Mapping Of QTLs And Cloning Of Genes Related To The Culm Mechanical Strength In Rice(Oryza Sativa L.)

Rice is an important food crop in China,thus it is essential to keep the high yield to meet the requirement from both national and consumer's levels.Rice lodging is a major factor that can seriously affect grain yield.Although many genetic factors are involved in the lodging resistance of rice,the mechanical strength of culm is the primary factor.In detail,culm diameter,culm wall thickness,number of vascular and cell wall composition are proved to the factors that involved in affecting the culm mechanical strength.As we known,all the above traits are typical quantitative traits that are controlled by multiple genes.By using the method of QTL mapping,we can mine the favor alleles that involved in controlling the culm mechanical strength in natural germplasms,which may provide the novel genes/alleles that can be directly used for breeding lodging resistant variety.Meanwhile,as the ideal materials on the study of the genetic mechanism for the mechanical strength and cell wall biosynthesis,many brittle culm(BC)mutants have been identified.What's more,the genetic mechanism of the culm mechanical strength is prelimary ayalyzed based on the study of the function of brittle culm genes.However,the genetic mechanism is still not clear,thus further effort is needed to identify more brittle genes in order to further reveal the genetic mechanism of the culm strength.In present study,many QTLs for culm diameter,culm wall thickness,number of vascular and cell wall composition were identified by using the chromosome segment substitution lines(CSSLs)which were developed by using the indica variety 9311 as donor parent and japonica variety Nipponbare as receipt parent.Besides,we identified three brittle culm mutants and then analyzed the factors that caused the brittle culm trait.Subsequently,the three genes were fine mapped via map-based cloning method,and among which a new brittle gene was cloned and its function was verified through complemention experiment.1.QTL analysis for the traits related to culm strength and mapping of QTL-qWTh5In this study,we totally detected 31 QTLs.In detail,a total of nineteen QTLs for the content of cellulose and four major neutral sugars and XA ratio(Xylose/Arabinose ratio)have been identified on nine chromosomes.Probably because cellulose biosynthesis is modulated by multiple genes with small effects,only one QTL for cellulose content(qCel6)with positive additive effect was mapped and could explain 10.9%phenotypic variance.Three QTLs for xylose content were detected,in which qXyl7 and qXyl10 showing negative effect were concentrated on chromosomes 7 and 10,whilst qXyl8 that has positive effect was located on chromosome 8.The variance explained by these QTLs varied from 8%to 13.6%.Four QTLs for arabinose content showing positive effect were mapped on chromosomes 4,7 and 8 with around 6.4%-12.5%variance.Similarly,four QTLs for XA ratio with positive additive effect located on chromosomes 7 and 8,in which qXA7-1 and qXA7-2 are major QTLs for XA ratio as they explained 59.2%and 17.7%phenotypic variance.Within the five QTLs for glucose content,qGlcl-1 and qGlcll explained 17.7%and 15.6%phenotypic variance.Finally,two QTLs for galactose content were mapped on chromosomes 4 and 8,respectively.Meawhile,the effect of qXyl10 and qXA7-1 was further verified.The results suggest that qXyl10 and qXA7-1 were stable genetic QTLs,which are worthy for further study.Using the same approach,a total of twelve QTLs for culm morphological traits were found to locate on five chromosomes with positive and negative additive effect.When the CSSL planted in Yangzhou University experiment field was used as material,seven QTLs for culm morphological related traits were identified,in which three QTLs for culm diameter were found to locate on three chromosomes that could explain the variance varied from 9.23%?13.63%.Beside,one QTL for no.of vascular and culm wall thickness were also identified,respectively.Among two QTLs for culm wall thickness,QTL-qTh2x that could explain the variance about 20.4%maybe as the major QTL.Compared with culm wall thickness,two QTLs for no.of vascular that were located on chromosomes 2 and 9 showed negative effects.Meanwhile,when the CSSL planted in Yangzhou Jiudian experiment fields was used as material,five QTLs for culm morphological related triats were also identified,in which three QTLs for the culm wall thickness and one QTLs for culm diameter and no.of large vascular,respectively.Compared with the results of the QTL mapping for the culm morophological traits,it was found that the same QTL for culm wall thickness was located on the chromosome5,which indicated that this QTL might be a stable genetic QTL with hardly be impacted by environmental condition.In addition to some QTLs that were mapped in the closely physical location were also found.Although the location of these QTLs is not exactly in the same physical location,according to the QTL positioning analysis method,it can be concluded that the adjacent QTL is presumed to be the same QTL,which are worthy for further study.Subsequently,based on the results of QTL mapping,we set out to define the localization of qWTh5 gene.Through the measurement of the culm wall thickness of 144 individual plants with different genotypes,qWTh5 gene was preliminary located on the substitution segment from chromosome 5 in N22 CSSL.Ultimately,according to information of the recombinant of the substitution chromosome segment,the qWTh5 QTL was mapped into 1.4 Mb fragment between molecular markers STS5-9 and STS5-12,based on the reference sequence of the Nippnobare genome.Then,a near isogenic line(NIL-qWTh5)containing 1.78 Mb fragment was constructed by molecular marker assisted selection.The results of the investigation of culm wall thickness,heading date and plant height between Nipponbare and NIL-qWTh5 showed that the culm wall thickness of NIL-qWTh5 was significantly thicker than that of Nipponbare,.but there was no significant change in heading date and plant height.These results suggest that the gene qWTh5 has the potential to be a high quality lodging resistance gene that may be directly used in lodging resistance breeding of rice.In conclusion,these results provide genetic support for molecular marker assisted selection of lodging resistance breeding,and lay foundation genetic basis for fine mapping or cloning of qWTh5 gene.2.Identification and gene cloning of the brittle culm mutant bcl-wu3,bc-wy7 and bc19Three brittle culm mutants were selected,bcl-wu3(brittle culm 1 from Wuyujing3)and bc-wy7(brittle culm from wuyunjing7)were obtained from the japonica varieties Wuyujing 3 and Wuyunjing7 after 60Co-? induced mutagenesis,respectively.bcl9(brittle culml9)was obtained from japonica variety Zhonghua11 after EMS induced mutagenesis.The leaf,leaf sheath and culm of bc1-wu3,bc-wy7 and bc19 exhibited brittle during the whole growing stage.The physicochemical mechanism of brittle culm were analyzed based on measurement of the cell morphology of culm and components of cell wall.Then,three brittle genes were mapped by a map-based cloning method.In compared with wild type(WT),the panicle length and grain length of bcl-wu3 decreased significantly,but the gain width presented a significant increase.There was a significantly decrease of the cellulose content in cell wall of culm,but the contents of xylose,glucose and arabinose increased significantly in the cell wall of bcl-wu3.The number of sclerenchyma cell layers and the thickness of sclerenchyma cell wall were also significantly decrease.Genetic analysis showed that the brittle trait in bcl-wu3 was controlled by one single recessive nuclear gene,and the locus was mapped to?57 Kb genomic region on chromosome 3 between molecular markers MK12 and MK18.In the mapped region the cloned brittle culm gene BC1(LOC_Os03g30250)was included,Sequence analysis revealed that there was a single-base substitution(G-T)in the second exon of BC1 gene in bcl-wu3 mutant,leading to a residue substitution from cysteine to phenylalanine.The data from qRT-PCR indicated that there was significant decrease in the expression of BC1 gene in the culm of the mutant.Based on these results,we speculate that bcl-wu3 gene mapped in this study is allelic to BC1 gene.These results would deepen our understanding for the function of BC1 gene and help us to clarify the genetic mechanisms of culm strength.In terms of mutant bc-wy7,the plant height,panicle length and grain number of panicle of bc-wy7 showed significantly decreased,and the heading date appeared significantly delayed compared with WT.Meanwhile,it was also showed that there were significant difference in cell wall composition between WT and mutant.The cellulose and glucose content also decreased significantly in the culm of mutant.Besides the number of sclerenchyma cell layers and their cell wall thickness were both decreased in culm of the mutant,the number of parenchymal cell increased in the bc-wy7.Genetic analysis implied that the brittle trait in bc-wy7 was controlled by one single recessive nuclear gene.Ultimately,the mutated locus was delimited to?180 Kb genomic region on chromosome 2 between the molecular markers S2-52 and S2-56.In the mapped region,there is a cloned gene named Brittle Culm 3(BC3)that can cause the brittle phenotype appeared when that gene is mutated.Hence,the sequence of BC3 were sequenced in WT and mutant,respectively.The results showed that there were two nucleotides deletion at the 9th exon in the mutant,resulted in a frame shift and premature termination of translation.Meanwhile,the data from qRT-PCR indicated that there was significant decrease in the expression of BC3 gene in culm and leaf of the mutant.Taken together,we can confirm that bc-wy7 mapped in this study is allelic to BC3 gene.In terms of bc19 mutant,both in Yangzhou and Hainan field growth conditions,plant height,tiller number,panicle length,seed setting rate and thousand-grain weight of the bc19 mutant were significantly lower than that of WT.Meanwhile,we found that there were significant difference in cell wall composition between WT and bc19 mutant.Similar to the mutant bc-wy7,the cellulose and glucose content also decreased significantly in the culm of mutant.Besides the thickness of cell wall also significantly decreased in culm of the bc19 mutant.Genetic analysis showed that the brittle trait in bc19 was controlled by one single recessive nuclear gene,and the locus was mapped to?47 Kb genomic region on chromosome 5 between molecular markers X31 and X56.On the mapped region,there are 6 open reading frames(ORFs),in which the ORF3 encoding the o-acetyltransferase that has been reported in Saccharomyces cerevisiae involved in cell wall biosynthesis was sequenced.Sequence analysis revealed that there was a single-base insert in the 2572?2573 bp of BC19 gene,resulted in a frame shift and premature termination of translation.Expression analysis showed that BC19 expressed in all tissues of bcl9 mutant,while the expression amount of BC19 was significantly decreased in bc19 mutant compared to WT.So,ORF3(LOC_Os05g05200)might be the candidate gene of bc19.The comparative analysis of the amino acid sequence of the BC19 protein revealed that the protein encoded by the BC19 gene was conserved in tobacco and yeast species.The results of yeast complemention experiment showed that the complementary vector with HA tags could well complement the phenotype of the corresponding yeast mutant.Thus,it implied that the function of BC19 gene may be similar to the GUP1 gene of yeast which also encoded o-acetyltransferase that was involved in the process of GPI-ANCHOR remodeling.Once this gene mutated,the localization of GPI-ANCHOR protein would be affected owing to alter the modification of GPI and terminally affect the physiological and biochemical processes of yeast cell wall.In rice BC1 like the gene encoding GPI anchored like protein.According to the research in yeast,the BC19 gene may be involved in modifying the GPI anchor of the GPI anchor protein that encoded by BC1 gene to regulate the biosynthesis of rice cell wall.These results provide an important reference for understanding the genetic mechanism of BC19 gene and corresponding protein in the regulation of rice cell wall synthesis.