| (chl) is one of the most important physiological traits as it is closely related to leaf photosynthesis and crop yield potential. Chl metabolic pathway and series of genes involved in pathway has been well characterized and identified in Arabidopsis, which provide a good base to characterize chl related genes in maize. Among the known genes in pathway, Non-Yellow Coloring 1 (NYC1) gene which translates chlorophyll b reductase (CBR) plays an important role to adjust the ratio of chla (chlorophyll a) to chlb (chlorophyll b) which is an essential index for plant healthy growth, and to degrade chlb during leaf senescence. Candidate-gene based association mapping is study of the correlation between phenotypic variation and DNA polymorphisms, and it is very efficient to identify the new and useful alleles for plant improvement. The objective of this study is to investigate favorable polymorphisms of ZmNYC1 gene associated with chl content at seedling stage and mature stage under natural and induced conditions using association mapping.Material and Methods:An association mapping panel consisting of 150 maize (Zea mays L.) inbred lines was used in both laboratory and field experiments. Each inbred was prepared two replicate whereas six healthy seedlings represents one experimental unit, and grown under control condition. When the seedlings reached two-leaf-stage (V2), one replicate of each inbred was subjected to light treatment and the other was exported to darkness for four days. By using SPAD-502 chl meter, relative chl content was determined at the first and second leaves under light and dark treatments and at ear leaf after pollination with three time intervals. Besides, chl was extracted from the green part of three seedlings of each genotype and was analyzed by the method described by Arnon,1949. Genotyping of ZmNYC1 re-sequencing was conducted using the four pairs of primer for each inbred in panel. Association analysis was performed by using TASSEL software and confirmed the results via mRNA analysis.Results and Discussion:Natural variation in the chl content (SPAD values) at seedling stage and mature stage was examined over two-fold in the association panel. Meanwhile, the broader variation of the chl concentration (over ten-fold) in dark treatment than in light (three to seven-fold) at seedling stage exhibited the differential response of the natural maize population to dark. We therefore, identified some inbreds that were sensitive (over 70% chl degradation) or insensitive (below 40% chl degradation) to dark. On average, chl content of insensitive inbreds was over two-fold higher than sensitive inbreds in dark treatment. Through candidate-gene association analysis, it was detected that 19 favorable polymorphisms out of 105 polymorphic sites in ZmNYC1 gene were significantly associated with chl related traits in association panel. This result points out that a critical role of ZmNYCl gene and its polymorphisms affect chl content in maize. Polymorphisms such as S320, S463 and S2951 explained higher phenol typic variation ranging from 5.34% to 8.89% for chlb and chl ratio under light condition, whereas S3355 and S3901 exhibited phenotypic variations from 5.18% to 7.01% for chl_ratio, chla deg (chl a degradation) and total_chl deg (total chl degradation) under dark. This suggests that different gene regulatory network may exit for different traits in different environment. The three polymorphic sites at S463, S3355 and S3645 detected in sensitive and insensitive to dark individuals explained phenotypic variation ranging from 47% to 89% with 1- to 5.8-fold change between favorable and unfavorable alleles. The results indicate that strong validation on association of ZmNYC1 gene with chl content. Besides, expression differences in this two inbred groups also designate that the gene associated with chl content at the transcript level.Conclusion:Although, the natural variation in chl content at both seedling and mature stages exhibit similar trend in association panel, amount of chl in the leaf tissues varies with the age of the plants at different developmental stage. We filtered inbred lines that were sensitive or insensitive to dark treatment depending upon their differential response to dark evaluated by chl concentration at seedling stage. Polymorphisms in ZmNYC1 gene contributed more variation in chlb, chl ratio, and chl degradation; however different gene regulatory network may exit for different traits in different environment. The expression differences between two inbred groups (sensitive or insensitive to dark) suggest that ZmNYCl gene affect the phenotypic variation at the transcript level. The results of our study will not only support better understanding of genetic basic of chl, but also assist the molecular breeding of chl content through the identified polymorphisms. |
