Positional Cloning And Functional Validation Of Reduced Height Gene Rht8 In Wheat

In 1960s,the widespread application of green revolution genes Rht1 and Rht2 has greatly improved the lodging resistance and harvest index in wheat,which resulted in significant increase of grain yield.However,gibberellin-insensitive dwarfing genes Rht1 and Rht2 still have adverse effects on coleoptile length and thousand grain weight.Therefore,identification of gibberellin sensitive dwarfing genes play an important role in breeding high lodging resistant wheat varieties.By using the dwarfing mutant je0199（renamed as Rht8-2 according to the mapping region）without negative effects on coleoptile length and grain yield as a material,we fine mapped and cloned the gibberellin sensitive dwarfing gene Rht8.Through gene editing,subcellular localization,gene expression,exogenous gibberellin treatment and endogenous gibberellin content determination,we validated and revealed the dwarfism mechanism of Rht8.Besides,we used a recombinant inbred line（RIL）population containing139 lines derived from a cross between the mutant Rht8-2 and the relatively taller wheat variety Nong Da5181（ND5181）to construct a high-density genetic linkage map by applying the Wheat 40K Panel.Combining with the phenotypic data,we mapped and identified the key QTLs that affected plant height and spikelet density,and revealed the effect of new allelic variation of Rht8-B1 on plant height.The main results were as follows:1.Positional cloning of reduced height gene Rht8 in wheat（1）Using an F₂ segregating population including 11670 individuals by crossing between wildtype winter wheat Jing411 and dwarfing mutant je0199,the reduced height gene was fine mapped to a 700 kb region on chromosome 2D,which contained a large gap region in the Chinese spring wheat reference genome.The Jing411 reference genome was assembled by third-generation sequencing technology.Combining with the assembled Jing411 reference genome and the published Fielder reference genome,the mutation of CG to T in one copy of tandem repeat of Traes CSU03G0022100 was identified,which resulted in premature stop codon and caused dwarfism in Rht8-2.Traes CSU03G0022100 encodes a ribonuclease protein containing a conserved ribonuclease-H domain.By cloning and sequencing analysis of Traes CSU03G0022100,the Rht8-original variety Akakomugi and the dwarf mutant Rht8-2exhibited the same mutation,implying Traes CSU03G0022100 was the Rht8 gene.（2）We analyzed plants with editing different functional copies of Rht8,and found that editing one functional copy of Rht8 reduced plant height by 9 cm,with 9.7%of plant height reduction,while editing two functional copies reduced plant height by 16 cm,with 17.2%of plant height reduction.The dwarfing gene Rht8 was mainly expressed in nodes and internodes of stem.The expression level in the mutant was significantly lower than that in the wild type,indicating that decrease of Rht8 gene expression in the mutant resulted in reduction of plant height.Subcellular localization suggested that Rht8 protein was localized in the nucleus.（3）Exogenous gibberellin treatment to the dwarf mutant Rht8-2 could restore the plant height of Rht8-2 to the level of wild type Jing411,indicating that the endogenous gibberellin synthesis may be inhibited in the mutant.The determination of endogenous GA content showed that the contents of bioactive GA₁ and GA₃ were significantly reduced in the dwarf mutant Rht8-2,and the contents of intermediates GA₅₃,GA₄₄ and GA₁₉ in the GA₁/GA₃biosynthetic pathway were synchronously reduced.The expression levels of GA13ox-2A,GA13ox-2B,and GA13ox-2D,which catalyze the conversion of GA₁₂ to GA₅₃,were significantly reduced,suggesting that the lower expression of GA13ox contributed to decrease bioactive GA1 and GA3 content in the dwarf mutant Rht8-2,which led to reduce plant height.（4）Cloning and sequencing of Traes CSU03G0022100 in Aegilops tauschii suggested that it only contained the CG type copy,indicating that the T type copy occurred after hexaploidization.And the sequencing marker S7 based on Rht8 mutation site was successfully developed and was used to genotype 365 modern wheat varieties and 87 Chinese landrace wheat varieties.The results showed that the dwarf allele Rht8 had the highest frequency of77.5%in the Yellow and Huai River Valleys Wheat Zone,followed by the Middle and Low Yangtze Valley Wheat Zone,with a frequency of 50.7%.The frequency of the dwarfing gene Rht8 in the Southwestern Autumn-Sown Spring Wheat Zone was 43.5%,and the frequency in the northern winter wheat zone was 17.1%,showing that Rht8 was widely used in Chinese wheat varieties.The frequency of dwarf gene Rht8 was account for 11.5%in 87 Chinese landraces,indicating that Rht8 was positively selected in modern wheat breeding.（5）In this study,the effects of dwarfing gene Rht8 on plant height,thousand grain weight and grain number per spike were analyzed using the Jing411/Rht8-2 F₆ population,the Rht8BC₃F₂ segregating population with Jing411 background and its BC₃F₃ lines.In the F₆ and BC₃F₂ populations,the plant height of Rht8 homozygous line was significantly reduced,but there was no significant difference in thousand grain weight and grain number per spike.In the BC₃F₃ lines,the Rht8 homozygous NILs had a 19%height reduction compared to their wild-type NILs,while the thousand grain weight and grain number per spike had no difference.2.Genetic mapping of Rht8-B1 that regulates plant height in wheat（1）We used a recombinant inbred line（RIL）population with 139 lines derived from a cross between the mutant Rht8-2 and winter wheat Nong Da5181（ND5181）to construct a high-density genetic linkage map by applying the Wheat 40K Panel.We identified seven stable QTLs for PH（three）and SC（four）in two environments using the RIL population,qPH2B.1,qPH2D,qPH4B,q SC1B,q SC2B.1,q SC2D.1,and q SC7D.By developing molecular marker,qPH2B.1 was delimited to a 3.5 Mb region,in which included the Rht8 in the B genome（Rht8-B1）.Analysis of sequences between the two parent lines of the Rht8-B1 gene revealed that the GC bases（Rht8-B1a）at positions of 524^th-525^th in ND5181 changed to TT in Rht8-2（Rht8-B1b）.In the RIL population,the effects of Rht8-B1b on reducing plant height were 6.2%and3.6%,spike length were shortened by 6.6%and 4.7%,and spikelet compactness was increased by 5.8%and 6.3%in the two environments,respectively.（2）The reduction effects of Rht8-B1 and Rht8-D1 were compared by gene editing plants in the Fielder background.The impact of Rht8-B1 and Rht8-D1 on reduction of plant height were 5.6%and 17.5%,respectively,indicating that the reduction effects of Rht8-B1 was much lower than that of Rht8-D1.Rht8-B1 and Rht8-D1 were highly expressed in the nodes or internodes of stems at jointing stage.The expression level of Rht8-D1 was significantly higher than that of Rht8-B1.In the Rht8-D1 mutant,the expression of the Rht8-B1 gene was significantly increased.（3）We used a total of 305 worldwide accessions for the distribution analysis of Rht8-B1a and Rht8-B1b,and found that 68 Chinese accessions（35.2%）contained the Rht8-B1b TT alleles,compared with only 6 accessions（5.4%）in other countries.Of the Chinese accessions,20.3%of the 118 modern varieties and 58.7%of the 75 landrace accessions had Rht8-B1b alleles.These results suggest that Rht8-B1b alleles were not widely used historically for wheat breeding.In conclusion,we fine mapped and cloned the Rht8 gene that is important for plant height reduction in wheat,revealed that Rht8 regulated bioactive gibberellin content and thus reduced plant height,and demonstrated that Rht8 evolved after hexaploidization and was positively selected during modern wheat breeding.Besides,we identified 7 stable QTLs for plant height and spikelet density,and cloned Rht8-B1,the target gene of qPH2B.1 with minor effect on height reduction.This work deepens our understanding on genetic regulation of plant height and facilitates development of novel wheat varieties with low plant height and high lodging resistance.