Mapping And Cloning Of Qbr.sau-5A:A Major Locus For Brittle Rachis In Tibetan Semi-wild Wheat

Tibetan semi-wild wheat(Triticum aestivum ssp.tibetanum Shao)is a unique subspecies of the hexaploid wheat in China.Its typical primitive and classified characters are spontaneous spike disarticulation(brittle rachis)and tough glume(Non-free-threshing).Brittle rachis and tough glume(Non-free-threshing)are two of most important domestication traits during wheat domestication.The identification of such genes in Tibetan semi-wild wheat not only can provide new insights on the understanding of the genetic basis of domestication traits,but also contribute to reveal the origin and evolution of Tibetan semi-wild wheat.Previous genetic studies have shown that the phenotype of brittle rachis and tough glume(Non-free-threshing)likely are controlled by multiple major genes.However,due to its technical limits in previous studies,none of genes has been isolated and cloned.In this study,we identified the major loci controlling brittle rachis and tough glume from Tibetan semi-wild wheat.And then,a major brittle rachis gene on 5AL was isolated based on map based cloning.The key mutation,origin and distribution of the gene were identified and analyzed.It has revealed that Tibetan semi-wild wheat is originated from common wheat during de-domestication.Here,our results not only provided a new insight on evolution and classification of common wheat,but also showed a unique case for understanding on the genetic basis of crop de-domestication.The main results are as follow:1.A whole-genome linkage map of T.aestivum ssp.tibetanum was constructed using a recombinant inbred line population(Q1028×ZM9023)with 186 lines,564 diversity array technology markers,and 117 simple sequence repeat markers.Phenotypic data on brittle rachis and threshability,as two quantitative traits,were evaluated on the basis of the number of average spike rachis fragments per spike and percent threshability in 2012 and2013,respectively.Quantitative trait locus(QTL)mapping performed using inclusive composite interval mapping analysis clearly identified four QTLs for brittle rachis and three QTLs for threshability.However,three loci on 2DS,2DL,and 5AL showed pleiotropism for brittle rachis and threshability;they respectively explained 5.3%,18.6%,and 18.6%of phenotypic variation for brittle rachis and 17.4%,13.2%,and 35.2%of phenotypic variation for threshability.A locus on 3DS showed an independent effect on brittle rachis,which explained 38.7%of the phenotypic variation.The loci on 2DS and3DS probably represented the effect of Tg1²DD and Br1^3D,respectively.The locus on 2DL has never been reported in common wheat but was prominent in Tibetan semi-wild wheat Q1028.The locus on 5AL was in very close proximity to the Q gene,but the sequencing of miRNA172 target region showed that the Q gene of Tibetan semi-wild wheat was same as domesticated type and different from the predicted q.2.Using the near isogenic lines(NILs),we successfully isolated a major brittle rachis and threshability locus(Qbr.sau-5A)on 5AL chromosome from polygenic genetic background in Q1028.The NILs clearly showed that Qbr.sau-5A has a pleiotropism for rachis fragility,threshability,spike length and spike density.Microscopic observation revealed the cytological mechanism of Qbr.sau-5A related to brittle rachis,resulting in the lower degree of tenacity in brittle type of spikes.And then,using a NILs derived population including 4810 lines and newly designed markers,Qbr.sau-5A was mapped within 0.04 cM genetic distance or 34 kb physical distance.Among of the four predicted genes,Q gene is most likely to underlie the locus.3.The results of molecular cloning showed that the Q allele of Q1028(Q^t)have a161-bp transposon insertion within exon 5.RT-PCR and qRT-PCR shown Q^t was normally expressed at the RNA,but Q^t encodes a deduced peptide with 318 residues(only 211residues same as Q protein),and lack some key features of APETALA2 family of transcription factors leading to lose the normal ability of interaction with other proteins.Further,by mutation comparison and transgenic overexpression,we confirmed that Q^t re-acquired the wild ability of brittle rachis by loss of function in Q.4.The Q gene region(147 kb)of 22 wheat accessions was analyzed by target-region capture sequencing,and the identified SNPs were used to construct a neighbor joining tree.It showed that Q^t should be originated from the mutation of Q in hexaploid wheat.Subsequently,the genetyping of 282 accessions of brittle rachis hexaploid wheat clearly showed Q^t only distributed in Tibetan semi-wild wheat population and had very high distribution frequency(about 69%).Geographical distribution analysis showed that the distribution frequency of Q^t is closely related to the geographical and climatic environment.The severe environment likely promoted the positive selection of Q^t,and the good environment for agricultural production might inhibite this selection.