Haplotype And Promoter Analysis Of TaSAP1Gene From Wheat (Triticum Aestivum L.)

Drought, salinity and low temperature are the major abiotic stresses that dramatically threaten thefood supply in the world. Wheat is one of the most important staple food crops in the world, drought isconsidered to be the major limiting factor seriously impacting the wheat production. Exploringdrought-response gene resources in germplasm is a fundamental approach for improving droughttolerance in wheat. There are abundant allelic variations in wheat germplasm. Identifying thesefunctional alleles for drought tolerance will help to further understand the drought-resistancemechanisms. Functional markers can also be developed based on the identified functional variations,which provide the basis for marker-assisted selection for drought improvement. Association mappingpossesses the advantages of higher mapping resolution and shorter research time, therefore it isconsideredas a powerful approach for identifying functional alleles in germplasm resources.In this study, TaSAP1gene was chosen as the candidate gene to perform two aspects of research.(1)Genomic region flanking TaSAP1was obtained by a BLASTsearch against the Aegilops tauschii draftgenome sequence. Thirty wheat accessions were selectedfor polymorphism analysis.Based on thevariations in promoter region of TaSAP1-A1, molecular markers were developed for genotyping.Association analysis were performed to identifyfunctional alleles and haplotypes;(2) The function ofTaSAP1-A2was preliminarily studied by using transient expression and stable expression. The mainresults are summarized as follows:1.Two fragments, designated as TaSAP1-A1(5.1kb)and TaSAP1-A2(4.1kb), were obtained by aBLAST search against the Aegilops tauschii draft genome sequence. Both of them had a coding regionof510bp. Sequence analysis indicated that the difference between TaSAP1-A1and TaSAP1-A2wasmainly located in the promoter region.Thesequences were3.4kb and2.5kbrespectively. Alignment ofthe cDNA andgenomic DNA sequence revealed two introns in the5′UTR of TaSAP1. Intron-1was153bp in length and located between-967bp and-814bp from ATG, and intron-2was758bp locatedbetween-771bp and-13bp.2.Forty-six variants in the entire region of TaSAP1-A1, comprising of43SNPs and3indels, wereidentified among the30accessions. These include39polymorphisms in thepromoter region, two SNPsin intron-2in the5′UTR, and five variants, including four SNPs and one indel, in the3′flanking region.No variation was observed in the coding region. Sliding-window analysis indicated the highest variationoccurred in the upstream promoter region(-3,499bp to-1,498bp)where the nucleotide diversitywas0.00631, followed by the3′flanking region(π=0.00133) and intron-2(π=0.00100).3.Based on three polymorphic sites in the promoter region, including InDel5-1810, SNP-2606andInDel39-1637, three marker T7AM5, T7AM2606and T7AM39were developed. T7AM5andT7AM2606were CAPS markers, and T7AM39was an allele-specific PCR marker. TaSAP1-A1wasmapped to a region flanked by Xwmc530(2.1cM) and Xbarc174(13.9cM) on chromosome7A usingmarker T7AM39. A total of six haplotypes (HapI-HapVI) were detected among the300wheataccessions based on the three markers. Among them, HapI and HapII were the main haplotypes, accounting for45.3%and31.5%of accessionsrespectively. The proportion of HapVI was the lowest(2.9%).4.Marker-trait association indicated T7AM5, T7AM2606and T7AM39were significantlyassociated withfive traits including spike length, peduncle length, number of spikelets per spike, numberof grains per spike and1000-grain weight. T7AM5was significantly associated with spike length in fourenvironments among which three were drought-stressed. It was again significantly associated withpeduncle length in six environments, four of which were drought-stressed. T7AM39and T7AM2606were mainly associated with1000-grain weight. Moreover, haplotype significantly affected someagronomic traits, such as1000-grain weight, spike lengthand number of spikelets per spike. HapIIImade a positive contribution to TGW and SL, and the average TGW of HapIII was the highest in allenvironments with the pattern of HapIII>HapII>HapI.The frequency of HapIII was an increasingtrend during the breeding process,showed it was positivly selected. The geographical distribution ofHapIII in China indicated the use of HapIII was not limited byecological environments, such as lightand temperature.5.Sequence analysis revealed several putative stress-response cis-acting regulatory elements inTaSAP1-A2promoter, such as ABRE, MBS and ARE. Transient expression in wheat calli proved thatthepromoter of TaSAP1-A2was the stress-inducible promoter. Introns in5′UTR of TaSAP1-A2wereinvolved in stress-induction. Five5′deletions were evalued by stable expression in Arabidopsis. Nopromoter activity was detected in Arabidopsis for all the fragments, except for P6. Further studyindicated a fragment named P(5-6) inhibited the promoter activity of TaSAP1-A2. P6possessesconstitutive transcriptional activation, and its activity was comparable to that of CaMV35S. The activityof P-(5-6) could be enhanced by drought and low temperature. The results showed that P6and P-(5-6)promoters have potential value in genetic engineering of crop plants.