| Leaf shape and its growth posture are main factors influenced crop canopy structure and important for ideal plant breeding. Soybean [Glycine max (L.) Merr.] with abundant diversity of leaf shape, among which broad and narrow are the two typical types. The division of soybean leaf shape in traditional breeding relies mainly on the method of visual identification, but it is with higher artificial bias. Leaf shape index (the ratio of length to width), as a classical quantitative parameter, is also with lower distinguish ability while used to identify large number of germplasms or leaf shape segregated populations (with intermediate leaflet plants). Map-based cloning and function dissection of genes conferring leaf shape are calling more exact identification of the leaf shape phenotype. Soybean with narrow leaves tend to have higher ratio of four-seeded pods and represent a certain ecological distribution, while the mechanism of leaf shape development and its relationships with the trait of number of seeds per pod and with ecological distribution are still unclear. Here, we defined and analyzed leaf shape parameters using mathematics method and selected the optional leaf shape parameter through actual data analysis. Then we identified the key candidate gene of Ln by fine mapping and haplotype association analysis, then the candidate gene was validated through gene silencing and over-expression. A series of experiments were performed focus on the different proteins encoding by four mainly transcripts of the Ln locus, including prediction of protein secondary structure, subcellular localization, transcript repression activity detection and the influence analysis on the trait of number of seeds per pod. Then analysis of transcriptome, proteome, stress and physiological detection were performed using one pair of near isogenic lines. Based on this, the molecular and physiological basis of Ln on leaf shape, number of seeds per pod and the ecological adaptability were discussed. In addition, a new soybean leaf shape mutant alf(abnormal leaf and flower) were studied through gene mapping and differentially expressed protein analysis. The results are as follows:1. Leaf shape variation and main leaf shape parameters of cultivated soybean germplasmTwo new leaf shape parameters, widest relative position (the ratio of the distance between the maximum leaf width and the leaf apex to the distance between the maximum leaf width and the leaf basis, λ) and leaf apex opening angle (the angle formed by two straight lines that linking leaf apex and the two endpoints of the maximum width, a) were defined. Based on this definition, three leaf parameters of leaf apex opening angle, leaf shape index (the ratio of leaf length to leaf width, s) and widest relative position were linked together:α=2arccot[2λs/(1+λ)] 。α, λ and s all showed bi-modal distribution in 242 cultivated soybeans and the corresponding normalized data indicating two separate underlying populations broad and narrow. Among them, leaf apex opening angle represented the highest distinguish ability, which were also be validated in a F3:4 leaf shape segregated population. This study suggests using the parameter of leaf apex opening angle to divide soybean leaf shape into two subpopulations of broad and narrow, among them, the broad subpopulation including all of the round, oval, long oval, elliptical and partial elliptical soybeans named in traditional breeding, the narrow subpopulation including all of the lanceolate, long leaf, pointed leaf and partial elliptical soybeans.2. Map-based cloning and validated of the Ln gene conferring leaf shape(1) The In gene conferring narrow leaf in NT-1 was fine mapped into a 48 kb physical region on the chromosome 20 of soybean through three F2 populations (NT-1 × NN1138-2, NT-1 × Forrest, NT-1 × KF-1). Sequencing analysis of three candidate genes showed that there were five SNPs in the first exon and upstream promoter region of ORF3 between broad and narrow parents.(2) Haplotype (the region that SNP1-SPN5 located) sequencing analysis of 55 represent materials selected from 242 germplasm showed that SNP1(Gâ†'C) and leaf shape was with highly related. Then the result was validated in F5:6 segregated population, recombinant plants of mapping population and other cultivated soybeans with CAPS (cleaved amplification polymorphism sequences) markers derived from SNP1. And the ORF3 gene that SNP1 located in was determined as the key candidate gene of Ln.(3) The whole sequence length of ORF3 was obtained by RACE method and found that the gene encoding a C2H2 zinc finger protein, which harboring an EAR (Ethylene-responsive element binding factor-associated Amphiphilic Repression) motif L/FDLNL/F(x)P. SNP1 just localized in the EAR motif and resulted in a mutation of aspartic acid/D to histidine/H. Sequencing analysis of RACE experiment also showed that there was an alternative splicing position in the gene and it resulted in six base pair in intron changed into the exon. RACE sequencing analysis also showed that there was an alternative splicing position, in which a 6 bp intron segment changed into a exon segment and resulting in two amino acid insertion (Valine and Alanine) but with no frameshift. So the Ln locus can encoding four proteins:Ln (the main transcript of broad leaf soybean with no alternative splicing), LnVA (the transcript with an VA insertion of alternative splicing in broad leaf soybean), ln (the main transcript of narrow leaf soybean with no alternative splicing), lnVA (the transcript with an VA insertion of alternative splicing in narrow leaf soybean)。In addition, there was an phenomenon of alternative polyadenylation in the 3’ end of the gene. Seven positions of alternative polyadenylation were found, positionl to position7, in which the fourth position showed the highest frequency of alternative polyadenylation. This suggesting that microRNA may be involved in the expression regulation of Ln gene.(4) ORF3 VIGS (virus induced gene silencing) analysis induced by BPMV (bean pod mottle virus) on soybean showed that the silenced plants growth abnormally from the second trifoliolate leaf. In detail, the leaflet couldn’t expanded normally and leaf apex opening angle reduced, meanwhile, the leaf margin contour line became not smooth, which was also appeared when the ORF3 gene in narrow soybean was interferenced. These data indicated that the ORF3 gene was the Ln gene conferring leaf shape in soybean, but the In allele was not a null mutant but with some modification function, at least, during the leaf margin development. When the tobacco Ln homologous gene NbLn was interferenced by TRV induced VIGS, the LAO A phenotype also reduced and the angle between leaf and petiole became into an obtuse from a cartesian angle. In another way, over-expression of the Ln gene in Arabidopsis can improve growth and expansion of the transgenic leaves. These results indicated that the gene family had a significant role in the regulation of leaf shape development and the function was conserved between species.3. The effects of the allelic variation of Ln (Lnâ†'ln) and its alternative splicing on protein secondary structure, subcellular localization, transcription repression activity, and their relationships with the phenotype of number of seeds per pod(1) Protein structure prediction indicated that the Ln locus could generate four proteins Ln, LnVA, ln and lnVA because of the point mutation and the alternative splicing, their secondary structure were ββαββα, ββα,α and ββα, respectively. Subcellular localization analysis showed that the four proteins were all localized in nucleus. Dual luciferase analysis indicated that Ln really has repression activity to the transcription and the repression activity of LnVA was reduced by 50%, ln and lnVA were both lost their repression activity.(2) Tissue specific analysis with Ln NILs (NIL-broad, NIL-narrow) showed that there was no significant differences in shoot apex meristem and leaf between the NILs, but with significant differences in flower and young pods. The level of alternative splicing in narrow soybean was higher than it in the NIL-broad which suggesting that different transcripts may have effect variation on the phenotype of number of seeds per pod. In order to investigate the relationship of alternative splicing and the phenotype of number of seeds per pod, we then detected the expression pattern of a broad leaf shape soybean SLJ which with more than 85% of four-seed pods. The results showed that the expression pattern were similar between SLJ and NIL-narrow (with about 10% four-seeded pods), and SLJ with a higher level of alternative splicing than NIL-narrow. Over-expression of the four transcripts (Ln, LnVA, ln, lnVA) revealed that the alternative splicing tend to increased the number of seeds per pod. These data indicating that narrow leaf soybean tend to has a higher percent of number of seeds per pod is result from the higher level of alternative splicing which was initially induced by the mutation of Lnâ†'ln.4. Ln allele mutation (Lnâ†'ln) influence on the genome-wide transcription and translation(1) Transcriptome analysis by Affymetrix chip detected 2044 (4.4% of the predicted gene in whole genome) up-regulated and 2003(4.3% of the predicted gene in whole genome) down-regulated genes in SAM tissue between NILs. This result is similar to the mutation of JAG to jag, i.e.4.4%/4.3% vs 4.0%/3.5%. Among them,16 random selected genes were verified by qRT-PCR and higher level of consistency (R2=0.898). In the differentially expressed genes the ratio of the transcription factors were the highest type, among them the expression of leaf regulator ARF (Auxin response factor), C2C2-YABBY were all up-regulated, AS1 (ASYMMETRIC LEAVES1) were down-regulated. Histone ATse, Histone, Chromatin redmodeling, cleosome assembly and so on the factors involved in chromatin state were all appeared to co-up-regulated. These data indicating that Ln may regulate expressions of the down stream genes by regulating the expression of genes involved in chromatin state.(2) 991 differentially expressed proteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation) technique. Among them,478 were down-regulated and 513 were up-regulated. These proteins are mainly involved in protein modification and metabolism, RNA metabolism, photosynthesis, cell signal transduction. Transcriptome and proteome data integration analysis revealed that the Ln gene participated regulation processes of post-transcription, translation and post-translation, and most of overlapping genes involved in salt, cold and other abiotic stress response and signal transduction processes.5. Leaf shape control of Ln and the molecular and physiological basis of ecological adaptation of soybean with narrow leaves(1) GO enrichment and cellular response (MapMan) analysis of differentially expressed genes revealed that genes associated with cell division and cell cycle were co-up-regulated. These genes were mainly involved in cell cycle of G1/S, G2/M and GO. Jasmonic acid (cell cycle inhibitor) metabolism related genes represented a significant up-regulated trend, auxin metabolism related genes expression represented a down-regulated trend. Phenotypic characterization of NILs revealed that the lateral veins increased, leaf area reduced, but cell size with no change in NIL-narrow. So we speculated that Ln may controlled the leaf morphogenesis through regulation of cell cycle and it’s maybe related to the auxin distribution. When JAG, the Arabidopsis homologous of Ln, was interferenced by VIGS method induced by TRV on the DR5-GUS transgenic background, the silenced plants represented the similar phenotype with jag mutant, and the distribution of the auxin was changed. In detail, the auxin was tend to gather a maximum in the jagged tips which resulted from the JAG interference, meanwhile, the distribution of auxin in the middle vein was reduced. This indicating that the JAG-like genes are involved in the regulation of auxin transport which influenced the cell division and expansion and thus affecting the leaf shape development.(2) GO enrichment analysis of the down-regulated genes revealed that they were notably enriched in processes of abiotic stress. Through the analysis of 48 C2H2 zinc-fingers from more than a dozen species, we found that most of these genes involved in development regulation and meanwhile responding to environmental stresses, in which the EAR domain always playing an important role. The physiological experiments including salt, base, aluminum, cold, dry and SMV environmental stresses revealed that NIL-narrow with more sensitive to SMV infaction but with higher resistance to cold and salt than NIL-broad. Chlorophyll content and fluerescence feature test showed that the NIL-narrow had more chlorophyll and Fo value, but with lower Fm value, this photosynthetic characteristics were more efficiency in weak light and long day areas. Phenotype and the genealogy analysis of 2250 soybeans in the Soybeans Varieties of China revealed that the narrow leaf soybean widely distributed in the northeast of China, Heilongjiang province (186,55.7% of the whole) is the highest, Inner Mongolia(17,48.6%) and Jilin (101,38.7%) are followed as behind. The distribution tendency has a consistent trend with the salt distribution trendy and the distribution of cold damage area in China. These results provided molecular and physiological basis for the geographical distribution of soybean with narrow leaf in China.6. Preliminary study of a new soybean leaf shape mutant alfalf is a new soybean leaf shape mutant. The mutant leaf width was about 1/4 of the wild type, but the length with no obvious changes. In addition, the flowers of this mutant were abnormal. In this study, genetic analysis and gene mapping were performed, then residual heterozygous lines of the target segment were selected for the subsequent fine mapping of the gene; and comparable proteomic analysis were also performed.(1) 44 DEPs between NILs were identified by 2-E technique, which mainly localized in plastid and participating abiotic stress responses, with molecular founctions of redox activity and peptide and cofactor binding. So we proposed that the mutant gene maybe a factor involved in information transduction coupling pathways of environment stress responses and growth development.(2) Four F2 groups (alf×NN1138-2, alf×N2899, alf×Williams82, alf× KF-1) were developed to performed genetic analysis of the mutant phenotypes. The results revealed that the mutant phenotypes of leaf and flowers inherited stably in different genetic backgrounds. In F2 populations, the wild type and mutant type plants represented 3:1 ratio, and progeny test represented 1:2:1 ratio, which indicated that the mutant phenotype were controlled by a single recessive gene or tight linkage genes. The gene were further delimited to approximately 1 Mb physic distance on the short arm of A1 linkage groups used SSR (Simple Sequence Repeat) markers. Residual heterozygous individuals were selected based on the mapping results and progeny test. The results provided foundation of materials and information for further position cloning and dissection of the key gene. |
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