Cloning TaTGW6 And TaTGW-7A Associated With Grain Weight And Developping Functional Markers In Common Wheat (Triticum Aestivum L.)

Wheat is one of the three major crops in China,and the production is closely related to food security in China.The grain weight of wheat is primarily determined by grain size and grain filling,is one of the three elements of wheat yield components.The sustained improvement of grain yield is one of main targets of wheat breeding.Identifying genes and developing functional markers associated with grain weight using molecular molecular biology play an important role during multi-gene aggregation breeding and study of genetic mechanism.However,Wheat is allohexaploid with complex chromosome structure,high repeat sequences and possesses a huge genome,making direct map-based cloning difficult.Therefore comparative genomics and high throughput sequencing technology to clone functional genes is a simple and efficient way.In rice,the OsTGW6 gene determining grain weight was selected as a candidate gene to clone ortholog of wheat,TaTGW6.By using BSA combining with SLAF-seq approach,TaTGW-7A was also isolated in this research.Some functional markers were developed according to the relationship of their variations with grain weight.The markers function was analyzed and verified using including recombined inbred lines?RIL,Jing 411×Hongmangchun 21?,natural population and Chinese wheat mini-core collections of wheat.In addition,the relationship of expression level of TaTGW6 and TaTGW-7A with IAA content and grain weight was also analyzed.The results in this research were as followed:1.TaTGW6 located on chromosome 4AL was closely linked to Xbarc1047 with 6.62 cM distence.The gene only contained one exon,encoding a protein with indole-3-acetic acid?IAA?-glucose hydrolase activity.The TaTGW6 coding sequence was 71 % identical to that of OsTGW6 cDNA of rice,and the deduced amino acid sequences was found to be 68 % identical between wheat and rice.Several homologous genes were founded in common wheat,mainly distributed in 2,3,4,7 homogenous groups.2.Three alleles of TaTGW6,TaTGW6-a?TaTGW6-b and TaTGW6-c,were detected between high-and low-grain-weight varieties.TaTGW6-b and TaTGW6-c were found to associate with higher grain weight,compared with TaTGW6-a.A 6 bp-InDel was identified between the two alleles,TaTGW6-a and TaTGW6-b,resulting in two amino acid insertion,but no frameshift mutation.The TaTGW6-c was a null allele.3.The function of TaTGW6 on grain weight was verified using a RIL,nature population and Chinese mini-core collections of wheat.The results showed that TaTGW6,like OsTGW6 of rice,was tightly associated with thousand grain weight?TGW?.TaTGW6-b and TaTGW6-c had positive correlation with TGW,but TaTGW6-a negatively affected TGW.The TaTGW6 locus could explain 15.8-21.0% of phenotypic variation of TGW in RIL population in four environments.The association analysis indicated that 7.7-12.4% phenotypic variations of TGW were estimated by the gene in nature population and Chinese mini-core collections.The positive alleles for TGW?TaTGW6-b and TaTGW6-c?showed lower distribution in Chinese wheat growing regions,relative to TaTGW6-a so that they had important effect on improving grain weight during wheat breeding.4.The significant difference in relative expression was detected in immature grains sampled from 10,20,30 days post anthesis?DPA?and mature grains between TaTGW6-a and TaTGW6-b.The further analysis showed that TaTGW6-a had higher expression level,compared with TaTGW6-b.The higher IAA content in immature grains from 20 and 30 DPA was found in these varieties possessing TaTGW6-a.5.The specific-locus amplified fragment sequencing?SLAF-seq?and bulked segregant analysis?BSA?were performed by high-throughput sequencing technology in the two bulks along with two parents with distinct difference in grain weight.Twenty-five polymorphic SLAFs were predicted on chromosome 2AS,2DL,7AS,and 7BL,and possibly related to TGW by association and gene-enrichment analysis.The 25 polymorphic SLAFs were further extended about 50 Kb forward and reverse for functional annotation and gene/transcript set/pathway enrichment analysis respectively,a total of 169 genes were obtained and possibly related to TGW.In particular,a putative gene?Traes₇AS₃78A12AA9.1?with a TIM barrel fold related to indole-3-glycerol-phosphate synthase?IGPS?was significantly associated with TGW,and thus suggested as a candidate gene underlying QTL region on chromosome 7AS,designed as TaTGW-7A.6.Sequence analysis revealed the TaTGW-7A gene contained nine exons and eight introns.Comparative analysis showed that the four single-nucleotide substitutions of TaTGW-7A were found in varieties with significant difference in TGW.Only one single nucleotide mutation in the first exon of the TaTGW-7A gene was identified constantly between the high-and the low-TGW phenotypes,resulting in an alanine codon?TGC?in the low-TGW phenotype changing to a valine codon?TGA?in the high-TGW phenotype.A cleaved amplified polymorphism sequence?CAPS?marker?TGW7?was developed to distinguish the two alleles,TaTGW-7Aa and TaTGW-7Ab associated with grain weight.The CAPS marker showed good polymorphism in RIL,nature population and Chinese mini-core collections.We further extended and sequenced the downstream of 7AS₂00₄248784?15Kb?about lengths of 3 Kb,and an InDel was detected between Jing411 and Hongmangchun 21 allele.An InDel marker TG9 nearly adjacent to TGW7 was also a stable and reliable codominant marker.7.Both the association and linkage analysis indicated that TaTGW-7A was closely related to TGW,grain length and width.TaTGW-7Aa could significantly increase grain weight,compared with TaTGW-7Ab.21.7-27.1% of phenotypic variations of TGW were explained by TaTGW-7A locus in RIL under 5 environments.7.3-12.3% of phenotypic variations of TGW were also explained in nature population and Chinese mini-core collections under 4 environments.8.Five hundred and one wheat varieties,as well as 180 accessions from Chinese wheat mini-core collections were used for testing the geographic distribution of TaTGW-7A alleles.The results indicated that TaTGW-7Aa showed higher frequency in Chinese main wheat growing regions,compared with TaTGW-7Aa;however in Chinese mini-core collections,the allele showed lower frequency in the Northern Winter Wheat Region and Southwestern Winter Wheat Region.The distribution characteristic revealed the preferred TaTGW-7Aa has undergo strongly positive selection in breeding processes.9.The expression profile of TaTGW-7A was analyzed in immature grains from 10,20,30 DPA and mature grains.The results revealed TaTGW-7Aa had higher expression level in grains from 20 and 30 DPA,compared with TaTGW-7Ab.On the contrary,IAA content of grain from 20 and DPA of varieties possessing TaTGW-7Aa was significant lower than that of TaTGW-7Ab.The result showed high level IAA in grain at late stage of grain filling was negative for improvement of grain weight.10.By the technology of SLAF-BSA,TaTGW-2A and TaTGW-2D,located on 2A and 2D respectively,were cloned and significantly associated with thousand grain weight.11.In this study,six genes had been reported associated with the grain weight,in addition to TaCWI-4A was significantly related to grain weight,the other five genes were extremely significant associated with grain weight.The order of six genes for grain weight was TaGS5-3A> TaTGW-7A> TaGS1a> TaCWI-5D> TaGW2> TaCWI-4A.There were a reciprocal effect between TaGW2 and TaGS5-3A,as well as TaGS1 a and TaTGW-7A.There was a significant interaction between TaGW2 and TaGS5-3A,and a relatively weak interaction effect between TaGS1 a and TaTGW-7A.