Mapping Of QTLs Controlling Seed Low Temperature Germinability And Fine Mapping And Transcriptome Analysis Of QLTGR3-1 In Rice

The direct seeding of rice is highly popularized in recent years.One of the important factor to ensure the safety of direct seeding is seed low temperature germinability.Therefore,the study of the genetic mechanism of low temperature germination of rice will help to improve seed low temperature germinability and cultivate new varieties suitable for direct sowing.It is of great practical significance for the increase of rice production and the spread of direct seeding.In this study,208 rice chromosome single segment substitution lines（SSSLs）derived from the receptor parent huajingxian 74（HJX74）were selected as materials.Seed germination percentage for six or seven days under 15℃were used as phenotypic index to identify quantitative trait loci（QTLs）controlling low temperature germinability.The stable expression QTL,qLTGR3-1,was fine mapped and analyzed by RNA-Seq.Our work obtained the following results:1.In the early and late season of 2016,the germination rate of 208 SSSLs at 15℃was continuously distributed,the early season germination rate was distributed from 12.7 to93.3%,and the late season germination rate was distributed from 8.7 to 79.3%,which showed typical quantitative characteristics.2.The germination rate at low temperature was phenotype index,and 23 SSSLs exhibited significant difference compared with HJX74 in 2016.Based on substitution mapping,a total of 14 low temperature germinability QTLs were detected on 10chromosomes except the chromosome 10 and 12 with the average length of 15.84 cM.The additive effects of these QTLs changed from-0.21 to 0.22,and the additive effect contributions ranged from-38.4 to 66.8%.Among them,the stable expression QTLs in early and late season were qLTGR1,qLTGR3-1,qLTGR5,qLTGR8-2,qLTGR11-1 and qLTGR11-2,and the positive QTLs were qLTGR3-1,qLTGR5,qLTGR8-2 and qLTGR3-1.3.Using HJX74 and S5 with main QTL qLTGR3-1 as materials,the changes of low temperature germinablity,dry weight,fresh weight and water content during seed development were investigated.S5 showed stronger low temperature germinablity than HJX74 during the whole development process,and the difference reached the maximum at35 d after heading.At the early stage of development（10-15 d）,the dry and fresh seeds of S5 were significantly higher than that of HJX74;at the late stage of development（30-40 d）,S5 showed faster dehydration rate than HJX74.4.The germination tests of S5（containing qLTGR3-1）under different stress conditions showed that the germination rates of S5 at normal temperature（30℃）and osmotic stress（1%and 3%mannitol）were significantly higher than that of HJX74,but there was no significant difference under high temperature and high salinity conditions.5.S5（containing qLTGR3-1）was backcrossed with HJX74 to develop F₂ populations,and 12 kinds of recombinant plants were screened by new molecular markers on the substitution fragment of S5,which developed F₃ populations after self-crossing.By co-segregation analysis and substitution mapping of the F₃ subtypes and genotypes,qLTGR3-1 was fine mapped in the region of approximately 319 kb between the molecular markers P7 and P8 which contained 51 candidate genes.In addition,12 homozygous secondary SSSLs with shorter substitution fragments were developed from the F₃population,which were named from SS1 to SS12 and the estimated lengths of substitution segments were from 851.22 to 4214.66 kb.6.After 35 d heading,the seeds of S5（qLTGR3-1）and HXJ74 were treated by dry heat for dormancy breaking,then transcript analysis was carried out with 5 d-imbibed seeds at 15℃.A total of 692 differentially expressed genes were detected,of which 315 genes were down-regulated and 377 genes were up-regulated.KEGG enrichment analysis showed that differential genes mainly involved in the metabolism and biosynthesis processes,such as diterpene compounds biosynthesis,amino sugars and nucleotide sugar metabolism,galactose metabolism,phenylpropanoid biosynthesis,starch and sucrose metabolism andα-linolenic acid metabolism.This suggests that qLTG3-1 may regulate the seed germination at low temperatures by affecting the metabolism of diterpenoids,carbohydrates and amino acids.7.Through fine mapping and transcriptome analysis,three differentially expressed genes were found in the fine mapping interval,including Os03g0213700,Os03g0214200,and Os03g0215400.These genes are important candidate genes for qLTGR3-1.In summary,this study identified some QTLs for low-temperature germination of rice and preliminary revealed the genetic basis of rice seed vigor;qLTGR3-1 was fine mapped and analyzed by RNA-Seq.The regulatory network further clarified the molecular mechanism of low temperature germination of rice seeds,which laid the foundation for the cloning of qLTGR3-1 and breeding rice variaties with strong low-temperature germinability by molecular design method.