Floral-promoting GmFT homologs trigger photoperiodic after-effects: An important mechanism for early-maturing soybean varieties to regulate reproductive development and adapt to high latitudes.

Soybean (Glycine max) is a typical short-day plant, but has been widely cultivated in high-latitude long-day (LD) regions because of the development of early-maturing genotypes which are photoperiod-insensitive. However, some early-maturing varieties exhibit significant responses to maturity under different daylengths but not for flowering, depicting an evident photoperiodic after-effect, a poorly understood mechanism. In this study, we investigated the postflowering responses of 11 early-maturing soybean varieties to various preflowering photoperiodic treatments. We confirmed that preflowering SD conditions greatly promoted maturity and other postflowering developmental stages. Soybean homologs of FLOWERING LOCUS T (FT), including GmFT2a, GmFT3a, GmFT3b and GmFT5a, were highly accumulated in leaves under preflowering SD treatment. More importantly, they maintained a high expression level after flowering even under LD conditions. E1 RNAi and GmFT2a overexpression lines showed extremely early maturity regardless of preflowering SD and LD treatments due to constitutively high levels of floral-promoting GmFT homolog expression throughout their life cycle. Collectively, our data indicate that high and stable expression of floral-promoting GmFT homologs play key roles in the maintenance of photoperiodic induction to promote postflowering reproductive development, which confers early-maturing varieties with appropriate vegetative growth and shortened reproductive growth periods for adaptation to high latitudes.

Genome-wide association study for temperature response and photo-thermal interaction of flowering time in soybean using a panel of cultivars with diverse maturity groups.

KEY MESSAGE: A total of 101 QTNs were found to be associated with soybean flowering time responses to photo-thermal conditions; three candidate genes with non-synonymous substitutions were identified: Glyma.08G302500 (GmHY5), Glyma.08G303900 (GmPIF4c), and Glyma.16G046700 (GmVRN1). The flowering transition is a crucial component of soybean (Glycine max L. Merr.) development. The transition process is regulated by photoperiod, temperature, and their interaction. To examine the genetic architecture associated with temperature- and photo-thermal-mediated regulation of soybean flowering, we here performed a genome-wide association study using a panel of 201 soybean cultivars with maturity groups ranging from MG 000 to VIII. Each cultivar was grown in artificially controlled photoperiod and different seasons in 2017 and 2018 to assess the thermal response (TR) and the interactive photo-thermal response (IPT) of soybean flowering time. The panel contained 96,299 SNPs with minor allele frequencies > 5%; 33, 19, and 49 of these SNPs were significantly associated with only TR, only IPT, and both TR and IPT, respectively. Twenty-one SNPs were located in or near previously reported quantitative trait loci for first-flowering; 16 SNPs were located within 200 kb of the main-effect flowering genes GmFT2a, GmFT2b, GmFT3a, GmFT3b, GmFT5a, GmFT5b, GmCOL2b, GmPIF4b, and GmPIF4c, or near homologs of the known Arabidopsis thaliana flowering genes BBX19, VRN1, TFL1, FUL, AGL19, SPA1, HY5, PFT1, and EDF1. Natural non-synonymous allelic variations were identified in the candidate genes Glyma.08G302500 (GmHY5), Glyma.08G303900 (GmPIF4c), and Glyma.16G046700 (GmVRN1). Cultivars with different haplotypes showed significant variations in TR, IPT, and flowering time in multiple environments. The favorable alleles, candidate genes, and diagnostic SNP markers identified here provide valuable information for future improvement of soybean photo-thermal adaptability, enabling expansion of soybean production regions and improving plant resilience to global climate change.

Effects of high night temperature on soybean yield and compositions.

Introduction: Soybean is sensitive to light and temperature. Under the background of global asymmetric climate warming. Methods: The increase of night temperature may have an important impact on soybean yield. In this study, three varieties with different level of protein were planted under 18°C and 28°C night temperatures for investigating the effects of high night temperatures on soybean yield formation and the dynamic changes of non-structural carbohydrates (NSC) during the seed filling period (R5-R7). Results and discussion: The results indicated that high night temperatures resulted in smaller seed size, lower seed weight, and a reduced number of effective pods and seeds per plant, and thus, a significant reduction in yield per plant. Analysis of the seed composition variations showed carbohydrates were more substantially affected by high night temperature than protein and oil. We observed "carbon hunger" caused by high night temperature increased photosynthesis and sucrose accumulation in the leaves during the early stage of high night temperature treatment. With elongated treated time, the excessive carbon consumption led to the decrease of sucrose accumulation in soybean seeds. Transcriptome analysis of leaves after 7 days of treatment showed that the expression of most sucrose synthase and sucrose phosphatase genes decreased significantly under the high night temperature. Which could be another important reason for the decrease of sucrose. These findings provided a theoretical basis for enhancing the tolerance of soybean to high night temperature.

Geographic distributions and the regionalization of soybean seed compositions across China.

As one of major food crops, soybean is grown over a broad ecological region in China with considerable variations in environmental conditions, and the seed compositions of soybeans are diverse among different regions. To clarify the spatial patterns of soybean seed compositions, crude oil, protein, and 11 categories of functional components were quantified in 1792 soybean samples collected from a vast range of soybean planting regions across China spanning from 2010 to 2017. The Kriging interpolation maps presented a clear north-to-south (high latitude to low latitude) increasing trend in contents of crude protein and dietary fiber and decreasing trend in contents of crude oil, phospholipids, saponins, and carotenoids. Soybeans with high-level of total oligosaccharide were concentrated in the central region. Based on the geographical distribution of soybean nutritional components, weather conditions, and cultivation systems, the soybean production areas in China were divided into three regions and 10 subregions. This study highlights the geographic distribution of soybean nutritional compositions and provides scientific evidence for guiding the construction of high-quality edible soybean production bases in China.

Can Soybean Cultivars with Larger Seed Size Produce More Protein, Lipids, and Seed Yield? A Meta-Analysis.

Increasing soybean production and ensuring greater access to soybean protein and lipids is critical for global food security and human health. Seed size (i.e., seed weight) is one of the most important agronomic traits of soybean, which not only determines the seed yield, but can also affect the yield of protein and lipids. In China, farmers favor soybean cultivars with large seeds, which they believe produce more protein and lipids; however, experimental evidence supporting this belief is lacking. Therefore, we conducted field experiments from 2017 to 2020 at 35 locations across the Huang-Huai-Hai region (HHH) of China with 64 soybean cultivars. The seed yield, seed protein content, and seed lipids content of soybean, and their relationship with seed size were investigated. The highest seed yield (i.e., seed weight per unit area) was 2996.5 kg ha−1 in the north of HHH. However, the highest seed protein content was found in the south of HHH (42.5%) for the higher temperature, which was significantly higher than that of the middle (41.7%) and north of HHH (40.2%). In contrast, the highest seed lipids content was 20.7% in the north of HHH. Temperature, which had a path coefficient on seed yield of 0.519, can promote soybean seed yield. The correlation analysis indicated that the selection of the large seed size cultivar did not increase seed yield, and even led to a reduction of seed yield under high-yield environmental conditions. The seed protein content of soybean was not increased in the cultivars with large seed sizes. In addition, under different levels of seed lipids content (<20.30% or >20.30%), a significantly negative relationship was found between seed lipids content and hundred seed weight. Therefore, it is recommended that farmers choose to plant cultivars with smaller soybean seed sizes, so as to ensure high and stable soybean seed yield and obtain more vegetable protein and lipids per unit area.

QTL analyses of soybean root system architecture revealed genetic relationships with shoot-related traits.

KEY MESSAGE: The genetic basis of soybean root system architecture (RSA) and the genetic relationship between shoot and RSA were revealed by integrating data from recombinant inbred population grafting and QTL mapping. Variations in root system architecture (RSA) affect the functions of roots and thus play vital roles in plant adaptations and agricultural productivity. The aim of this study was to unravel the genetic relationship between RSA traits and shoot-related traits in soybean. This study characterized RSA variability at seedling stage in a recombinant inbred population, derived from a cross between cultivated soybean C08 and wild soybean W05, and performed high-resolution quantitative trait locus (QTL) mapping. In total, 34 and 41 QTLs were detected for RSA-related and shoot-related traits, respectively, constituting eight QTL clusters. Significant QTL correspondence was found between shoot biomass and RSA-related traits, consistent with significant correlations between these phenotypes. RSA-related QTLs also overlapped with selection regions in the genome, suggesting the cultivar RSA could be a partial consequence of domestication. Using reciprocal grafting, we confirmed that shoot-derived signals affected root development and the effects were controlled by multiple loci. Meanwhile, RSA-related QTLs were found to co-localize with four soybean flowering-time loci. Consistent with the phenotypes of the parental lines of our RI population, diminishing the function of flowering controlling E1 family through RNA interference (RNAi) led to reduced root growth. This implies that the flowering time-related genes within the RSA-related QTLs are actually contributing to RSA. To conclude, this study identified the QTLs that determine RSA through controlling root growth indirectly via regulating shoot functions, and discovered superior alleles from wild soybean that could be used to improve the root structure in existing soybean cultivars.

Genomic Dissection and Diurnal Expression Analysis Reveal the Essential Roles of the PRR Gene Family in Geographical Adaptation of Soybean.

Pseudo-response regulator (PRR) family members serve as key components of the core clock of the circadian clock, and play important roles in photoperiodic flowering, stress tolerance, growth, and the development of plants. In this study, 14 soybean PRR genes were identified, and classified into three groups according to phylogenetic analysis and structural characteristics. Real-time quantitative PCR analysis revealed that 13 GmPRRs exhibited obvious rhythmic expression under long-day (LD) and short-day (SD) conditions, and the expression of 12 GmPRRs was higher under LD in leaves. To evaluate the effects of natural variations in GmPRR alleles on soybean adaptation, we examined the sequences of GmPRRs among 207 varieties collected across China and the US, investigated the flowering phenotypes in six environments, and analyzed the geographical distributions of the major haplotypes. The results showed that a majority of non-synonymous mutations in the coding region were associated with flowering time, and we found that the nonsense mutations resulting in deletion of the CCT domain were related to early flowering. Haplotype analysis demonstrated that the haplotypes associated with early flowering were mostly distributed in Northeast China, while the haplotypes associated with late flowering were mostly cultivated in the lower latitudes of China. Our study of PRR family genes in soybean provides not only an important guide for characterizing the circadian clock-controlled flowering pathway but also a theoretical basis and opportunities to breed varieties with adaptation to specific regions and farming systems.

GmFT3a fine-tunes flowering time and improves adaptation of soybean to higher latitudes.

Onset of flowering of plants is precisely controlled by extensive environmental factors and internal molecular networks, in which FLOWERING LOCUS T (FT) is a key flowering integrator. In soybean, a typical short-day plant, 11 FT homologues are found in its genome, of which several homologues are functionally diversified in flowering pathways and the others including GmFT3a are yet unknown. In the current study, we characterized GmFT3a, which is located on the same chromosome as the flowering promoters GmFT2a and GmFT5a. Overexpression of GmFT3a significantly promoted flowering of Arabidopsis under the inductive long-day (LD) photoperiod. GmFT3a over-expressed soybean also flowered earlier than the control under LD, but they were not significantly different under inductive short-day (SD) conditions, indicating that GmFT3a acts as a flowering promoter in the non-inductive photoperiod in soybean. Compared with other GmFT homologues, GmFT3a exhibited a slighter effect in flowering promotion than GmFT2a, GmFT5a and GmFT2b under LD conditions. GmFT3a promoted flowering by regulating the expression of downstream flowering-related genes and also affected the expression of other GmFTs. According to the re-sequencing data, the regional distributions of two major haplotypes in 176 soybean varieties were analyzed. The varieties with GmFT3a-Hap2 haplotype matured relatively early, and relative higher expression of GmFT3a was detected in early maturing varieties, implying that Hap2 variation may contribute to the adaptation of soybean to higher latitude regions by increasing expression level of genes in metabolism and signaling pathways. The early flowering germplasm generated by overexpression of GmFT3a has potential to be planted at higher latitudes where non-inductive long day is dominant in the growing season, and GmFT3a can be used to fine-tune soybean flowering and maturity time and improve the geographical adaptation.

CONSTANS Polymorphism Modulates Flowering Time and Maturity in Soybean.

CONSTANS (CO) plays a critical role in the photoperiodic flowering pathway. However, the function of soybean CO orthologs and the molecular mechanisms in regulating flowering remain largely unknown. This study characterized the natural variations in CO family genes and their association with flowering time and maturity in soybeans. A total of 21 soybean CO family genes (GmCOLs) were cloned and sequenced in 128 varieties covering 14 known maturity groups (MG 0000-MG X from earliest to latest maturity). Regarding the whole genomic region involving these genes, GmCOL1, GmCOL3, GmCOL8, GmCOL9, GmCOL10, and GmCOL13 were conserved, and the remaining 15 genes showed genetic variation that was brought about by mutation, namely, all single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels). In addition, a few genes showed some strong linkage disequilibrium. Point mutations were found in 15 GmCOL genes, which can lead to changes in the potential protein structure. Early flowering and maturation were related to eight genes (GmCOL1/3/4/8/13/15/16/19). For flowering and maturation, 11 genes (GmCOL2/5/6/14/20/22/23/24/25/26/28) expressed divergent physiognomy. Haplotype analysis indicated that the haplotypes of GmCOL5-Hap2, GmCOL13-Hap2/3, and GmCOL28-Hap2 were associated with flowering dates and soybean maturity. This study helps address the role of GmCOL family genes in adapting to diverse environments, particularly when it is necessary to regulate soybean flowering dates and maturity.

Establishment of a novel experimental system for studying the photoperiodic response of short-day dicots using soybean 'cotyledon-only plant' as material.

Soybean is an important model crop for photoperiodic response studies in plants and contributes significantly to the study of plant development and physiology in the past century. Because soybean plant is much bigger in size and longer in life cycle than Arabidopsis, it needs much more space for growth and time for investigation, which significantly hamper the efficiency of research. In the current study, we tested the photoperiodic response of a distinctive artificially-made cotyledon-only plant (COP) using a photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and other varieties with diverse sensitivity to photoperiod. ZGDD COPs flowered 39.4 ± 2.5 d after emergence under short-day conditions but maintained vegetative growth under long-day and night break conditions, which is similar to the case in the intact ZGDD plants. The COPs of early-maturing and medium-maturing soybean varieties also grew and flowered normally under natural day-length conditions. At the molecular level, the key genes in the photoperiodic pathway such as E1, GmFT1a, GmFT2a, and GmFT5a in the COPs also showed the same photoperiod sensitivity as in the intact plants. In addition, a simpler material of COP with only one cotyledon and root was generated and found to be sensitive to photoperiod as well. Notably, the COPs are only one-fifth the height of intact plants and one-third the maximum diameter of the intact plants grown in chambers 30 d after emergence. Based on COPs, we established a novel experimental system characterized by an entire photoperiodic response and longer longevity of cotyledons in addition to small plant size, ensuring the consistency, reliability, and stability of plant materials. COPs have the potential to be a novel model material for studies of the developmental biology of soybean and other dicots.

Speed-Breeding System in Soybean: Integrating Off-Site Generation Advancement, Fresh Seeding, and Marker-Assisted Selection.

Speed breeding by artificial control of photothermal conditions facilitates generation advancement but was limited in scale and cost. In this study, we demonstrated a cost-saving off-site summer nursery pattern, taking full advantage of shorter daylength and higher temperature with lower latitude compared to the origin of the soybean cultivars used in the study. This substantially reduced the generation cycles under totally natural conditions. Using this approach, two generations of soybean cultivars from Northeastern Spring Planting Region (NE) and Yellow-Huai-Hai Valleys Summer Planting Region (YHH) were successfully obtained in Beijing and Hainan, respectively, compared to one generation in origin. Fresh-seeding method was also used to further shorten the generation duration by 7-10 days, thereby allowing at least four generations per year. Using DNA markers to define haplotypes of maturity genes E1-E4, we proposed a model to predict the optimum adaptation region of the advanced generation lines. Taken together, we present a speed-breeding methodology combining off-site nursery, fresh-seeding method, and marker-assisted selection, aimed at accelerating soybean improvement.

Cotyledons facilitate the adaptation of early-maturing soybean varieties to high-latitude long-day environments.

Soybean (Glycine max), a typical short-day plant (SDP) domesticated in temperate regions, has expanded to high latitudes where daylengths are long from soybean emergence to bloom, but rapidly decrease from seed filling to maturity. Cotyledons are well known as the major storage organs in seeds, but it is unclear whether developing cotyledons store flowering substances at filling stage in SD for upcoming seedlings, or instead respond to photoperiod for floral induction after emergence of matured seeds in long-day (LD). Here, we report that cotyledons accelerate flowering of early-maturing varieties not resulting from stored floral stimuli but by perceiving photoperiod after emergence. We found that light signal is indispensable to activate cotyledons for floral induction, and flowering promoting gene GmFT2a is required for cotyledon-dependent floral induction via upregulation of floral identity gene GmAP1. Interestingly, cotyledons are competent to support the entire life cycle of a cotyledon-only plant to produce seeds, underlying a new photoperiod study system in soybean and other dicots. Taken together, these results demonstrate a substantial role for cotyledons in flowering process, whereby we propose a 'cotyledon-based self-reliance' model highlighting floral induction from emergence as a key ecological adaptation for rapid flowering of SDPs grown in LD environments at high latitudes.

GmNMHC5, A Neoteric Positive Transcription Factor of Flowering and Maturity in Soybean.

The soybean (Glycine max (L.) Merr.) is an important oil and food crop. Its growth and development is regulated by complex genetic networks, and there are still many genes with unknown functions in regulation pathways. In this study, GmNMHC5, a member of the MADS-box protein family, was found to promote flowering and maturity in the soybean. Gene expression profiling in transgenic plants confirmed that the 35S:GmNMHC5 T3 generation had early flowering and precocity. We used CRISPR-Cas9 to edit GmNMHC5 and found that late flowering and maturity occurred in Gmnmhc5 lines with stable inheritance. Remarkably, in the 35S:GmNMHC5 plants, the expression of flowering inhibitors GmFT1a and GmFT4 was inhibited. In addition, overexpression of GmNMHC5 in ft-10 (a late flowering Arabidopsis thaliana mutant lacking Flowering Locus T (FT) function) rescued the extremely late-flowering phenotype of the mutant A. thaliana. These results suggest that GmNMHC5 is a positive transcription factor of flowering and maturity in the soybean, which has a close relationship with FT homologs in the flowering regulation pathway. This discovery provides new ideas for the improvement of the flowering regulation network, and can also provide guidance for future breeding work.

Allele combinations of maturity genes E1-E4 affect adaptation of soybean to diverse geographic regions and farming systems in China.

Appropriate flowering and maturity time are important for soybean production. Four maturity genes E1, E2, E3 and E4 have been molecularly identified and found to play major roles in the control of flowering and maturity of soybean. Here, to further investigate the effect of different allele combinations of E1-E4, we performed Kompetitive Allele Specific PCR (KASP) assays based on single nucleotide polymorphisms (SNPs) at these four E loci, and genotyped E1-E4 genes across 308 Chinese cultivars with a wide range of maturity groups. In total, twenty-one allele combinations for E1-E4 genes were identified across these Chinese cultivars. Various combinations of mutations at four E loci gave rise to the diversity of flowering and maturity time, which were associated with the adaptation of soybean cultivars to diverse geographic regions and farming systems. In particular, the cultivars with mutations at all four E loci reached flowering and maturity very early, and adapted to high-latitude cold regions. The allele combinations e1-as/e2-ns/e3-tr/E4, E1/e2-ns/E3/E4 and E1/E2/E3/E4 played important roles in the Northeast China, Huang-Huai-Hai (HHH) Rivers Valley and South China regions, respectively. Notably, E1 and E2, especially E2, affected flowering and maturity time of soybean significantly. Our study will be beneficial for germplasm evaluation, cultivar improvement and regionalization of cultivation in soybean production.

Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b, an ortholog of FLOWERING LOCUS T.

Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a 'weight' model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.

Natural variation and CRISPR/Cas9-mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean.

Flowering time is a critical determinant of the geographic distribution and regional adaptability of soybean (Glycine max) and is strongly regulated by photoperiod and temperature. In this study, quantitative trait locus (QTL) mapping and subsequent candidate gene analysis revealed that GmPRR37, encoding a pseudo-response regulator protein, is responsible for the major QTL qFT12-2, which was identified from a population of 308 recombinant inbred lines (RILs) derived from a cross between a very late-flowering soybean cultivar, 'Zigongdongdou (ZGDD)', and an extremely early-flowering cultivar, 'Heihe27 (HH27)', in multiple environments. Comparative analysis of parental sequencing data confirmed that HH27 contains a non-sense mutation that causes the loss of the CCT domain in the GmPRR37 protein. CRISPR/Cas9-induced Gmprr37-ZGDD mutants in soybean exhibited early flowering under natural long-day (NLD) conditions. Overexpression of GmPRR37 significantly delayed the flowering of transgenic soybean plants compared with wild-type under long photoperiod conditions. In addition, both the knockout and overexpression of GmPRR37 in soybean showed no significant phenotypic alterations in flowering time under short-day (SD) conditions. Furthermore, GmPRR37 down-regulated the expression of the flowering-promoting FT homologues GmFT2a and GmFT5a, and up-regulated flowering-inhibiting FT homologue GmFT1a expression under long-day (LD) conditions. We analysed haplotypes of GmPRR37 among 180 cultivars collected across China and found natural Gmprr37 mutants flower earlier and enable soybean to be cultivated at higher latitudes. This study demonstrates that GmPRR37 controls soybean photoperiodic flowering and provides opportunities to breed optimized cultivars with adaptation to specific regions and farming systems.

Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean.

Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean (Glycine max) to diverse latitudes and farming systems. GmFT2a and GmFT5a have been extensively identified as flowering activators and integrators in soybean. Here, we identified two quantitative trait loci (QTLs) regions harbouring GmFT2a and GmFT5a, respectively, associated with different genetic effects on flowering under different photoperiods. We analysed the flowering time of transgenic plants overexpressing GmFT2a or GmFT5a, ft2a mutants, ft5a mutants and ft2aft5a double mutants under long-day (LD) and short-day (SD) conditions. We confirmed that GmFT2a and GmFT5a are not redundant, they collectively regulate flowering time, and the effect of GmFT2a is more prominent than that of GmFT5a under SD conditions whereas GmFT5a has more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions. The ft2aft5a double mutants showed late flowering by about 31.3 days under SD conditions and produced significantly increased numbers of pods and seeds per plant compared to the wild type. We speculate that these mutants may have enormous yield potential for the tropics. In addition, we examined the sequences of these two loci in 202 soybean accessions and investigated the flowering phenotypes, geographical distributions and maturity groups within major haplotypes. These results will contribute to soybean breeding and regional adaptability.

A Combined Linkage and GWAS Analysis Identifies QTLs Linked to Soybean Seed Protein and Oil Content.

Soybean is an excellent source of vegetable protein and edible oil. Understanding the genetic basis of protein and oil content will improve the breeding programs for soybean. Linkage analysis and genome-wide association study (GWAS) tools were combined to detect quantitative trait loci (QTL) that are associated with protein and oil content in soybean. Three hundred and eight recombinant inbred lines (RILs) containing 3454 single nucleotide polymorphism (SNP) markers and 200 soybean accessions, including 94,462 SNPs and indels, were applied to identify QTL intervals and significant SNP loci. Intervals on chromosomes 1, 15, and 20 were correlated with both traits, and QTL qPro15-1, qPro20-1, and qOil5-1 reproducibly correlated with large phenotypic variations. SNP loci on chromosome 20 that overlapped with qPro20-1 were reproducibly connected to both traits by GWAS (p < 10-4). Twenty-five candidate genes with putative roles in protein and/or oil metabolisms within two regions (qPro15-1, qPro20-1) were identified, and eight of these genes showed differential expressions in parent lines during late reproductive growth stages, consistent with a role in controlling protein and oil content. The new well-defined QTL should significantly improve molecular breeding programs, and the identified candidate genes may help elucidate the mechanisms of protein and oil biosynthesis.