Difference Analysis Of Physiological Characteristics And Gene Expression During Senescence Between Different Stay-green Wheat Varieties (Lines) And QTL Mapping For Stay-green Traits

Wheat is very important food crop which human being lives on,and about 35 %-40 % of the population consumes it as a staple food all over the world.In recent years,with the change of climate,premature senescence of the wheat leaf,especially flag leaf,occurs frequently,which seriously affects the yield and quality of wheat.The stay-green wheat varieties could effectively delay the senescence of flag leaf after anthesis and prolong the duration of the effective photosynthetic time,which can provide more photosynthetic products for grain filling,and finally increase the wheat yield.At present,the researches on wheat leaf senescence mainly focus on physiological characteristics,while the molecular mechanism is relatively rare.In this study,difference analysis of physiological characteristics and gene expression during senescence between different stay-green wheat varieties(lines)were performed using non-stay-green wheat varieties,Jinmai 39 and Hanxuan 3 hao,and stay-green wheat cultivars(lines)Tailv 113 and Yannong19.And QTLs for stay-green traits were mapped using two BC3F3 introgression line(IL)popilations,Lumai 14 / Jing 411 and Lumai 14 / Shaanhan 8675.The mian results are as follows:(1)Yield-related traits of two kinds of wheat varieties were analyzed and found that thousand-grain weight,grain yield and harvest index of both stay-green wheat varieties were higher than those of both nonstay-green wheat varieties.(2)The chlorophyll content,super oxide dismutase(SOD)activity,peroxidase(POD)activity,soluble protein content,the malondialdehyde(MDA)content,photosynthetic parameters and chlorophyll fluorescence parameters of flag leaf at 0 day,10,18,22,26,30 and 34 days after anthesis in two kinds of wheat cultivars were analyzed.The results showed that the onset of senescence was at about 26 days after anthesis in both kinds of wheat varieties.During senescence,the rate of senescence and MDA content of stay-green wheat varieties(lines)were always lower than those of non-stay-green wheat varieties.While,the chlorophyll content,net photosynthetic rate(Pn),the actual photochemical efficiency of photosystem ?(Y(?)),the rate of photosynthetic electron transport(ETR),antioxidant enzyme activity and soluble protein content of flag leaf were always higher than those of non-stay-green wheat varieties.It showed that the staygreen wheat varieties under protective enzyme systems,such as SOD and POD,effectively removed ROS and protected the photosynthetic organs in the late stage of senescence.Compare to non-stay-green wheat varieties,the flag leaves in the stay-green wheat varieties maintain higher photosynthetic capacity by higher chlorophyll content in the late stage of senescence,which provided a reasonable physiological basis in terms of superior yield-related traits among stay-green wheat varieties(lines).(3)High-throughput transcriptome sequencing was performed on flag leaves at 0 day,26 and 30 days after anthesis in non-stay-green wheat variety Jinmai 39 and stay-green wheat line Tailv 113.A total of 20513 and 5762 differentially expressed genes(DEGs)were detected in 26 d vs 0 d,30 d vs 0 d and 30 d vs 26 d from Jinmai 39 and Tailv 113,respectively.Among them,1655 and 1196 genes were up-regulated and down-regulated in the two wheat varieties(lines),respectively.600 DEGs were only detected from 26 days to 30 days after anthesis of Jinmai 39(in JM₃0 d vs JM₂6 d),among them,the genes involved in photosynthesis PS?,PS? and photosynthetic electron transport pathways were significantly down-regulated.While only 61 specific DEGs were identified from 26 days to 30 days after anthesis of Tailv 113(TI₃0 d vs TL₂6 d),of these DEGs,the genes encoding ATP synthase in photosynthesis were significantly downregulated.During leaf senescence in both wheat varieties(lines),the expressions of nine enzyme genes involved in chlorophyll biosynthetic pathway and genes(GID1 and IAA6)related to plant hormone signal transduction pathway and transcription factor NAC30 were down-regulated,but the expressions in non-staygreen wheat variety Jinmai 39 were lower than those in stay-green wheat line Tailv 113.Four enzyme genes related to chlorophyll degradation,including NOL,PPH,PAO and RCCR,and genes related to plant hormone signal transduction pathways,including AHP4,ARR3,ARR6,IAA30 and LAX1,and transcription factors encoding NAC100,NAC22,NAC29,MYB4,WRKY6 and WRKY75,were up-regulated,with higher expression levels in non-stay-green wheat variety Jinmai 39 than in stay-green wheat line Tailv 113.(4)Seedlings of both kinds of wheat varieties were treated with darkness,6-BA,ABA and ETH.And the expression levels of key enzyme genes for chlorophyll degradation,including Ta CLH1,Ta CLH2 and Ta PPH at 1 day,2,3 and 4 days after treatment and at 0 day,10,18,22,26,30 and 34 days after anthesis were determined.It showed that the expression levels of Ta CLH1 and Ta CLH2 were very low in both kinds of wheat varieties(lines),while,the expression level of Ta PPH increased during natural senescence and senescence induced by various treatments,and the expression level of Ta PPH of non-stay-green wheat varieties were significantly higher than that of stay-green wheat varieties(lines).(5)In the above DEGs detected,we found that the expression level of the transcript annotated as SAG39 was very low in leaves at 0 d after anthesis in different stay-green wheat varieties(lines),while high in senescence leaves,and the expression level of SAG39 in non-stay-green wheat variety Jinmai 39 was higher than that in stay-green wheat line Tailv 113,suggesting that SAG39 was related to the senescence of wheat leaves.The transcript was used as target sequence to align against wheat genome from URGI website,and the candidate sequences of Ta SAG39 of chromosome 5A,5B and 5D were obtained,respectively.According to the differences among three candidate genomic sequences,specific primers were designed to clone the gene Ta SAG39-5A,Ta SAG39-5B and Ta SAG39-5D,with the length of the coding region of 1041 bp,1050 bp and 1038 bp,respectively.The expression pattern of Ta SAG39 was detected by q RT-PCR,showing that the expression level of Ta SAG39 increased during natural senescence and senescence induced by different treatments.(6)QTL mapping for chlorophyll content at 0 day,15 and 20 days after anthesis,flag leaf length,flag leaf width and flag leaf area were conducted in 2016-2017 and 2017-2018.A total of 14 and 15 QTLs for stay-green traits were detected in Lumai14 / Jing411 and Lumai14 / Shaanhan8675,respectively.Among them,QCCS2-3A,QCCS3-1A,QFLL-4A and QFLL-6A were repeatedly identified under two environments.Besides,QFLW-6A and QFLA-6A were detected in Lumai14 / Jing411 under E2 and in Lumai14 / Shaanhan8675 under two environments.Genome-wide scanning and QTL mapping found that 18 lines and 44 lines from Lumai 14 / Jing 411 and Lumai14 / Shaanhan8675 contained QFLW-6A,respectively.Among them,the IL 124 from Lumai14 / Jing411 and the IL 59 and IL 127 from Lumai14 / Shaanhan8675 had no more than five donor fragments which carried no other QTL controlling flag leaf width(FLW)than QFLW-6A.On the other hand,the FLW of these lines were significantly greater than Lumai 14 under two environments.So,these lines and their donor parent can be regarded as potential near-isogenic lines.