Cloning Of The TaCRTISO Gene Modulating Kernel Length And Carotenoid Content In Common Wheat

Wheat is an essential cereal in the world’s food supply and accounts for 20%of the total calories in the diet.As the world’s population grows and arable land becomes scarcer,higher yields from limited arable land are required.Current breeding efforts are primarily aimed at improving both yield and nutritional value.Therefore,wheat breeders have placed a high priority on identifying and determining the functions of superior alleles linked to yield and quality traits.Kernel size is an important trait that contributes to yield potential.Carotenoids serve as the precursor of vitamin-A and several plant hormones that govern plant development and reaction to poor growth conditions.Accordingly,identifying candidate genes related to yield is urgently needed to meet the desired level of crop productivity.The present study identified the TaCRTISO-1B gene regulating wheat kernel size by map-based cloning and found its association with carotenoid content.We further revealed that TaCRTISO-1B strongly interacted with TaPAP6 involved in JA regulation.The main progress is summarized as:（1）Fine mapping of QKL.hau-1B:Linkage mapping in the RIL population UC1110/PI610750（UP）identified two stable QTLs associated with kernel length in common wheat.The QTL,QKL.hau-1B on chromosome 1B was selected for fine mapping.A secondary F₂subgroup containing 1200 individual plants was derived by crossing between two parents（UC1110 x PI610750）,for screening the recombinant individuals.The genome-resequencing data was used to develop 43 markers,and 11 of them showed polymorphism with the kernel length in the target 7.5 Mb region of QKL.hau-1B.By counting recombinant events of the closest flanking marker,the QKL.hau-1B was finally mapped between markers Ta90100 and Ta90400,covering two open reading frames,Traes CS1B02G090200 and Traes CS1B02G090300.（2）QKL.hau-1B candidate gene prediction and genetic validation:According to the co-segregation and gene expression analysis,Traes CS1B02G090300（TaCRTISO-1B）,which encodes carotenoid isomerase,was predicted as a candidate gene for QKL.hau-1B and was further validated by genotyping with the Ta90300 marker for QTL re-analysis and GWAS.（3）TaCRTISO-1B functional verification:EMS-Mutants in tetraploid wheat significantly reduced the kernel length,whereas TaCRTSIO-1B overexpression lines in hexaploid wheat greatly enhanced the kernel length and thousand kernel weight（TKW）.（4）Influence of TaCRTISO-1B on carotenoid content:To evaluate the pleiotropic effect of CRTISO,a linkage mapping for carotenoid content（CC）was carried out in the UP-RILs.A stable QTL QKL.hau-1B flanked by comparable markers w Pt-5279 and w Pt-1251 was identified on chromosome 1B.QTL re-analysis and GWAS verified TaCRTISO-1B functional maker Ta90300.To further ascertain the contribution of TaCRTISO-1B to carotenoid accumulation,we measured CC in transgenic overexpressed lines and EMS-Mutants.EMS-Mutants in tetraploid wheat showed significantly reduced carotenoid content,while TaCRTSIO-1B overexpression lines in hexaploid wheat significantly enhanced carotenoid content.（5）Mechanism of TaCRTISO-1B regulating kernel size and carotenoid content:The PAP/fibrillin＿dom protein（TaPAP6）was screened as strongly interacted with TaCRTISO-1B by yeast two-hybrid,and the interaction was further verified by the spilt luciferase system.Protoplast localization showed that both proteins were localized in the chloroplast.Since PAP6,as a member of the fibrillin family,was possibly involved in jasmonic acid（JA）regulation,the transcriptomes of TaPAP6-OE and WT were sequenced to illustrate the association of TaPAP6with JA.The RNA-Seq assay in TaPAP6-OE lines identified the five sharply upregulated genes involved in JA synthesis with three suppressed JAR1（jasmonic acid-amino synthetase）genes.The suppression of JAR1 by the improved expression of TaPAP6 could result in the accumulation of JA through the hindering of the degradation of JA into various JA conjugates.Measurement results showed that JA content in TaPAP6-OE lines was significantly increased by 36.23%compared with WT,whereas JA content in mutant lines was decreased by 27%.These results suggested that TaCRTISO positively regulates KL and CC,possibly because of improving TaPAP6 expression to repress JAR1 and thereby reducing degradation of JA.As JA levels rise,carotenoid content can rise as well,which contributes to the enhanced photosynthesis that could be a pool’s energy source for kernel size.（6）Domestication of TaCRTISO-1B in polyploid wheat:The Ta90300 marker identified two alleles,TaCRTISO-B1a and TaCRTISO-B1b,associated with KL and CC.The relatively superior allele TaCRTISO-B1b with the In Del possessed 4.16%,49.16%,and 71%in landraces,historical cultivars,and modern cultivars of Chinese wheat,respectively.It suggested that the superior allele TaCRTISO-B1b has been artificially domesticated in common wheat.（7）Identification in eight types of tetraploid wheat showed that the percentage of TaCRTISO-B1b allele is 67.85%in T.durum,32.72%in T.diciccon,29.26%in T.dicoccoides,16%in T.polonicum,7.40%in T.turgidum,6.66%in T.carthlicum,and 10%in T.turanicum.While TaCRTISO-B1b was not detected in T.timopheevii.T.durum possessed the significantly highest frequency of the TaCRTISO-B1b allele among eight types,suggesting that a preferential selection of TaCRTISO-B1b was performed in tetraploid wheat.