| Wheat(Triticum aestivum L.)is one of the most important food crops,and is one of important sources of protein and microelements for human.Grain quality has become an important goal for wheat breeding.Studies have shown that the NAM gene plays an important role in the transfer of wheat nitrogen from nutrient organs to grains during the grouting process,thereby promoting the accumulation of proteins and trace elements such as zinc and iron in grains,which in turn affects the grain quality of wheat.At the same time,the NAM also affects the expression of senescence related enzymes during wheat grouting,which affects the aging process of wheat.Therefore,the research of NAM gene in wheat varieties has been paid more and more attention,but so far the wheat varieties in the middle and lower reaches of the Yangtze River have not been involved.In this study,we used totally 238 wheat varieties and 11 emmer wheats form both China and abroad to identify the genotype of NAM-B1.Further,NAM-B1 homology genes including NAM-A1,NAM-D1,NAM-A2,NAM-B2 and NAM-D2 were cloned using gene specific primers in 32 wheats to screen SNP sites in their sequences.On this basis,the haplotype of NAM gene was studied in 112 wheat varieties in the middle and lower reaches of the Yangtze River.The correlation between SNP-site and grain related traits was analyzed,and the SNP-site related to grain traits was discovered.Six different haplotype wheat materials were selected to compare the nitrogen metabolism and senescence characteristics during grain development.The main findings of this study are as follows:1.Among 238 wheat cultivars,136 wheats lose NAM-B1 gene sequences,accounting for 57.2%of the total wheats;91 wheats contain mutated NAM-B1 gene sequences,accounting for 38.2%of the total wheats;11 wheats have the functional NAM-B1 gene,accounting for about 4.6%of the total wheats.In the 192 wheat cultivars that originated from China,122 wheats lose NAM-B1 gene sequences,occupied by 63.5%,70 wheats contain mutated NAM-B1 gene sequences,occupied by 36.5%,and no wheat were found to have the functional NAM-B1 gene.In 11 emmer wheats,only 3 wild emmer wheats contain functional NAM-B1 gene,other 5 including 4 wild emmer wheats and 1 cultivated emmer wheat contain mutated NAM-B1 gene,and the left 3 cultivated emmer wheats lose NAM-B1 gene.Multiple sequences alignments indicateds 2 SNP sites exist in the mutated and functional NAM-B1 gene sequences and linkage heredity is found between the 2 SNP sites.2.Sequence cloning of NAM-A1,NAM-D1,NAM-A2,NAM-B2 and NAM-D2 in 32 wheat cultivars indicated that NAM-D1,NAM-B2 and NAM-D2 sequences in 32 wheats are identical and no SNP sites were found.Wheras 2 and 1 SNP sites were found in NAM-A1 and NAM-A2,respectively.In NAM-A1 gene sequence,both of 2 SNP sites locates in exons of 722 bp(C/T)and 1509bp(A/-)from starting codon.The former SNP site causes amino acid changes from Ala to Val,and the second SNP site result in frameshift mutation and lead to a truncated protein.The only 1 SNP site of NAM-A2 locates in exon of 110bp(C/G)from starting codon and result in amino acid changes from Pro to Arg.Afterwards,97 copies of wheat materials were added to NAM-A1 for KASP classification identification and NAM-A2 gene was positively sequenced.A total of 14 haplotype combinations were found in 129 materials identifying the gene.3.The identification of NAM genotypes of 112 wheat materials in the middle and lower reaches of the Changjiang River contained a total of 13 haplotype combinations,of which 4 haplotypes had a probability of appearing in 112 wheat materials of less than 1%.Therefore,the other 9 haplotype combinations were associated with grain protein content,grain hardness,grain diameter,thousands of grains weight,wet gluten content,and stability time.The SNP1 point on NAM-A1 is related to the grain protein content,and the N9 haplotype combined grain protein content is the optimal haplotype combination.The SNP1 and SNP2 point on NAM-Al and the absence of NAM-B1 fragments are related to the grain hardness,and the N7 haplotype combined grain hardness is the largest.The absence of the SNP1 bit on the NAM-A1 and the loss of the NAM-B1 fragment are all related to the grain diameter,and the N7 haplotype combined grain diameter is the optimal haplotype combination.Whether the NAM-B1 fragment is missing is related to the thousand-grain weight and the N6 haplotype combination of thousand-particle weight is the optimal haplotype combination.The SNP-1 point on NAM-A1 is associated with the wet gluten content and the N9 haplotype combination wet gluten content is the optimal haplotype combination.The SNP2 bit on the NAM-A1 is associated with the stabilization time and the N9 haplotype combination has the longest stabilization time.4.Variation of nitrogen concentration during grain-filling stage,and nitrogen and yield related indexes were analyzed in 6 different genotype wheat cultivars.Results indicated that nitrogen concentration in stem and leaves decreased with the process of grain-filling stage,but differences exist in different wheat backgrounds,especially between different grain protein content wheat.Compared to differnet grain protein content wheat showed earlier nitrogen remobilization and have stronger nitrogen remobilization ability,which result in more nitrogen accumulate in grain.Different from Ningmai 13,Zhenmai 8,Shengkang 1,Emai 25 and Xiangmai 25,nitrogen concentration in leaves of Yangmai 15 at harvest stage was higher than that of 30 day after anthesis.For wheat material with relatively close nitrogen content and protein content of grains Ningmai 9 and Yangmai 15,the similar low nitrogen harvest index and significant differences in harvest index enable us to speculate that the lower nitrogen remobilization ability in Ningmai 9 and Yangmai 15 may result from different mechnisms.Nitrogen concentration in grain was found highest at flowering stage and decreased later in grain-filling stage indicated the synthetical effects of nitrogen and photosynthetic products in transportation to grain.In analysis of yields of 6 wheat cultivars,seeds number per panicle and seed weight palys more important roles than panicle number in Yangmai 15 yield formation.5.The dynamic variation of soluble protein and sensily-related enzyme activity in the leaves of the flag of Ningmai 13,Yangma15,Zhenmai 8,Shengkang 1,Emai 25 and Xiangmai 25 during grain-filling stage were analyzed.It was found that the content of soluble protein and SOD activity decreased with the grain-filling stage and the single peak of POD and CAT activity increased first and then decreased.MDA activity gradually increased,and the later stage of the increase accelerated.The soluble protein content of the leaves of Ningmai 13,Shengkang 1,and Yangmai 15 dropped rapidly 10 days after flowering,and the SOD activity increased at 10 days.It may be that plant at this time have many oxide radicals that need SOD and POD to resist together.The changes of SOD and CAT activity in Zhenmai 8 during grouting process were small.The soluble protein content and CAT activity in the leaves of Ningmai 13 and Yangmai 15 were higher than those of other wheat materials 30 days after flowering,indicating that Ningmai 13 and Yangmai 15 had a longer grouting period.According to the records of the grouting period,Ningmai 13 and Yangmai 15 are about 1-4 days long for other wheat materials.The SOD activity decreased and the POD activity increased of Xiangmai 25.It maybe that photosynthesis and nitrogen transport were sufficient in the early stages of grouting.The decrease in SOD activity increased the POD activity to maintain normal metabolic activity of the cells.The soluble protein of Yangmai 15 in the flag leaf decreased significantly at 10 days after flowering,and SOD activity was higher than that in anthesis.It is possible that at this stage the plant cell accumulates a large amount of oxide free radicals that need POD and SOD to work together to maintain balance. |
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