Cloning And Functional Analysis Of A Major QTL QME1 For Mesocotyl Elongation Of Rice

With the transfer of social labor and the increasing shortage of natural resources,traditional and intensive transplanting cultivation of rice has been unable to meet the demands of the modern rice production.The direct seeding cultivation which is characterized by labor saving,low labor intensity and low production cost has been widely applied.However,there are still some problems in direct seeding of rice,such as difficult and irregular emergence,many weeds in seedling stage and poor lodging tolerance.To solve the problem of seedling emergence,it is urgent to follow up scientific research.Rice planting under a certain depth of soil has the following advantages:strong ability of rooting,high efficiency of soil nutrient absorption,water conservation,protection of seedlings at low temperature,and prevention from birds and mice eating seeds.Research showed that the elongation of rice mesocotyl plays a key role in the rapid breaking of soil and provides the main power source.Therefore,it is of great theoretical and practical significance to illuminate the mechanism of mesocotyl elongation during seedling emergence,and to identify the genes that control mesocotyl elongation.This research focused the QTL scanning of mesocotyl elongation of backcross recombinant inbred lines?BIL?between Kasalath and NIP,and a major QTL qME1 was finely mapped and cloned,the research results are as follows:1. There is a wide range of mesocotyl length among varieties,of which the variation range of indica rice is more extensive.The emergence rate of materials with long mesocotyl is significantly higher than those of short mesocotyl materials.Mesocotyl elongation mainly depends on cell elongation,and mesocotyl middle upper cell elongation provides the main power source.2. The QTL qME1 controls mesocotyl elongation in Chr 1,and LOD value is 5.2,contribution rate is 16.9%.QTL q ER1?quantitative emergence rate 1?and qME1 locus were coincident,suggesting that the same QTL locus simultaneously controls mesocotyl length and seedling emergence rate.3. The F₂ population was constructed by the substitution line?SL-5?of Kasalath fragment in the background of NIP where qME1 was located,and qME1 was finely located between Indel marker lv-7 and lv-8,with an interval size of about 47.5 kb.ORF1 encodes Os GA20ox2,namely is the dwarf green revolution gene SD1.The sd1 mutants in the background of Kasalath showed a phenotype of shorter mesocotyl and lower emergence rate,indicating that qME1 is SD1 gene.4. Sequencing analysis showed that there are three main genotypes of SD1:the SD1^EQtype,represented by Geng?japonica?with a short-or no-mesocotyl.The SD1^GRtype,represented by Kasalath and Peta,is mainly a pre-green revolution varieties and wild rice,with a longer mesocotyl.There is also one kind of green revolution varieties,represented by IR8 and Guangluai4,which is SD1^Nulltype,with a short mesocotyl.The pre-green revolution varieties with SD1^GRgenotype generally had longer mesocotyl and higher stems,suggesting that the mesocotyl elongation trait was gradually lost along with the tall height trait during the process of dwarf breeding.5. We found that the synthesis of ethylene increases during the growth of rice seedlings covered with different depth of soils.Ethylene signal might mediate GA synthesis pathway to promote mesocotyl elongation and seed emergence.The expression level of SD1 was induced by ethylene and soil depth.Biochemical and genetic evidence showed that Os EIL1 could promote the promoter activity of SD1 gene and regulate mesocotyl growth.6. The protein level of SLR1,a core inhibitor of GA signal,was inhibited by soil mechanical pressure,and the SLR1 mutant showed longer mesocotyls and higher stems.SLR1 interacts with Os PIL13 in rice,and the over-expression plants of Os PIL13 also shows a phenotype similar to that of slr1.In addition,SLR1 inhibits the activition of Os PIL13 on downstream expansion gene Os EXPA4,and over-expressing lines of Os EXPA4 also exhibited a long mesocotyl phenotype.7. qME1 can promote rapid emergence under water-resistant conditions and increase the germination rate of direct seeding under dark condition,indicating its application value in direct seeding of rice.The promoters with high specific expressions levels in the mesocotyl and coleoptile of the etiolated seedlings were selected to construct ectopic expression vectors.We hope that we can construct the excellent breeding materials with long mesocotyl length,high seeding emergence rate,strong lodging-resistance in the future.