Cloning And Breeding Application Of Rbh1 From A Rice Spaceflight-induced Mutant With Red-brown Hull

Rice（Oryza sativa L.）is the most important food crop in China,accounting for more than 50%of Chinese total grain output.Maintainace of rice production is of great strategic significance for food security and sustainable development in China.In order to meet the huge demand of rice and solve the problem of food security,it is necessary to change the existing mode of production and create novel germplasm to achieve a new breakthrough in rice breeding.In this study,a spaceflight-induced reddish-brown mutant rbh1 was studied including phenotypic investigation,mutation gene cloning,and development and application of functional molecular markers.The main conclusions are as follows:1.The red-brown marker character of the mutant rbh1 appeared gradually in the internode of stem at the late jointing stage,and the red-brown color displayed gradually in the leaf sheath and vein at the booting stage;the red-brown color appeared in the hull at the late booting stage,and deepened gradually in the filling stage until the seed matured.Whereas,rbh1 had no significant difference in other agronomic traits and rice quality except for reduced grain width and 1000-grain weight.The total flavonoids and anthocyanins in the hull of mutant rbh1 increased significantly,while the lignin content decreased significantly.It was suggested that the red-brown of the hull of rbh1 might be due to the increase of total flavonoids and anthocyanins in glume shell and the decrease of lignin content.2.The whole genome re-sequencing was carried out using the mutant rbh1 and its wild-type H9808.It revealed 24295 single base substitutions（SBSs）and 2615 In Dels were detected in rbh1 in comparison to the wild-type.Most of the mutation sites were located in the intergenic region.The main types of SBSs are T:A>C:G and C:G>T:A.The amount of SBSs distributed on the chromosomes was positively correlated with chromosome size.1-2-bp insertionsor deletions,accounted for about 60%.The distribution of In Dels on each chromosome was not even.3.The red-brown was controlled by a single recessive gene,which was was located in the48kb region of the short arm of chromosome 2 through map cloning.The sequencing revealed that the No.1001 base of the coding region of Os CAD2（LOC＿Os09g09490）was mutated from T to C,which caused led the change of leucine（Leu）into proline（Pro）.Transgenic complementary experiments showed that the phenotype of the red-brown hull of rice mutant rbh1 was caused by Os CAD2,a new allele of the previously reported Os CAD2controlling the golden glume and internode of rice.The expression of Os CAD2 in internodes and young panicles increased gradually,and reached to the highest in internodes and young panicles at heading stage.The expression levels of Os CHI and OsF3’H,as well as OsDFR which directly involved in anthocyanin biosynthesis,increased significantly in rbh1,while the expression levels of Os CHS1,Os F3’H and Os ANS1 did not differ significantly at booting stage.In addition,four genes related to lignin biosynthesis were highly expressed in glumes of the mutant.By subcellular localization,RBH1was located on the nucleus and cell membrane.The three-dimensional protein structure prediction showed that two alpha helixes occurred in rbh1 comparing with that of RBH1.4.A functional molecular marker RBH-T/C for red-brown character was developed based on HRM technology according to the mutation site in rbh1,and was successfully applied in genotyping.Furthermore,the marker was used to identify the homozygous male sterile line and maintainer line in F₁seeds of hybrid rice of Huaxiang series to realize rapid and efficient identification of rice hybrid seeds.5.Three target genes,rbh1,fgr and Pi2 were successfully pyramided through HRM technology,anther culture,high-pressure screening of rice blast nurseries,and rice quality testing,Seven maintaining lines with significantly enhanced blast resistance were developed,with the leaf blast grade 0-1 and panicle neck blast grade 1 at rice blast nurseries.6.Three new CMS lines with reddish-brown marker traits including Jinhua A,Huaxiang2A and Huaxiang7A,were developed based on the mutant rbh1.Furthermore,13 new hybrid rice conbinations based on Huaxiang A and Jinhua A,such as Huayou 230 and Huayou 357,were approved in Sichuan,Yunnan,Guizhou and the upper reaches of the Yangtze River.The increased yield of Huaxiang A series hybrid combinations mainly came from（1）increased their daily yield and effective panicles;（2）appropriately delayed growth period;（3）reduced plant height and panicle length.The increased rice quality was mainly owed to moderately reduced ratio of grain length to width so as to improve their grain filling efficiency.The results may be helpful for the breeding of subsequent hybrid combinations derived from Huaxiang series.