| Soybean [Glycine max (L.) Merr.] is an important food and cash crop, but its yield is relatively low. High yield is the most important breeding objective, which can be achieved through improving yield and its component traits, or developing ideal plant type, etc. It can also be considered the cause of improving the efficiency of solar energy utilization. Leaf is the place for photosynthesis in plant through chlorophyll, and therefore understanding the law of growth and development of soybean leaf morphology will help to mine elite genes for breeding practices. Mutants are good materials for developmental biology and metabolic pathway studies. The chlorophyll deficient (leaf color) mutant can play a significant role in revealing the plant photosynthesis and chlorophyll synthesis and metabolic pathway studies. There is only a few studies in soybean, so in-depth study of the genetic bases and molecular mechanisms of soybean leaf yellowing of new mutants, will help to understand the developmental laws of soybean leaf morphology, and provide valuable reference for high light efficiency breeding. In the present study, five newly discovered yellow leaf mutants were used to reveal their photosynthetic physiology and genetic machnism. Main results are as follows:1. Five new yellow leaf soybean mutants can be divided into three types:1) NTvl-M, NTv2-M mutants are virescence, the cotyledons of their seedlings are green, but the unifoliolate and followed trifoliate leaves perform yellow when they are young. The leaves become green when full maturity; 2) NTyl-M is a green-yellow leaf type, trifoliate leaves are light green at beginning, and turn to yellow afterwards. The plants show whole yellow after R3 stage; NTy2-M also transfers from green to yellow mutation leaves. It performs normal at vegetative growth stage, but appears from green to yellow at pod-set stage; 3) dlm-YY is a yellow and rugose leaf mutant, from the first trifoliate leaf, it performs yellow leaves with the rugose morphology similar to the symptoms by mosaic virus infection. Compared to wild-type plants, the chlorophyll a, b and total contents of two virescence mutants, NTvl-M and NTv2-M, were significantly reduced. NTyl-M leaves are more yellow, indicating its lower chlorophyll content than NTv2-M. The relative chlorophyll content of dlm-M was also significantly lower than that of the wild type plants (P＜0.01). The photochemical activity and PSII maximum efficiency of the virescence mutants and yellow leaf mutants are significantly lower than those of the wild-type plants. Genetic analysis showed that F1 hybrid plants between NTvl-M, NTv2-M mutants and normal parents had normal green leaves, NTvl-M×Nannong1138-2 plant numbers of the F2 populations fitted a 3 (normal):1 (mutant) phenotypic segregation ratio, suggesting that virescence trait controlled by a single recessive gene. The (NTvl-M × NTv2-M) F1 plants showed normal, F2 population fitted a 9 (normal):6 (yellow):1 (albino) phenotype segregation ratio, indicating that the two virescence genes are non-allelic. NTy1-M mutants with normal parental F1 hybrid plants showed normal green leaves, F2 segregating population fitted the phenotypic segregation ratio of 3 (normal):1 (mutant), the number of F2:3 lines from F2 normal plants fitted 1(homozygous green leaves):2 (segregated) genotype proportions, and the F2:3 population from F2 heterozygous plants fitted the ratio of 3:1. It indicated that these three leaf coloris were controlled by a single recessive mutant gene, respectively.2. Leaf morphology of F1 hybrid from the crosses between rugose and yellow leaf mutant dlm-YY and normal parents Nannongl 138-2, Williams 82 were normal, indicating that mutation trait was controlled by a recessive gene; two F2 populations of dlm-YY× Nannong1138-2 and Williams82×dlm-YY fitted the 15 (normal):1 (yellow and rugose leaf) phenotypic segregation ratio. The number of F2:3 lines from F2 normal plants of dlm-YY×Nannong 1138-2 fitted the theoretical ratio of 7(normal):8(mutational trait), and the F2:3 population from F2 heterozygous plants fitted 27 (normal):5 (mutational trait) segregation ratio, suggesting dlm-YY yellow and rugose leaf traits was controlled by two duplicate recessive genes. The dlm-YY × Nannong1138-2 and Williams82 × dlm-YY F2 populations were used to map target genes using SSR markers, and nine polymorphic SSR markers were screened out by BSA. One of the target gene (dlm1) controlling leaf yellowing was mapped on the end of 18 Chromosome (G linkage group).3. Differentially expressed genes (DEGs) of leaves between dlm-YY mutant and its wild-type dlm-W were detected by Affymetrix gene chip technology. A total of 2,469 DEGs were identified through random variance model (RVM) method. Among them,1,565 DEGs were up-regulated and 904 appeared down-regulated. Total 600 genes involved significant difference function was analyzed using hierarchical, average linkage algorithm. These genes performed similar trends between the experimental results and control groups among three replicates, and could be obviously divided into two classes. Intersection between the genes in significant function (GO-Analysis) and those in significant pathway was taken to build coexpression networks. Twenty-seven DEGs on chromosome 18 were found showing great degree of difference. Combined with the result of dlml gene mapping, eight differentially expressed genes at the target chromosome region were selected and carried on the preliminary analysis of gene structure and function. |
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