Fine-mapping Of Brittle Rachis Locus Qbr.sau-2DL From Tibetan Semi-wild Wheat And A Preliminary Study On Its Mechanism

Tibetan semi-wild wheat（Triticum aestivum ssp.tibetanum Shao）was originated by de-domestication of common wheat.It is a unique wheat germplasm in China and has broad prospects in identifying valuable genes and in wheat breeding.Brittle rachis is one of the most critical traits in wheat domestication and de-domestication,which directly affects the continuation of crop generations in nature.It was found that Tibetan semi-wild wheat accession“Q1028”contains three main brittle rachis loci,i.e.Qbr.sau-2DL,Qbr.sau-3D and Qbr.sau-5A.In this study,to fine map Qbr.sau-2DL,the monogenic line“QZ110”（only containing Qbr.sau-2DL,derived from“Q1028”×commmon wheat cultivar“Zhengmai”9023）was crossed with common wheat cultivar“Shumai482”to construct F₂,F_2:3and F₃population.Moreover,the preliminary mechanisms of Qbr.sau-2DL on brittle rachis was investigated by using cytological and transcriptome analysis.The main findings are as follows:1.Brittle rachis occurred as early as the heading stage（GS57）.“QZ110”（brittle rachis）and“Shumai 482”（non-brittle rachis）were sampled from the booting stage（GS49）to the yellow ripening stage（GS92）.It was found that their brittle rachis rates began to show differences at GS49,and reached extremely significant at GS57,and then increased stably until the yellow ripening stage（GS87）.The results demonstrated that Qbr.sau-2DL functions earlier than GS49 and continuously enhanced the brittleness before GS87.2.Fine mapping of Qbr.sau-2DL.Using Bulked Segregant Exome Capture Sequencing（BSE-Seq）data of 15 brittle rachis wheat plants and 15 non brittle rachis individuals of F₂,Kompetitive Allele Specific PCR（KASP）markers were developed between the 570 Mb to 620 Mb region on chromosome 2D.A total of 226 F₂ individuals were used for genotyping.Qbr.sau-2DL was initially located between the markers K-55 and K-69 within the 594.5 Mb to 604.1 Mb region of 2D chromosome of Chinese Spring RefSeqv1.1.Based on the Next-generation sequencing（NGS）data of Tibetan semi-wild wheat Q1028,Single Nucleotide Polymorphisms（SNP）within the initial mapping interval were identified,and KASP markers were designed to genotype the F_2:3 and F₃ population.Subsequently,Qbr.sau-2DL was located between the KASP markers K-18 and K-24 within the 595.9 Mb to 596.7 Mb（836.4 kb in total）.3.Prediction of candidate genes.According to the genome sequencing data of Chinese Spring RefSeq v1.1,there were 12 genes between the 836.4 kb interval.It was found that Treas CS2D02G502900 was the homologous gene of OsLG1 affecting rice shattering,and was highly expressed in wheat panicles,when searching the gene annotation information and wheat gene expression database.Quantitative real-time PCR analysis showed that the relative expression level of Treas CS2D02G502900 was significantly higher in the developing spikes of“QZ110”than in that of“Shumai482”.However,through the comparison of genomic data between Tibetan semi-wild wheat“Q1028”and“Chinese Spring”,it was observed that the coding regions of the remaining 11 genes remained unchanged.Therefore,Treas CS2D02G502900 was selected as the candidate gene for Qbr.sau-2DL.4.Cytological mechanism of Qbr.sau-2DL on brittle rachis.Scanning electron microscope（SEM）was used to observe the section of the rachis at GS87.To observe the region where the brittleness occurred（the separation layer）,cytological experiments such as semi-thin sections of Abscission Zone（AZ）and freehand sections were performed.It showed that the volume of the cells in this region of“QZ110”was small,and the secondary cell wall was thin,and the lignin content of the fiber cells near the spikelet was decreased,which led to brittle rachis.The cytological mechanism of brittle rachis of Qbr.sau-2DL is similar but still different from that of Btr1 and Btr2 in barley.5.Molecualr pathways of Qbr.sau-2DL in regulating brittle rachis.Comparison of the transcriptome of AZ region of"QZ110”and“Shumai482”showed that a large number of genes related to cell wall synthesis were down-regulated in“QZ110”.The expression level of genes related to cellulose,xylan and fasciclin-like arabinogalactan protein synthesis in the secondary cell wall was decreased,which led to thinning of the secondary cell wall.At the same time,downregulation of the expansin genes in the cell wall may result in the inability of the AZ cells to elongate sufficiently,resulting in a reduced cell size.Under these regulatory pathways,the cell wall of the AZ cells becomes thinner and the cell volume becomes smaller,which eventually leads to brittle rachis in“QZ110”.