Fine Mapping And Cloning Of QTL For Cold Tolerance At The Booting Stage In Rice

Rice is a cold-sensitive crop that originated from tropical and sub-tropical areas.With the continuous enlargement of cultivation areas of rice,low temperature is a major limiting factor in rice production.Rice can be suffered from cold injury during all the growth stage.Cold injury at the booting stage is an important factor in rice production because spikelet fertility is irreversibly reduced resulting in loss of yield in this stage.Therefore,cloning cold related genes and exploiting beneficial gene resources are very significant to develop cold-tolerant rice cultivars and to guarantee the stability of rice yield.In our previous work,we obtained a series of cold-tolerant near-isogenic lines(NIL)by crossing a cold-tolerant cultivar KMXBG and a cold-sensitive cultivar Towada.In our study,we fine mapped two QTL qCTB10-1 and qCTB10-2 that confers cold tolerance at the booting stage in rice.On the other hand,we selected NIL1913 that also exhibit cold tolerance at the booting stage to cross with Towada to construct mapping population and finally cloned a new gene CTB4b and elucidate the mechanism involved in regulating cold tolerance in rice.The main findings are as follow:1.Fine mapping of QTL qCTB 10-1 and qCTB10-2 conferring cold tolerance at the booting stageWe constructed a population from NIL-ZL31(cold-tolerant)and Towada(cold-sensitive).Because there were two QTL in this region,we further analyzed them,respectively.The QTL qCTB10-1 was fine mapped and delimited to 17 kb on chromosome 10.At the same time,we developed another population and fine mapped the QTL qCTB10-2 into 132.5 kb distance.There were 17 candidate genes in which only five genes could encode protein.After analyzed the expression pattern of these genes under cold stress,we found that four genes were cold-induced.Based on these results,we concluded that these four genes would be the candidate genes related to cold tolerance.2.Cloning and molecular mechanism analysis of CTB4bWe constructed another population from NIL1913 and Towada and fine mapped the QTL to 56.8 kb on chromosome 4.Within this region,these four ORFs were predicted by RGAP(Rice Genome Annotation Project).After sequencing four ORFs between NIL1913 and Towada,only two ORFs had different sequences in two parents.One of the ORFs,CTB4a was cloned by our lab and we named the other as CTB4b.Sequence analysis showed two SNPs in the coding region of CTB4b between NIL1913 and Towada,in which only one SNP resulted in amino acid changes.After evaluating the cold tolerance between parent and different transgenic lines several years,we found that CTB4b was a positive regulator of cold tolerance at the booting stage in rice.Expression pattern analysis demonstrated that CTB4b was highly induced by cold stress and mainly expressed in panicles.CTB4b encodes an UDPG sterol glucosyltransferase and the mutation is existed in protein conserved domain.Enzematic analysis in vitro showed that CTB4b could promote to generate sterol glycoside.Also CTB4b had impact on accumulation of sterol glucoside and acyl sterol glucoside under cold stress in vivo which had important functions on cell membrane permeability and formation of pollen wall.Associated with the measurement of ion leakage and the observation of pollen grain and pollen wall,it indicated that CTB4b improved cold tolerance at the booting stage by mediating generation of sterol glucoside and maintaining cell membrane permeability and the structure of pollen wall.3.Evolutionary analysis of CTB4b and CTB4aWe did haplotype analysis of CTB4b using re-sequencing data and phenotype of 118 cultivars under cold stress.The results indicated that rice cultivars harboring the functional SNP of CTB4b exhibited higher cold tolerance at the booting stage.Furthermore,phylogenetic and minimum spanning tree analysis showed that the functional SNP was originated from wild rice and specifically existed in temperate japonica located in high latitude and altitude area with lower yearly temperatures.Nucleotide diversity,Tajima’s D and MLHKA analysis demonstrated that a strong artificial selection occurred on the CTB4b locus during temperate japonica domestication.We also analyzed the united haplotype of CTB4a-CTB4b and found that the result was same like CTB4b.CTB4a-CTB4b also experienced aritificial selection during temperate japonica domestication.Different combinations with CTB4a and CTB4b in rice cultivars showed that CTB4b was originated from wild rice during early cold adaptation and rice harboring CTB4b became moderate cold tolerance.New allele of CTB4a was also generated from rice having CTB4b under higher cold selective pressure.Because rice harboring both genes exhibited stronger cold tolerance and could be better adapted the low temperature environment,CTB4a-CTB4b was selected and conserved in temperate japonica.