A Study On Protein Components And Subunit Grouping Of Chinese Soybean Germplasm And Their Variability, Inheritance And QTL Mapping

Soybean[Glycine max(L.) Merr.],originated from China,is one of the most important sources of vegetable protein and edible oil world-widely.Soybean protein is rich with essential amino acids for human bodies.Soybean protein quality depends on magnitude of 11S and 7S proteins and their subunits.In the past,protein component and its content were determined by ultracentrifuge,basic dissolve,acid-precipitation and gel filtration in large sample of a few materials.But it is needed a technology that can be used to analyze amount of materials in small sample for breeding and resource studying.Several studies have been done on SDS-PAGE analysis of protein component in soybean.It was a facility method. What's more,materials used in experiments were mostly from local regions,without representative,resulting in the limitation of reference to soybean breeding for very difference and even confliction on molecular weight of component and subunits.So,the range of materials analyzed should be extended,the material representative should also be improved,then the results studied will be used in improving protein characters of soybean. Soybean inbreeding men want a facility method to test resources and materials,to increasing effect,saving manpower,material resources and time.Before founding simple method,investigating variation and inheritance mechanism and QTL mapping of protein content and its variation range of soybeans,and some results obtained.But less concern was on relative content of 11S,7S components and their subunits.There existed few report on inheritance and QTL mapping of 11S,7S and their subunits.Herein above instance,the main objectives of this study were:(a) On the basic of former study,finding distribution law of electrophoresis bands of protein component and subunit,establishing a convenient method and molecular criterion for 11S,7S protein and their subunit in SDS-PAGE,by analyzing protein extracts of 640 soybean cultivars selected to represent different cultivar eco-regions for improving soybean protein component and their subunits,(b) With wild soybean,cultivated landraces and released domestic cultivars represent different cultivar eco-regions in China in the same experimental condition and directness test method,studying the effect of nature and artificial selection on content of protein,fat,total protein plus fat,relative content of 11S and 7S,analyzing variation characteristic of various germplasm in different eco-regions in China,screening out elite accessions for breeding purposes,(c)Revealing inheritance mechanism of protein content, 11S and 7S relative content of soybeans,mapping QTLs of them related genes with NJRIKY and NJBIEX population offering by Soybean Research Institution of Nanjing Agriculture University.The results of this study will be helpful for soybean protein quality breeding.The mostly results obtained in this study are as follows:1.Protein extracts of 640 soybean cultivars and landraces,mainly from China and a few from the US,were analyzed for their components and subunits based on distribution patterns of bands with varying molecular weights(MW) under SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The number and molecular weight of the bands in SDS-PAGE varied among materials and showed a tendency of continuous distribution.Accordingly,referring to former results and situation of apex and vale of frequency distribution of bands,the SDS-PAGE patterns of the soybean protein extracts were divided into two regions:the region of bands with MW＜44 KDa and that with MW≥44 KDa.The first region containing mainly 11S proteins was divided into four parts, called subunit groups,i.e.11S-1(14.4-22 KDa),11S-2(22-26 KDa),11S-3(26-34 KDa) and 11 S-4(34-44 KDa).The second region containing mainly 7S protein was divided into six subunit groups,i.e.7S-1(44-49 KDa),7S-2(49-55 KDa),7S-3(55-67 KDa), 7S-4(67-73 KDa),7S-5(73-82 KDa) and 7S-6(82-91 KDa).The sum of relative contents of 11S-1～11S-4 was obtained as the relative content of 11S protein,those of 7S-1～7S-6 as that of 7S protein,and therefore,the 11S/7S ratio obtained.2.The content or relative content of traits relating to soybean protein component of 778 accessions,including 138 wild soybean(Glycine soja Sieb.et Zucc.),409 cultivated landraces(Glycine max(L.) Merr.) and 148 released domestic cultivars sampled from various eco-regions as a representative in China,along with 83 released foreign cultivars, were tested.The results showed that the range of protein content,fat content and total of both in wild soybean were 39.2～54.2%,7.5～17.5%,47.3～64.6%,those in cultivated landraces 38.8～51.5%,11.5～23.4%,55.6～70.6%,those in released domestic cultivars 41.7～49.4%,12.9～24.9%,55.6～72.0%,respectively.The increase of fat content and protein plus fat content after domestication,especially due to scientific breeding effort,and the decrease of protein content as well as its range implied that the improvement of fat content happened during the history.There existed large amount of genetic variation of protein content,fat content and total of both contents in various eco-regions in different kinds of materials,with elite accessions in each eco-regions,but the variation was not large enough among eco-region means.No significant correlation between the three contents and the original latitude of the materials was found in wild accessions,while significant positive correlation between fat content and latitude existed in cultivated landraces and released cultivars and significant negative correlation between protein content and latitude was found only in released cultivars.There was no significant correlation between protein content and fat content in wild soybeans,but significant negative correlation between the two contents found in cultivated land races and released cultivars.The reason for this kind of change should be the differential artificial selection among the eco-regions.11S relative content mean of 54.7%,64.8%and 71.7%,11S relative content range of 28.8-82.6%,38.8-79.4%and 48.8-88.9%,7S relative content mean of 44.7%,34.9%and 27.9%,7S relative content range of 20.6-71.2%,20.6-61.1%and 15.7-47.8%,11S/7S ratio mean of 1.4,2.0 and 2.7,11S/7S ratio range of 0.4-3.9,0.6-3.9 and 0.9-4.0 in wild soybean, landraces and released cultivars,respectively.After breeding cultivars by domestication wild soybean,11S relative content and 11S/7S ratio was step up,7S relative content was step down,with their variation range decreasing.The subunit groups 11S-2 and 11S-3 relative content were increased.The relative content of 6 subunit groups of 7S protein, particularly,7S-1 and 7S-6 were decreased.There existed abundance and choiceness variation of 11S and 7S protein relative content and 11S/7S ratio of three germplasm types in each Eco-region.There was no correlation between geography latitude of origin(or protein and fat content) and three traits in three germplasm types,respectively.In addition,from the tested accessions,10 most elite accessions for each of the three traits(protein content more than 50%,or fat content more than 23%,or total of both contents more than 68%),11S/7S ratio of 8 elite screened out from 778 accessions(about 1.03%) was more than 3.7,with their 11S relative content 78.9-88.9%.The relative content of elite accessions was more than 37%for 11S'subunit groups and 24%for 7S' subunits groups,respectively.There existed amount of materials lacked 11S-1,or from 7S-1 to 7S-6. They will be used for high output breeding purposes.3.Soybean germplasms,the NJRIKY population(KY,from Kefeng1hao and Nannong1138-2) and NJBIEK population(EX,from Essex and ZDD2315),with big differences in traits relating to protein component were used to reveal the genetic mechanism of them.Genetic analysis was performed under major gene+polygene mixed inheritance model.The resulted indicated that in KY population major gene heritability of protein content,11S relative content,7S relative content,11S/7S ratio,four 11S' subunit groups and six 7S' subunit groups were 31.3%,14.3%,34.5%,74.8%,45.2～77.9%,and 38.9～67.8%,respectively.Their polygene heritability were 53.7%,50.7%,45.1%,20.1%, 15.5～41.2%,and 29.2～45.5%,respectively.In EX population major gene heritability of protein content,11S relative content,7S relative content,11S/7S ratio,four 11S' subunit groups and six 7S' subunit groups were 40.9%,60.7%,44.1%,56.6%,45.4～67.6%,and 76.2～92.6%,respectively.Their polygene heritability were 37.2%,17.0%,21.6%,10.1%, 22.6～53.4%,and 5.0～22.2%,respectively.The polygene heritability of protein content, relative content of 11S and 7S were higher than major gene heritability of them in KY.It was reverse for 11S' and 7S' subunit groups in KY.In EX,the major gene heritability of most traits was higher than polygene heritability of them.There existed difference in polygene heritability for KY and EX,but polygene play on an important role in major gene +polygene mixed inheritance model.4.Based on the genetic research above,QTL mapping for traits relating to protein component in soybean was carried out as following.The composite interval mapping(CIM) of the software Windows QTL Cartographer Version 2.5 were used to map QTLs.In KY, there were 2 QTLs(B1pr and Epr1) for protein content with R²(R² means accumulative total account of the phenotypic variation) of 16.5%,2 QTLs(A211S and D1a11S) for 11S relative content with R² of 13.3%,2 QTLs(I7S1 and I7S2) for 7S relative content with R² of 12.7%,3 QTLs(D1arat,Irat1 and Irat2) for 11S/7S ratio with R² of 19.8%.r²(account of the phenotypic variation) of 11S' and 7S'subunit groups was below 10%,respectively. In EX,there were 1 QTL(Epr2) for protein content with r² of 10.6%,2 QTLs(E11S and B211S) for 11S relative content with R² of 23.5%,3 QTLs(E7S1,E7S2 and D1b-27S) for 7S relative content with R² of 38.3%,1 QTL(Erat) for 11S/7S ratio with r² of 14.3%.r² (account of the phenotypie variation) of 11S' and 7S'subunit groups was about 8.7～21.9% and 8.2～16.3%,respectively.The highest r² of 21.9%was QTL M11S-11 of 11S-1 in EX. There were 3 QTLs from marker GMKF008b to GMKF008a on linkage group D1a in KY (D1a11S,D1arat and D1a7S-2) and from marker sat₃80 to satt263 on linkage group E in EX(E11S,E7S1 and Erat),respectively.QTL with high account of the phenotypic variation was not found.There existed a few QTL with low account of the phenotypic variation, about 10%,for the traits relating to protein component.So,polygene was important in inheritance of them.In this study,the molecular criterion and method about 11S,7S protein components and subunit groups of protein extracts were demonstrated to be simple,stable and feasible. It had been used to study protein component and subunits of soybean germplasm resources and inheritance analysis and QTL mapping in this study.The elite accessions for protein content,11S/7S ratio,11S relative content 78.9-88.9%11S'subunit groups and 7S' subunits groups screened out from 778 accessions will be used for high output breeding purposes. The inheritance of traits relating to protein component was controlled by a few low effect major gene and lots of polygene.It was consistent with analysis results of inheritance. Based on the results,it is inferred that the markers linked to the detected QTLs should be useful for marker-assisted selection for 11S,7S and related traits in soybean.