Construction Of Mutant Population In Soybean (Glycine Max (L.) Merr.) And Study On Curled-Cotyledon Mutation Related Genes

Soybean [Glycine max (L.) Merr.] is one of the most economically important crops in the world. However, complex organization of genomic DNA and low efficiency on genetic transformation restricts the advancement of functional genomics research. With the completion of soybean genome sequencing, the research of gene function will become the research hotspot. Mutants are the important materials for the advancement of functional genomics research, and are the basic materials for soybean genetic improvement.Therefore, in this research; soybean cv. "Nannong 86-4", cv. "Nannong 87c-38" and cv. "Nannong 94-16" were treated with NaN3,60Co y rays and EMS to construct the mutant population. Meanwhile, a systematic research for curled cotyledon mutant has been developed. Main results are as follows:1. Construction of mutant populationPhenotypic characters of M2 and M3-generation mutants were investigated for various mutants including morphological characters such as leaf, stem, flower and seed. There are 185 mutants identified from "Nannong 86-4" including 120 for leaf,24 for stem,26 for flower,9 for seed,6 for maturity and fertility, respectively. There are 62 mutants identified from "Nannong 87c-38" including 10 for leaf,4 for stem,3 for flower,3 for seed,42 for maturity and fertility, respectively. There are 120 mutants identified from "Nannong 94-16" including 84 for leaf,3 for stem,10 for flower,21 for seed,2 for maturity and fertility, respectively. Proteins and the oils of partial mutants have been studied. The protein content of mutants in "Nannong 86-4" was in the range of 39.5%-52.5% and the oil content range was 15.3%-22.9%. While in "Nannong 94-16" the protein and oil content range variation was 39.1%-48.1% and 17.3%-22.2%, respectively. 2. Identification and analysis of a curled cotyledon mutant in soybean1) Characters of agronomic and qualityFirst, overall investigation of agronomic characters of curled cotyledon mutant suggested the mutant cotyledons curled outwards and seed coats were partially shrunken. Plants raised from mutant seeds were found to mature 9 days later than the wild type. The result of observation showed significant difference in morphology between the mutant and its wild type at the seed formation stage. Analysis of quality characters indicated a 3% higher protein content in mutant than wild type. Analysis of 17 kinds of amino acids in mutant indicated that serine content was not affected, while, proline, threonine and tyrosine were reduced. The rest however, showed increased, levels especially the methionine which increased by as much as 33% in the mutant. Analysis of free amino acid content in mutant was 74% higher than that of wild type.2) Genetic analysisGenetic analysis of the curled-cotyledon mutant by hybridization indicated that it was a double recessive mutant based on nuclear inheritance.3) Analysis in genome levelTo further address the difference in genomics between curled-cotyledon mutant and wild type, SSR marker research was developed, altogether 957 pairs were screened, and result showed difference on 9 pairs of markers. These markers were mainly concentrated in three linkage groups namely B1, D2, and G. According to the previous findings, QTL for the protein and the oil was located on B1 linkage group nearby marker Satt426. QTL for the seed character was located among cluster markers Satt163, Satt038, Sat₁68, Satt570 and AW734137 on linkage group GFurthermore, in order to search the difference between mutant and wild type in the genome level, CGH array was used. Regarding a certain specific value of 1.2 times as the threshold,19 different genes were obtained. The ten genes with stronger signal in mutant than that in wild type, among 7 genes encode unknown protein, and 3 genes encode known protein. Functions of the known protein are showed as follows:aspartate-tRNA ligase, UDP-glycosyltransferase, EF-hand Ca2+-binding protein CCD1. The nine genes with stronger signal in mutant than that in wild type, among 3 genes encode unknown protein, and 6 genes encode known protein. Functions of the 6 known proteins are as follows:leucine amino peptidase, translation initiation factor, structural constituent of ribosome, Dof zinc finger protein, pyruvate dehydrogenase, and cytochrome P450 monooxygenase. To discover the possible gene that closely related to cotyledon, the validation of RT-PCR with seeds obtained within 30 days after flower indicates that among the 10 genes with stronger signal there is only one gene complied with the result of CGH. It is Gma.15488. Among the 9 genes with weaker signal there is only one gene complied with the result of CGH. It is TC208897. The result of research indicates that there are intimate relationship between 2 genes above and the cotyledon shape. This viewpoint also needs to carry on further confirmation, such as to obtain full length gene and transgene so on.4) Influence of curled cotyledon on seed storage protein in soybeanIn order to study the influence of cotyledon shape difference to the mature seed storage protein components in soybean, three kinds of curled-cotyledon mutants was searched by 2-D electrophoresis. The research results showed that there were 3 common proteins which are different in mutant and wild type. The increased two protein spots in mutants are both globulin subunit G3/AlaBlb, at meanwhile, the protein spot that decrease is lectin.3. The establishment and application of TILLING in soybeanStudies of optimizing the new method of TILLING have been conducted. CEL I enzyme that extracted from celery were used in TILLING The KNOX gene was studied using 75 mutants of leaf; as a result, one base change was found in this gene in one mutant.