The Genetic And Function Analysis Of Soybean Leaf Shape Mutant

Soybean(Glycine max L. Merr) belongs to Glycine genus, Faboideae subfamily,Leguminosae family. It is an important economical crop that provides a large amount of the plant protein and oil for human world widely. Soybean is an ancient polyploidy;the genome size is 1.1 Gb that has more than 46,430 protein coding genes, nearly 75%of which exist as multiple homologs. The complexity of the soybean genome and genetic transformation make it difficult to perform studies on gene functions. As the soybean genome sequence published, a new chapter for soybean genetic studies begins. In this study, we screened a gamma ray mutagenesis population of Hedou 12 and identified a crinkly leaf mutant. We re-sequenced Hedou 12 genome and obtained a large amount of INsertion/DELetion(INDEL) polymorphisms information by comparing sequence data with the Williams 82 reference genome. At the same time,we crossed the mutant with Williams 82 wild type to construct a mapping population,and then the map based cloning was performed. We did an allele-test by crossing different lines of mutants. Over-expression the gene in the mutant rescued the mutant phenotype. After the further investigation, we found the gene was involved in cuticle development of soybean leaf epidermis that affected mutant response to drought and pathogen stress. The gene will be important in the future for breeding more drought and pathogen resistant soybean cultivars. The results were as follows:1 Molecular marker screening among cultivars1) AFLP markers designing and screening among soybean cultivarsWe did an AFLP analysis among Hedou 12, Jilin 35 and Williams 82. We gained625, 619 and 622 PCR products by 27 pairs of primers. There were 410 length polymorphisms in the pairwise comparisons, including 138 polymorphisms betweenHedou 12 and Jilin 35, 145 polymorphisms between Hedou 12 and Williams 82, 127 polymorphisms between Jilin 35 and Williams 82.2) SSR markers designing and screening among soybean cultivars98 SSRs with a high likelihood of polymorphisms were collected from BARCSOYSSR. Polymorphisms were tested among Hedou 12, Jilin 35 and Williams82. There were 26(27%) polymorphisms between Hedou 12 and Jilin35, 16(16%)polymorphisms between Jilin 35 and Williams 82, and 41(42%) polymorphisms between Hedou 12 and Williams 82. There were more polymorphisms between Hedou12 and Williams 82, which indicated more genetic diversities and made them ideal parents for mapping population.3) INDEL markers designing and screening among soybean cultivarsIn order to gain enough anchor markers for map based cloning, we selected 248 candidate loci that were evenly distributed in soybean chromosomes. By PCR validation, there were 169 loci with polymorphisms; the success rate was 69%. The validated 169 INDEL markers were distributed throughout the whole genome. The number of validated INDEL markers ranged from 4-24 per chromosome. These markers can meet the basic need for preliminary mapping as the anchor markers.These validated markers were also tested in 12 other soybean cultivars. The results indicated that these INDEL polymorphisms were widely transferable between Chinese and American cultivars. After further analysis, we found that 77% validated markers gave a single PCR product, which is much higher than the false positive INDEL loci(41% PCR product). The result indicated that INDEL loci with homologs were with lower reliability, probably because the fragments were aligned to the homolog places incorrectly and the mismatches were treated as INDELs.2. Identification of crinkly leaf(cl) mutantWe screened the crinkly leaf mutant from our Hedou 12 gamma ray irradiated mutagenesis population. The tips of first leaflet become necrotic 18 days after seeding.As the other part of leaflet continue to develop, the mutant’s distinctive crinkly leaves merge. We crossed the mutant with Hedou 12 wild type in order to purify the mutant’s genetic background. The F1 generation showed the wild type phenotype, and~ 0.25 of the F2 population was mutant which was consistent with a single recessive mutation.3. Map based cloning of crinkly leaf mutantA mapping population was constructed by crossing crinkly leaf mutant with Williams 82. The mutants in F2 population were chosen to perform map based cloning. By preliminary mapping, the mutation was narrow down to 1.37 Mb and2.417 Mb in chromosome 7. We failed to amplify some PCR products in further mapping, which indicated that there was probably a deletion in this region. We then performed an inverse PCR to investigate the border of the deletion region. Sequencing of the inverse PCR product revealed that there is a 253 Kb deletion in the Gm07 from2,118,557 bp to 2,371,744 bp. Within this region, 27 genes were predicted by SoyBase, involved in fatty acid metabolism, glycerol metabolism, and auxin polar transport and so on.4. Conformation of the mutant geneWe screened other four lines of crinkly leaf mutants from our Williams 82 gamma ray irradiated and EMS mutagenesis population. They were numbered with MSN7442, SC5591, SC5962, and SC7321. The phenotype of F1 population from Hedou 12 mutant crossing with MSN7442 was mutant, which indicated that they were allelic mutants. Genomic DNA of MSN7442 was extracted. 27 genes that were deleted in Hedou 12 mutant were amplified and sequenced. There is a G to T transversion in the exon of Glyma.07G028600, which results in an amino acid coding change from aspartic acid to tyrosine. We sequenced Glyma.07G028600 in other three lines. The result showed that: there is a ten bp deletion(TCTTTTATCC) and one bp insertion(A) in the exon in MSN7442; there is an A to T transversion 2 bp in front of exon3 in SC5591, which might cause a mis-splicing of the m RNA; there is a G to A replacement in the exon which results in amino acid coding change from glycine to arginine. According to the sequencing results, four mutant lines all have non-synonymous mutation in Glyma.07G028600, MSN7442 and Hedou 12 mutant are allelic mutants. The over-expression vector of Glyma.07G028600 was constructed and transformed into MSN7442, and the mutant phenotype was rescued. All theseexperiments proved that mutations in Glyma.07G028600 caused the mutant phenotype.5. The expression pattern of Glyma.07G028600Glyma.07G028600 encodes glycerol kinase(GK); its ortholog in Arabidopsis is NHO1/GLI1. nho1 mutant compromises non-host resistance to pathogens.Glyma.07G028600 expresses all over the soybean tissues. Green pod and leaf let have the highest and second highest abundance of mRNA expression. We constructed over-expression vector of Glyma.07G028600 fused with eGFP and transformed Arabidopsis protoplast. The fused protein mainly located on cell membrane.Hybridization in situ showed that the mRNA was widely expressed in shoot apical meristem(SAM) and leaf primordium and so on.6. Characteristics of Glyma.07G028600The water loss rates of isolated leaves for the five lines mutants were one time higher than the wild types. We estimated that there were some defective in plant cuticles. Scanning electron microscope(SEM) of leaf epidermis showed that mutant had an abnormal wax crystal. Transmission electron microscope(TEM) of cross-section leaf showed a much thinner cuticle proper and less osmiophilic cuticular layer for the mutant. GC-MS of epidermic wax showed that there was no significant difference in wax total amount, but significant differences in amounts of compositions.We inoculated Pseudomonas syringae pv glycinea for MSN7442 and wild type to investigate if the mutant compromise the resistance to bacteria. The leaf samples were taken at four days post inoculation(dpi), the number of bacteria is six times more in the mutant leaves than in the wild type.7. The expression abundance of genes that involved in cuticle development are affected by Glyma.07G028600According to the SEM, TEM, and GC-MS results, we chose a series of genes that involved in cuticle biosynthesis, transform, assembling, and regulation for expression abundance analysis. The result showed that the expression abundance of genes involved in cuticle precursors transport had no significant differences; but the expression abundance of some enzyme genes such as GmLTL and some transcriptionfactors had significant differences between mutants and wild type. The result indicated that Glyma.07G028600 might play an important role in cuticle development.The innovations of this thesis:1. We developed a number of INDEL markers that are transferable among other cultivars. Almost fifty thousands of INDEL loci can provide great information for the soybean genetic study and molecular assistant breeding. The information has been uploaded into Soybase, which can fill the INDEL vacancy in the public database.2. By map based cloning, we identified Glyma.07G028600, which involved in soybean cuticle development. The ortholog gene in Arabidopsis play an important role in non-host resistance, but there is no record for this gene taking part in cuticle development.3. We uncovered the mechanism of molecular regulation for soybean cuticle development, which will avail breed more drought and pathogen resistant soybean cultivars.