Identification Of Rice Chromosome Segment Substitution Line Z481 With Seed Shattering And Fine Mapping Of The SH6(t)

Rice is one of the most important crops all around the word and it's a staple food of more than half the population in our country.Organ abscission is a ubiquitous phenomenon in the plant kingdom,of which seed shattering is a typical case.Seed shattering is closely related to mechanized harvesting.Moderate shattering is favorable to improve production efficiency by reducing rice production losses and mechanized harvesting.Therefore,identification of genes for seed shattering is important for rice mechanization production.Precious research has shown that seed shattering is mainly related to abscission layer,and development of the abscission layer is regulated by many genes.By cloning seed shattering genes such as qSH1,SHA1 and SH4,the molecular mechanism regulating shattering has been primarily known.However,specific regulatory networks were still not very clear.Identifying novel gene of seed shattering is important to reveal the molecular mechanism of seed shattering.In the study,a chromosome segment substitution line?CSSL?named Z481 with easy seed-shattering was identified deriving from advanced backcrosses between Nipponbare as recipient and excellent rice restorer R225 as donor by SSR maker-assisted selection?MAS?.Furthermore,a series of studies about Z481 was done,such as molecular identification,morphological analysis,evaluating of agronomic trait,observation by scanning electron microscope,genetic analysis and fine-mapping of the seed shattering gene,candidate gene analysis of SH6?t?.The main results were as follows: 1The identification of substitution segment of Z481On the basis of previous research,Z481 was indicated homozygosis by identification of substitution segments and detection of residual segments of donor with 62 SSR makers.Z481 contained four substitution segments which located on chromosome 1st,3rd,6th and 6th.They were RM11694-RM6292,RM5864-RM15709,RM6734-RM276 and RM3330-RM3187.The length of these substitution segments was 8.30,6.17,3.12 and 10.79 Mb,respectively,the total substitution length was 28.38 Mb,and the average length was 7.39 Mb.2 The morphological and agronomic characteristics of Z481The seed shattering ratio of Z481 was 5.3%,while Nipponbare was 94.1%,so Z481 belong to easily shattering while Nipponbare difficultly shattering,which reached 0.01 significant level.Compared with Nipponbare,plant height,panicle length,length of the 1st internode and width of the 2nd leaf in Z481 were significantly reduced,and width of the flag leaf and seed setting ratio were significantly increased.Other important agronomic traits such as length of the leaf,panicle number,spikelets per panicle,1000-grain weight displayed no significantly difference.3 Cytological analysis of abscission layerThe result of scanning electron microscope of abscission layer between sterile lemma and pedicle showed that abscission layer of Z481 was completely degraded at maturity stage,whereas that of Nipponbare was complete.4 The distribution of seed shattering in F2 population and genetic analysisThe seed shattering of F1 population from Nipponbare and Z481 was nearly the same with Nipponbare,displaying difficult seeding shattering,which indicated that the gene of seed shattering trait in Z481 was recessive heredity preliminarily.The seed shattering ratio in F2 population showed bimodal distribution,and by ?² test,the ratio of difficult shattering plants to easy shattering plants fitted completely to 3:1(?² = 0.87 < ?_?0.05,1?²=3.84).These results further showed that the easily seed shattering in Z481 was controlled by a single recessive nuclear gene.5 Mapping of the easy seed shattering geneSSR makers in the 4 substitution segments in Z481 were used for linkage analysis with recessive plants in F2 population crossed by Nipponbare and Z481.The results showed that RM6734 maker was linked to the easy shattering gene.Then,the target gene named SH6?t?was initially mapped between RM6734 and ZTQ53 on chromosome 6.By further developing new markers,the gene was finally mapped between RM235 and ZLY72,with the physical distance of 269 kb.6 Candidate gene prediction and sequencingCandidate gene prediction was done with related data base.There were 37 genes in the mapping region,where 5 genes possibly related to seed shattering were sequenced.Four genes were found no difference between Nipponbare and Z481.One gene encoded AP2 domain protein was found existing difference between Nipponbare and Z481.That is,6 bases?GCAGCA?was added behind the 56 th base G in the 2th exon,the 841 th base G in the 2th exon was replaced by A,and the 979 th base G in the 2th exon was replaced by A.Based on the above results,we preliminarily identified the gene as the candidate gene of SH6?t?.7 Analysis of expression pattern of SH6?t?The expression of candidate gene of SH6?t?in organs of Z481 in heading was analyzed by RT-PCR.It turned out that SH6?t?was universally expressed in various tissues including roots,stems,leaves,sheaths and panicles at different times,especially high in 6 cm of panicles.8 Expression analysis of seed shattering related genes of Nipponbare and Z481The expressions of relative genes to seed shattering were analyzed in Nipponbare and Z481 at heading stage by RT-PCR.The results showed that the expression level of these genes were all up-regulated to some extent in 3,6,12 cm of panicles,especially,the up-regulation of SH4 was very obvious,indicating that SH4 might be the same pathway gene.