| ABSTRACT Leaves are the principal photosynthetic organs of the plant,which are essential to the development of plant architecture and yield potential.The morphology of flag leaf closely related to yield components in common wheat.The leaf length,leaf width and leaf area have significantly positive correlations with kernel weight per spike.Thus,increasing the area of flag leaf will benefit the yield improvement.Moreover,flag leaf length,width,area and leaf angle also influence the reception of sunlight,and its photosynthetic characteristics determines the yield potential of common wheat in a large extent.Therefore,investigations on variation and its trend of flag leaf morphology of Chinese germplasms,and the genetic loci and candidate genes for flag leaf size,will facilitate the genetic improvement of flag leaf in common wheat.1.Variation trends on flag leaf morphology in Chinese wheat germplasmsIn 2014-2015 growing season,1500 wheat lines,including landraces and varieties released before or after the year of 2000 were grown in Beijing and Zhaoxian County,Hebei Province,and their flag leaf traits were measured with the Yaxin-1241 leaf meter at one week after anthesis.The data shows that the flag leaf areas range from 1306mm2 to 5483mm2,leaf length from 121 mm to 327 mm,and leaf width from 8.6 mm to 24.0 mm.In the landraces,the means of leaf area,length and width are 2528 mm2,240mm and 13.7 mm,respectively.In the varieties released before 2000,the average leaf length decreased to 220 mm,while the average leaf width increased to 16.0 mm,which resulted in an increase of the leaf area to 2671 mm2.In the varieties released after 2000,the trends of leaf-length decreasing and leaf-width increasing are preserved.These findings revealed that the newly bred varieties had shorter but wider flag leaves to ensure a larger leaf size for photosynthesis,indicating the vital role of the flag leaf width on yield improvement in common wheat.Further analysis provided the highly significant correlations between the flag leaf width and yield related components,positive correlations between the width and 1000 kernel weight(r = 0.345***),grain width(r=0.391***)and average kernel number(r=0.456***),but negative correlation with panicle number per plant(r=-0.440***),indicating that the synchronized selection of leaf width with the improvement of kernel weight and kernel number in wheat breeding.Moreover,the correlation analysis revealed lager effect of environments on flag leaf area and length,but the flag width was relatively stable between two locations.2.Genetic loci for flag leaf morphology in Chinese wheat germplasmsTo dissect the genetic control of the variations on flag leaf morphology,a natural population,including 342 wheat varieties/lines released in Huanghuai and Northern winter wheat region,was grown in Zhaoxian Country and Hengshui City in Hebei Province in 2014-2015 growing season.The flag leaf traits were screened at one week after anthesis,and the genotypes were collected with wheat660K SNP chip.The population structure analysis shows that the whole population could be divided into 7 sub-groups by the structure v2.3.4 software.The genomewide association study(GWAS)was performed with 383,780 polymorphic markers and flag leaf traits using TASSEL v5.2.2.The preliminary data showed that sixteen chromosome segments(1A,1B,2A,2B,3A,3B,3D,4A,4B,5A,5D,6A,6B,7A,7B and 7D)and forty genetic locus were associated with the variations of flag leaf traits.Among them AX-109920673(2B),AX-108806630(3B),AX-109688668(3D),AX-108916381(4A),AX-110537692(7A),AX-109372256(7A),AX-111235579(7A)and AX-110878687(7D)were significantly associated with leaf length and leaf area,while AX-95628815,AX-94909932,AX-111616054 and AX-111648402 markers on 5A significantly affected leaf width and leaf area.The variation of flag leaf morphology was mainly controlled by the genetic loci,though the environmental impact was observed.To establish efficient molecular markers and clone the genes will facilitate the improvement on flag leaf morphology and wheat breeding.3.The functional analysis of candidate gene conferring to flag leaf widthOsNALl gene has been proven to control the flag leaf width,tiller number,and panicle number in rice.Using comparative genomics,TaNALl,homologous to OsNAL1 gene is located on chromosome 5A,which harbor associated markers with flag leaf width in our GWAS data.In this study,using rice OsNAL1 gene as the reference sequence,three TaNAL1 sequences were cloned from Chinese Spring,which were located on chromosomes 5A,5B and 5D,and designated as TaNAL1-5A,TaNAL1-SB,and TaNAL1-5D respectively.Wheat TaNALl-5 gene is composed of five exons and four introns,with a 1803bp ORF in TaNAL1-5A and TaNAL1-5B,and a 1796bp ORF in TaNAL1-5D.This gene was cloned from 10 common wheat varieties to mine their allelic variations.Two haplotypes(Hap-A1 with 19bp insertion,and Hap-A2)of TaNAL1-5A were detected based on 9 SNPs and one 19bp Indel in the coding region,and 6 SNPs and a 24bp Indel in the promoter region,but TaNALl-5B/D were highly conserved in the analyzed samples.The SNPs in Hap-A1 and Hap-A2 of TaNAL1-5A were confirmed with cDNA sequences in different wheat varieties,which included a M-T transition at the 473rd amino acids and a H-P transition at the 480th amino acids.In order to clarify the relationship between the haplotypes and flag leaf morphology and other agrornomic traits,a functional marker,NA1F/NA1R was developed based on the 19bp Indel between Hap-A1 and Hap-A2,and this marker was used to screen a natural population composed of 115 accessions of common wheat.The data showed that the average leaf width of 55 Hap-A1 accessions was 14.7 mm,while that of 60 Hap-A2 accessions was 18.1mm,which was significantly higher than that of Hap-A1 type.Furthermore,1000 grain weight and spikelet number was also significantly higher in Hap-A2,indicating its positive role in the development of flag leaf and yield components.To elucidate its functional mechanism of different allelic variations,the expression patterns were investigated on different tissues of Chinese Spring and several Hap-Al and Hap-A2 accessions.TaNALl-5 was expressed in all the analyzed tissues of Chinese Spring with highest expression level in the leaves and stems.TaNAL1-5 was more abundant in Hap-A2 than Hap-A1,which demonstrated that the expression level of TaNAL1-5 contributed to the development of wider flag leaf.Phenotypic variations on wheat flag leaves were investigated in this study.Modern varieties were found to have shorter but wider flag leaves,and the increase of leaf width was along with the improvement of kernel number and 1000 kernel weight.The association analysis identified forty genetic loci associated with the variation of flag leaf traits.A leaf width related gene,TaNAL1-5,located in the associated region in the GWAS data,was analyzed in details.All the findings in this work will facilitate the oriental improvement of flag leaf and yield potential in common wheat. |
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