Fine Mapping And Candidate Gene Analysis Of New Gene Awn8 Conferring Long Awn In Wild Rice

Awn is commonly present in wild rice species,and awn can help seed dispersal,and prevent seeds from being eaten by birds or animals.Modern rice varieties are generally awnless or exceptionally short awn.The loss of awn character is a critical event in rice domestication.It is of great significance to study on function,development and genetic basis of awn and explored the process of rice domestication through mapping and cloning the genes related to the awn development and regulation.Awn is a visual morphological marker like plant height,panicle phenotypes and grain color.Understanding the genetic mechanism of awn’s morphological development will help to use morphological markers in rice breeding and provide theoretical bases of gene functional study of other speciesIn this study,one accession of Guangxi common wild rice(Oryza rufipogon Griff.)DP 15 as donor parent and an indica rice variety 9311 as receptor parent were used to construct a set of rice chromosome segment substitution lines(CSSLs)and a population containing high backcross generation(>BC5)under 9311 background was obtained,from which we selected a genetically stable line,XL138,with long awn phenotype.By constructing large segregating population and several cycles of molecular marker design,the gene conferring long awn character in XL 138 has been fine mapped and the candidate genes in the mapping region were predicted.It is discovered that the gene conferring long awn in XL138 is a new locus and therefore,is designated as Awn 8.The study results obtained are as follow:(1)Analysis of characters of CSSL XL138.By comparison with recurrent parent 9311 the phenotype of XL 138 was analyzed.It was shown that there was no significantly different in plant height,tiller number,and grain number per panicle between XL 138 and 9311.Awn length(AL)and percentage of awned spikelets per panicle(AP)were significantly larger in XL138 than these in 9311.And grain length,length-width ratio,and 1000-grain weight were significantly smaller than the 9311.Study data presented that the AL was 3.91±0.30 cm and AP was 98±1.2%in XL138,both 275.96%and 214.10%of these in 9311,respectively.Grain length,length-width ratio,and 1000-grain weight in XL138 were 28.71±0.28g,11.23±0.04mm and 3.40±0.03,respectively,and these were 7.03%,3.68%and 3.13%lower than these in the 9311.(2)Genetical analysis and mapping population construction of lone awn character.The CSSSL XL138 and 9311 were crossed to construct the segregating population for gene mapping of the long awn.The F1 plants was long awn,which was the same with the XL138.The F2 population was segregating with long and short awn phenotypes by ratio 981:306,which fitted 3:1 segregation ratio by Chi-square test and confirmed the long awn controlled by a pair of main function dominant genes.(3)Primary mapping of the long awn gene Awn8.The number of 415 polymorphic molecular markers between DP15 and 9311 were obtained from 1633 molecular markers covering the 12 chromosomes.These polymorphic markers were used to detect the genetic background of the XL138.It was found that there were only four substitution segments from the wild DP 15 which distributed only in chromosome 8 and 9 in XL138.Target gene was found between molecular markers M3 and M8 on chromosome 8,and the location region was about 0.89 Mb through linkage analysis.This gene is a new locus for long awn in rice and designated as Awn8 in this study.(4)Fine mapping of the gene Awn8.Because molecular porymorphism in the gene location region was deficient and earlier designed SSR and STS markers counld not used for fine mapping the Awn8,the genome of CSSL XL138 was sequenced to exploit SNP markers.The number of 8 new polymorphic markers was developed between the M3 and M8 by referring the result of genome sequence.Finally,the Awn8 was located to a 54-kb narrow region between markers M16 and M18 on chromosome 8 by using of an enlarged mapping population including 2816 individials with short awn phenotype.(5)Candidate analysis for long awn gene Awn8.There are 7 predicted genes in the 54-Kb genomic region according to rice genome annotation project.Through sequence alignment,it was found that sequence of three genes P1,P2 and P5 in the cultivated rice 9311,Nipponbare and CSSL XL138 genome was no different from each other,and therefore,these three genes would not be the candidate of the target gene.On the other hand,gene P4 in CSSL XL138 had two-base difference with high simility upto 99%of protein sequence as compared with the sequence of 9311.Also,the P4 was no much related the long awn character according to its molecular function as catalytic activityy,biotic stress and metabolic process.Two genes P6 and P7 were belonged to a C2H2 zinc finger protein family in rice.Currently reported plant C2H2 zinc finger proteins are mainly involved in plant growth and development of the various periods which lead to the change of the lemma of rice,and further affecting the growth and development of the awn in rice.Though predicted gene P7 belong to C2H2 zinc finger protein family,it could not be the candidate gene as it only present one-base samesense mutation.While the other gene P6 presented two-base no sense mutation and therefore was regarded as one of the candidate genes for the long awn,but it remained further funtional confirmation.There was no report about the function of gene P3,but it in the CSSL XL138 presented a 51-bp losed and a 12-base displacement,which led to the gene only encoding 81 amino acids,while the normal gene in the cultivars 9311 and Nipponbare encoded 267 amino acids.As there was large difference between the both parents CSSL XL138 and 9311 in the DNA and protein sequences,the gene P3 was much possible to be the candidate gene of long awn,but the speculation still needed further verification.