| Soybean[Glycine Max（L.）Merr.],as an important food and economic crop in the world,is a major source of protein for human beings and animals.Cotyledon is one of the important components of soybean seed embryo,and its function is mainly to supply seedling nutrients during the vegetative growth stage of the plant.Until now,many studies have indicated that the growth state of cotyledons directly affects the vegetative growth and reproductive growth of a plant,which will ultimately determine the traits related to resistance,yield and quality.Therefore,the study of cotyledon deficient mutant is of great significance for soybean breeding and improvement.In 2002,a soybean curled-cotyledon mutant（cco）was obtained in our lab by sodium azide（NaN3）and 60Coγ ray mutagenesis of a cultivar Nannong 94-16（WT）.Besides the typical curled cotyledons phenotype,cco also displays other traits,including significantly reduced hypocotyl length,germination rate,plant height,lateral root system and hundred grain weight;delayed growth period;increased protein and hydrolyzed amino acids content.Therefore,compared with WT,cco mutant has undergone many traits changes throughout the whole growth cycle.In this study,map-based cloning method and high throughput sequencing technology were used to map the loci controlling soybean curled cotyledon and analyze the genetic variation of cco,so as to detect some important genes related to soybean growth and development.The main results in this study are as follows:1.The investigation of seed,cotyledon,pollen vitality and quality traits of cco mutant indicated that:compared with WT,there are creases on the surface of cco seed coat,and the seeds appear in two shapes（round and wrinkled）;also,the storage cells of cotyledons in cco are wide,oval and loosely arranged;and the pollen vitality is decreased in cco;the contents of protein and hydrolytic amino acids（including sulphur amino acids）in cco mature seeds are significantly increased,while the oil content is decreased.Further,two gene loci controlling soybean curled cotyledons were identified based on the F2 generation hybridized by cco and Kefeng No.1 by using bulk segregant analysis（BSA）and SSR molecular marker assisted selection methods.The results showed that one gene is located between BARCSOYSSR050037 and Satt684 markers on chromosome 5,and the other is near Satt597 marker on chromosome 1 1.2.Genome-wide resequencing data showed that there exist many variations between WT and cco,including 120201 SNP sites,50228 Indel sites,2231 SV sites and 16648 CNV sites,which suggests that NaN3-60Coγ composite mutagenesis has greatly changed the genome of WT.As the variation located in genic region is more likely to cause the changes of encoding protein,thus the genes varied in genic region were statisticsed.There are 3339 genes induced by SNP,6704 genes induced by Indel,5530 genes induced by SV,and 1587 genes induced by CNV.GO and KEGG enrichment analysis showed that these different variant genes could participate in many biological processes,including organ growth and development,stress response and signal transduction,biosynthesis and metabolic processes.Therefore,it is speculated that these different variant genes might be directly or indirectly involved in the growth of cco mutant.Based on the preliminary gene mapping,31 variant genes from genic region were identified on chromosome 5,which is important for further selection of candidate genes controlling curled cotyledon in soybean.3.Genome-wide methylation sequencing technology was used to analyze the epigenetic regulatory network of cco.From the result,cco has more methylation sites on genome than WT,but the overall methylation level is lower than that in WT.There are 83366 significant differentially methylated regions（DMR）between WT and cco,of which,38429 DMRs（hyper）have higher methylation levels in cco,and 44937 DMRs（hypo）contain higher methylation levels in WT.Among the three methylation types including mCG,mCHG and mCHH,the mCHH is the most abundant in DMRs,thus its distribution on chromosome is the most intensive.Further,there were 8905 significant differentially methylated genes（DMG）identified between WT and cco.GO and KEGG functional enrichment analysis suggested that these DMGs are involved in cell function regulation,signal transduction,growth and development,biosynthesis and metabolism,stress response and transport.A total of 47 DMGs were identified on chromosome 5 based on preliminary gene mapping result,which will help to understand the regulation of cco development on methylation level.4.By using preliminary gene mapping result of cco mutant and its previous RNA-seq data,a significantly down-regulated gene in the early development of cco seed embryos was selected,which is named as Gmhdz20（Glyma.05G030000）.Also,the methylation sequencing data showed that the methylation level in the promoter region of Gmhdz20 in cco was significantly lower than that in WT.Gmhdz20 protein contains homeobox domain and leucine zipper motif,belonging to HD-Zip Ⅰ（Homeodomain-leucine zipper）subfamily.Tissue expression pattern indicated that Gmhdz20 expressed in all tissues examined in soybean,but the expression levels in leaves and cotyledons were relatively higher.Subcellular localization suggested Gmhdz20 is a nuclear localization protein.Overexpression of Gmhdz20 in Arabidopsis thaliana altered a series of phenotypes,including leaf and flower morphology,branch number,silique length and seed arrangement.These results suggest that Gmhdz20 plays important roles in both vegetative and reproductive developments.Through yeast two-hybrid assay,one gene from soybean leaf library and two genes from pod library were identified to interact with Gmhdz20,including Glyma.13G027600（ubiquitin carboxyl-terminal hydrolase 13-like）,Glyma.18G014900（homeobox protein 6）and Glyma.04G123900（alpha-glucosidase 2）.5.GmLBD12 was previous identified to participate in the root development of cco mutant based RNA-seq data and qRT-PCR examination;and its positive regulation of lateral roots has been demonstrated in transgenic Arabidopsis thaliana.Therefore,we investigated soybean LBD genes using a genome-wide approach and studied the expression patterns of these genes in various tissues and in response to different stressors.The results showed that there were 90 LBD genes identified in soybean genome,and 41 of them were supported by ESTs.Consistent with other plants,the soybean LBD genes were phylogenetically clustered into two classes（Ⅰ and Ⅱ）.The majority of the GmLBD genes were evenly distributed across all 20 soybean chromosomes,and 77（81.11%）of them were detected in segmental duplicated regions,indicating that segmental duplication appears to be the main driver of the expansion of LBD genes in soybean.Furthermore,gene structure and motif composition analysis indicated their conservation in nucleotide and protein sequences.A close phylogenetic relationship was identified between the soybean LBD genes and 41 previously reported genes of different plants in the same group,providing insights into their putative functions.Digital expression data and qRT-PCR analysis suggested that more than half of the LBD genes were expressed,with the two gene classes showing differential tissue expression characteristics;in addition,they were differentially induced by biotic and abiotic stresses. |
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