 464 SNP markers for the fine mapping of the main QTL MT05,and successfully added 193 SNP markers to the MT05 segment of chromosome V.Anintegration linkage map ofchromosome V was constructed(a total length of 42.07 cM and mean marker interval of 0.22 cM)and the one-LOD support interval of MT05 was reduced from the original 15 cM to 4 cM.Combining the QTL mapping results with the association analysis of markers and phenotypes,we reduced the physical segment of MT05 on the potato reference genome from the original 2 Mb to 0.7 Mb(47.3Mb-48.0 Mb),in which contain 78 coding genes.On the other hand,combined with previous BSA RNA-seq results and our localization results,we detected 22 differentially expressed genes located on chromosome V in the parent pool and mixed pool,and 97 genes in the QTL segment in different tubers.The amount of expression and expression pattern in the phenotypic material showed that seven candidate genes might related to potato tuber formation.Comprehensive QTL mapping phenotype-associated markers and quantitative gene expression test results of candidate genes,we finally proposed 22 genes as key candidate genes related to potato tuber formation.