Effect Of Dwarf Gene Rht14 On Key Agronomic And Quality Traits In Durun Wheat (Triticum Durum) And Its Fine Mapping

The application of dwarf genes Rht-B1b and Rht-D1b in the“Green Revolution”greatly improved the lodging resistance and yield of wheat,however,gibberellic acid insensitive genes Rht-B1b and Rht-D1b had adverse effects on coleoptile and root vigor,which limited their application on wheat dwarf breeding in arid areas.Therefore,it is very important to find new alternative gibberellic acid responsive dwarf genes for wheat dwarf breeding.Reciprocal crosses were made between two durum cultivars,Castelprziano（CP）,the donor of gibberellic acid responsive gene Rht14,and Langdon（L,no known dwarf gene）,and 25homozygous dwarf lines and 20 homozygous tall lines of the F_2:3,F_3:4 and F_4:5 generations were used for evaluating the effects of Rht14 on important agronomic traits and quality traits.At the same time,the responsiveness of Rht14 to exogenous gibberellin（GA₃）was investigated by applying exogenous GA₃ to the dwarf and tall lines of the reciprocal crosses to explore its dwarf mechanism.The responsiveness of Rht14 to osmotic stress was observed in the seedlings of the F_5:6 lines derived from CP×L treated with 15%and 25%PEG6000.In addition,Rht14 was fine mapped and the candidate gene was preliminarily predicted by the F₂ populations and its recombinants derived from reciprocal crosses.The main results were as follows:1.Effects of Rht14 on key agronomic traits and quality traits of durum wheatAgronomic traits and quality traits were investigated and compared between the F_2:3,F_3:4 and F_4:5dwarf and tall lines of the CP×L and L×CP populations.The plant height was significantly reduced by an overall average of 26.68%and 27.68%in the reciprocal crosses of thress years,respectively,and Rht14 reduced the plant height by significantly shortening the length of each internode.The diameter and thickness of each internode of dwarf line were remarkable wider and thicker,and the lodging index was significantly reduced,which contributed to the improvement of lodging resistance.Rht14 dwarf lines showed higher flag leaf area,SPAD value and net photosynthetic rate of flag leaf than tall lines.Rht14significantly reduced 1000-kernel weight,which might be related to the delaying of heading and flowering stage,but had no negative effects on plump grain size and other yield traits.Rht14 had no adverse effects on coleoptile length and seedling root traits.The sedimentation value,stability time and development time were significantly reduced in dwarf lines,indicating Rht14 might have some negative effects on wheat quality traits.2.Response of Rht14 to exogenous gibberellin（GA₃）It was found that Rht14 was more sensitive to GA than tall lines when applied with exogenous GA₃ on the F_2:3,F_3:4and F_4:5lines of CP×L and L×CP populations.The plant height of the dwarf and tall lines increased significantly 50%and 15%on average,respectively,after GA₃ treatment,and all the internodes of the dwarf plants while only the top three internodes increased significantly,meanwhile,the diameter and thickness of these internodes remarkably reduced.Exogenous GA₃ significantly reduced the leaf area,SPAD value and net photosynthetic rate of flag leaves of dwarf lines,but significantly increased the spikelet number spike^-1,grain number spike^-1,and 1000-kernel weight,which may be related to the earlier heading and flowering stages.Application of exogenous GA₃ significantly increased the coleoptile length,whereas notably shortened the max root length and total root length of the dwarf lines.The application of exogenous GA₃ can significantly increase the crude protein content,wet gluten content,sedimentation value,stabilization time and formation time of the dwarf lines.In conclusion,exogenous GA₃ could compensate the dwarf effects of Rht14,and make its agronomic and quality traits more similar to the tall lines,which indicated that Rht14 was a GA-deficient dwarf gene.3.The physiological characters and transcriptome analysis of Rht14 under osmotic stressAfter treated with 15%and 25%PEG 6000 for 24 h and 48 h,respectively,the relative leaf water content of dwarf plants in CP×L F_5:6generation declined slower than tall plants while the fresh weight/dry weight showed no differences,and the content of malondialdehyde,activity of peroxidase and content of proline in dwarf plants were higher than tall plants.Transcriptome analysis of tall and dwarf leaves treated with PEG 6000 for48h showed that the number of differentially expressed genes upregulated was more than downregulated genes in dwarf lines,while the opposite tendency was observed in tall lines.The GO and KEGG enrichment analysis of the differentially expressed genes indicated that the dwarf and tall lines had both the same and specific responses to osmotic stress.The up-regulation or down-regulation of key differentially expressed genes related to stress,hormone and transcription factors made the dwarf lines more resistant to osmotic stress.4.Fine mapping and candidate genes analysis of Rht14In order to map and clone Rht14 and explore the mechanism of dwarf,the KASP（Kompetitive Allele-Specific PCR）markers were developed after the Bulked Segregant Analysis（BSA）combined with the wheat 660K SNP array,and Rht14 was mapped in the312-454 Mbp of chromosome 6A by using the F₂ populations of CP×L and L×CP.Rht14was further fine mapped to the genomic region of 405-411 Mbp in Chinese Spring Ref Seq v.1.0（i.e.,402-408 Mbp in Durum Wheat Svevo Ref Seq Rel.1.0）using recombinants by indel markers.Rht14 and Rht18 are alleles,and the increased expression of the oxidase of gibberellin synthesis pathway GA2ox A9 resulted in the reduction of Rht18 plant height,while the expression of GA2ox A9 increased significantly in the internodes and nodes of Rht14 dwarf plant,and the content of bioactive GA₁ decreased.No sequence difference was observed in the promoter and coding region of GA2ox A9 between the dwarf and tall parent,while obvious DNA methylation difference was found in its promoter.Two methylation-related genes with high confidence located in the candidate region and expressed differently between the tall and dwarf ones in seedling leave while one gene expressed differently in peduncle of tall and dwarf ones may regulate the expression of GA2ox A9 by methylation in the promoter region of GA2ox A9,resulting in the decrease of plant height,but this conclusion needs further verification.It is speculated that Rht14 might regulate the expression of GA2ox A9 by DNA methylation in its promoter,and eventually lead to the reduction of plant height,but this conclusion needs further verification.