| ’Breeding by Design’ as a concept aims to bring together superior alleles for all loci/genes of agronomic importance from potential genetic resources. This might be achievable through association mapping of parental resources combined with linkage mapping of genetic populations. The alleles with genetic effects of QTL can be identified and estimated. From the obtained information, genome-wide QTL-allele matrices of breeding traits are therefore established. Based on them, the parental materials can be chosen and organized into two-way or multi-way crosses for complementary recombination among loci and alleles to make the crossing plans genetically optimized. This approach has provided a way to practice breeding by design.In this study, a representative sample composed of 366 accessions from the Chinese soybean landrace (CSLRP) were tested under three years including four environments for its 100-seed weight, oil, unsaturated fatty acid and protein content. The genetic diversity and the population structure were tested based on the genetic markers that obtained by re-sequencing and gel-based after PCR amplification. The genome-wide association study (GWAS) was conducted in new two stages method combined with phenotype and genetic data. The alleles with genetic effects of QTL can be identified and estimated at GWAS. From the obtained information, genome-wide QTL-allele matrices of traits were therefore established. Based on them, the parental materials can be chosen. In addition, a recombinant inbred lines population (NJRSXG) was also used, and a true cross for high protein content and the marker-assisted breeding were performed based on the results of the NJRSXG. The main results were as follows:1. The high phenotype variation of six seed traits in CSLRPThe great variation of 100-seed weight, oil, oleic acid, linoleic acid, linolenic acid and protein content were achieved in CSLRP, of which ranged from 4.59～40.35g, 15.81-24.14%,11.94～36.84%,44.10～60.82%,2.85～10.40% and 37.51～50.46%, respectively; The line and line X environment were all significant for all tested traits according to the joint ANOVA results, however, the variance of line X environment was more less than of line. There were wide correlation between the seed traits. The oil content positive correlated with 100 seed weight, while negative correlated with linolenic acid and protein content. The oleic acid content negative correlated with linoleic acid and linolenic acid content. The linoleic acid content positive correlated with linolenic acid content.2. The high-density genotype established in CSLRPA total 116,769 SNPs were detected based on the re-sequencing in CSLRP, and found the 53% SNPs that the minor allele frequency between 0.01 and 0.10. The SNPs were organized into 29,121 SNP linkage disequilibrium blocks (SNPLDBs) including 7,223 haplotype and 21898 free SNPs based on the soft of Haploview; The length of the haplotype ranged from 5bp to 200,000bp, included 2-130 SNPs and contained 2-12 alleles; haplotype in heterochromatin had longer length and more SNPs; Genetic diversity of CSLRP was analysis using 318 SSR$SV markers and 29,121 SNPLDBs, and found the landraces in Huanghuaihai double cropping, spring and summer planting eco-region (Ⅱ eco-region) contained more alleles and the highest genetic diversity; The LD decay in CSLRP was about 100-200kb at r2=0.5, and over 2000kb at r2=0.1 based on the 116,769SNPs; Phylogenetic tree and Principal component showed the near distance among landraces with the mean 0.18, and the widespread penetration among landraces in different eco-regions;3. The QTL-allele matrix of 100-seed weight established in CSLRPThe results from GWAS combined with ANOVA indicated that 98.45% of the phenotypic variance was accounted for by five major additive QTL (51.00%),46 small additive QTL (41.10%), and the collective unmapped minor QTL (10.39%) of oil content in CSLRP. The QTL×Environment variance was only 4.93% of the phenotype variation. From the second stage, the number of alleles of each QTL (range 2-10) in a total of 276 and the respective allele effects were obtained and organized into a 276×366 matrix as the 100-seed weight genetic constitution of CSLRP. Both negative and positive alleles existed in a landrace no matter in the largest or the smallest seed landrace, indicating great potential in recombination. The whole population matrix was separated into six eco-region matrices from which the genetic differentiation among eco-regions was found. Based on the matrices, potential optimized crosses among and within eco-regions were estimated, showing the predicted values more than the highest, even over 48.24 g in numerous crosses.4. The QTL-allele matrix of oil content established in CSLRPThe results from GWAS combined with ANOVA indicated that 91.29% of the phenotypic variance was accounted for by five major additive QTL (39.80%),28 small additive QTL (43.90%), and the collective unmapped minor QTL (3.55%) of oil content in CSLRP. The QTL×Environment variance was only 1.01% of the phenotype variation. From the second stage, the number of alleles of each QTL (range 2-9) in a total of 147 and the respective allele effects were obtained and organized into a 147×366 matrix as the oil content genetic constitution of CSLRP. Both negative and positive alleles existed in a landrace no matter in the highest or the lowest oil content landrace, indicating great potential in recombination. The whole population matrix was separated into six eco-region matrices from which the genetic differentiation among eco-regions was found. Based on the matrices, potential optimized crosses among and within eco-regions were estimated, showing the predicted values more than the highest, even over 26.99% in numerous crosses.5. The QTL-allele matrix of unsaturated fatty acid content established in CSLRPThe results from GWAS combined with ANOVA indicated that major additive QTL, small additive QTL, and the collective unmapped minor QTL explained 28.00-34.90%, 47.50～54.50% and 7.94～11.40% of oleic acid, linoleic acid and linolenic acid content in CSLRP, respectively. From the second stage, the effect of total 172 alleles of 43 QTL,171 alleles of 41QTL and 145 alleles of 38 QTL were obtained and organized into 172×366, 171×366 and 145×366 matrix for oleic acid, linoleic acid and linolenic acid content of CSLRP, respectively. The whole population matrix was separated into six eco-region matrices from which the genetic differentiation among eco-regions was found. Based on the matrices, potential optimized crosses among and within eco-regions were estimated, showing the landraces of I-W ecoregion could quieted higher oleic acid and lower linolenic acid content soybean.6. The QTL-allele matrix of protein content established in CSLRPThe results from GWAS combined with ANOVA indicated that 84.31% of the phenotypic variance of protein content in CLSRP was accounted for by four major additive QTL (27.80%),25 small additive QTL (44.80%) and the collective unmapped minor QTL (11.71%) with the QTL×Environment variance only 9.08% of the phenotype variation. From the second stage, the number of alleles of each QTL (ranged 2-10) in a total of 137 and the respective allele effects were obtained and organized into a 137×366 matrix as the genetic constitution of CSLRP. Both negative and positive alleles existed in a landrace no matter in highest or lowest seed protein content landrace, indicating great potential in recombination. The whole population matrix was separated into six eco-region matrices from which the genetic differentiation among eco-regions was found. Based on the matrices, potential optimized crosses among and within eco-regions were estimated, showing the predicted values more than the highest even over 51.16% in numerous crosses.7. Soybean with high protein content createdThe cross of WT133 and XG30 was performed based on phenotypic and genotypic, and the lines that with high protein content were achieved in three environments (generations). The Prot-l-08-1, The Prot-l-14-1 and The Prot-l-19-2 detected in NJRSXG showed the selection efficiency, especially for Prot-l-08-1 explained 28.83-43.83% of the phenotypic variation. The six lines of F2:5 were chosen based on the genotype of F2 that the Prot-l-08-1 was heterozygous, and the line with protein content as high as 54.75% was achieved.8. The comprehensive optimization design in CSLRPThe total 235 QTL of six seed traits were detected in CSLRP, and 21 regions (around 5Mb) that contained more than 3 seed traits QTL were obtained and contained 94 QTL; Due to the co-located QTL of seed traits, the crosses that oil content were high while protein content were not reduced, the protein content is high while the oil content is not reduced, the oil and oleic content were high while the linoleic and linolenic were not reduced were listed. |
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