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Soybean seeds contain high levels of oil and protein, providing 57 and 69% of a person's dietary requirements, respectively. Although many quantitative trait loci for the 100-seed weight (100SW), protein content (PRC), and oil content (OIC) have been reported, their genetic controls in soybeans remain unclear. The QTL–allele constitution of three traits in the Sichuan and Chongqing eco-regions population (SCLBP) was studied using a representative sample composed of 228 accessions. These were tested in four environments and analyzed using 135 simple sequence repeats (SSR) and 107,081 valid single nucleotide polymorphism linkage (SNP) markers. The range of 100SW, PRC, and OIC in SCLBP accessions were 4.82–33.35, 36.47–49.75%, and 14.68–21.77%, respectively. The heritability (h2) and genetic coefficient of variation (GCV) of the three traits were high. As a result, 26, 33, and 31 QTLs were found using SSR for 100SW, PRC, and OIC, respectively. The allele of Sat_260 for 100SW was detected in the four environments. In addition, 28, 198, and 250 loci for 100SW, PRC, and OIC, respectively, were found using SNP and mixed linear model (MLM). Further SNP haplotype analysis revealed that 13, 35, and 60 blocks were found for 100SW, RPC, and OIC, respectively. The block of Gm11_9895764-9,917,646 for 100SW was simultaneously detected in the four environments. Among these QTLs, 1, 5, and 7 for 100SW, PRC and OIC were found using two methods of SSR and SNP at the same time. A majority of these QTLs overlapped with the previously reported loci. However, 9, 11, and 9 loci for 100SW, PRC, and OIC using SSR; and 3, 5, and 8 for 100SW, PRC, and OIC hadn’t been reported using SNP. Moreover, the genes of Glyma.11g130800, Glyma.13g217000, and Glyma.08g122600 were considered the most likely genes controlling 100SW, PRC, and OIC, respectively. These findings provide evidence for mixed major plus polygenes inheritance for the three traits and an extended understanding of their genetic architecture for the molecular dissection and breeding of soybeans.

Legume plants form symbiotic relationships with rhizobia to convert N2 into ammonia, and the nodulation status can affect plant development including photosynthesis. However, the relationship between nitrogen fixation and photosynthesis during carbon and nitrogen metabolism remains unclear. This study was undertaken to unravel regulation of nodulation and photosynthesis using a spontaneous nonnodulated soybean mutant by grafting. The results of inheritance and gene mapping showed that the nonnodulated mutant was controlled by a recessive gene overlapped with the reported rj1 locus, and might be a new rj1 allele with 1 bp deletion in the fourth exon in comparison to the sequence of normal nodulation plants. According to grafting results, soybean nodulation is obviously determined by the roots, not the seedlings. Moreover, nitrogen content along with related metabolic enzyme activity, and photosynthetic capacity were enhanced by nonnodulated scions grafted with nodulated roots. Contrary results were obtained for nodulated scions grafted with nonnodulated roots. A total of 853 differentially expressed genes (DEGs) in the leaves and 1874 in the roots were identified by transcriptome analyses of the grafting treatments. We identified 285 differential gene ontology (GO) terms and 57 differential pathway terms identified in the leaves, while 856 differential GO terms and 207 differential pathway terms in the roots. Twenty DEGs interacting at translation level were selected, and the results of transcriptome analyses were verified by q-PCR. These findings indicated that the nodulation-related Nod allelic gene increases the nitrogen content of nonnodulated plants, which affects the enzymes involved in nitrogen metabolism, leading to changes in hormone levels and further regulation of photosynthesis and carbon metabolism.

Annual wild soybean (G. soja) is the ancestor of the cultivated soybean (G. max). To reveal the genetic changes from soja to max, an improved wild soybean chromosome segment substitution line (CSSL) population, SojaCSSLP5, composed of 177 CSSLs with 182 SSR markers (SSR-map), was developed based on SojaCSSLP1 generated from NN1138-2(max)×N24852(soja). The SojaCSSLP5 was genotyped further through whole-genome resequencing, resulting in a physical map with 1366 SNPLDBs (SNP linkage-disequilibrium blocks), which are composed of more markers/segments, shorter marker length and more recombination breakpoints than the SSR-map and caused 721 new wild substituted segments. Using the SNPLDB-map, two loci co-segregating with seed-coat color (SCC) and six loci for days to flowering (DTF) with 88.02% phenotypic contribution were identified. Integrated with parental RNA-seq and DNA-resequencing, two SCC and six DTF candidate genes, including three previously cloned (G, E2 and GmPRR3B) and five newly detected ones, were predicted and verified at nucleotide mutant level, and then demonstrated with the consistency between gene-alleles and their phenotypes in SojaCSSLP5. In total, six of the eight genes were identified with the parental allele-pairs coincided to those in 303 germplasm accessions, then were further demonstrated by the consistency between gene-alleles and germplasm phenotypes. Accordingly, the CSSL population integrated with parental DNA and RNA sequencing data was demonstrated to be an efficient platform in identifying candidate wild vs. cultivated gene-alleles.

Size and shape of soybean seeds are closely related to seed yield and market value. Annual wild soybeans have the potential to improve cultivated soybeans, but their inferior seed characteristics should be excluded. To detect quantitative trait loci (QTLs)/segments of seed size and shape traits in annual wild soybean, its chromosome segment substitution lines (CSSLs) derived from NN1138-2 (recurrent parent, Glycine max) and N24852 (donor parent, Glycine soja) and then modified 2 iterations (coded SojaCSSLP3) were improved further to contain more lines (diagonal segments) and less heterozygous and missing portions. The new population (SojaCSSLP4) composed of 195 CSSLs was evaluated under four environments, and 11, 13, 7, 15 and 14 QTLs/segments were detected for seed length (SL), seed width (SW), seed roundness (SR), seed perimeter (SP) and seed cross section area (SA), respectively, with all 60 wild allele effects negative. Among them, 16 QTLs/segments were shared by 2–5 traits, respectively, but 0–3 segments for each of the 5 traits were independent. The non-shared Satt274 and shared Satt305, Satt540 and Satt239 were major segments, along with other segments composed of two different but related sets of genetic systems for SR and the other 4 traits, respectively. Compared with the literature, 7 SL, 5 SW and 2 SR QTLs/segments were also detected in cultivated soybeans; allele distinction took place between cultivated and wild soybeans, and also among cultivated parents. The present mapping is understood as macro-segment mapping, the segments may be further dissected into smaller segments as well as corresponding QTLs/genes.

A wild soybean allele conferring 100-seed weight, protein content and oil content simultaneously was fine-mapped to a 329-kb region on Chromosome 15, in which Glyma.15g049200 was predicted a candidate gene. Annual wild soybean characterized with small 100-seed weight (100SW), high protein content (PRC), low oil content (OIC) may contain favourable alleles for broadening the genetic base of cultivated soybeans. To evaluate these alleles, a population composed of 195 chromosome segment substitution lines (SojaCSSLP4), with wild N24852 as donor and cultivated NN1138-2 as recurrent parent, was tested. In SojaCSSLP4, 10, 9 and 8 wild segments/QTL were detected for 100SW, PRC and OIC, respectively. Using a backcross-derived secondary population, one segment for the three traits (q100SW15, qPro15 and qOil15) and one for 100SW (q100SW18.2) were fine-mapped into a 329-kb region on chromosome 15 and a 286-kb region on chromosome 18, respectively. Integrated with the transcription data in SoyBase, 42 genes were predicted in the 329-kb region where Glyma.15g049200 showed significant expression differences at all seed development stages. Furthermore, the Glyma.15g049200 segments of the two parents were sequenced and compared, which showed two base insertions in CDS (coding sequence) in the wild N24852 comparing to the NN1138-2. Since only Glyma.15g049200 performed differential CDS between the two parents but related to the three traits, Glyma.15g049200 was predicted a pleiotropic candidate gene for 100SW, PRC and OIC. The functional annotation of Glyma.15g049200 indicated a bidirectional sucrose transporter belonging to MtN3/saliva family which might be the reason that this gene provides a same biochemical basis for 100SW, PRC and OIC, therefore, is responsible for the three traits. This result may facilitate isolation of the specific gene and provide prerequisite for understanding the other two pleiotropic QTL.

Jasmonates (JAs) are key phytohormones involved in regulation of plant growth and development, stress responses, and secondary metabolism. It has been reported that treatments with JAs could increase the contents of Amaryllidaceae alkaloids in Amaryllidaceae plants. Jasmonate ZIM (zinc-finger inflorescence meristem) domain (JAZ) proteins are key components in JA signal processes. However, JAZ proteins have not been characterized in genus Lycoris. In this study, we identified and cloned seven differentially expressed JAZ genes (namely LaJAZ1-LaJAZ7) from Lycoris aurea. Bioinformatic analyses revealed that these seven LaJAZ proteins contain the ZIM domain and JA-associated (Jas, also named CCT_2) motif. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that these LaJAZ genes display different expression patterns in L. aurea tissues, and most of them are inducible when treated with methyl jasmonate (MeJA) treatment. Subcellular localization assay demonstrated that LaJAZ proteins are localized in the cell nucleus or cytoplasm. In addition, LaJAZ proteins could interact with each other to form homodimer and/or heterodimer. The findings in this study may facilitate further functional research of the LaJAZ genes, especially the potential regulatory mechanism of plant secondary metabolites including Amaryllidaceae alkaloids in L. aurea.

Plant height (PH) and the number of nodes on the main stem (NN) serve as major plant architecture traits affecting soybean seed yield. Although many quantitative trait loci for the two traits have been reported, their genetic controls at different developmental stages in soybeans remain unclear. Here, 368 soybean breeding lines were genotyped using 62,423 single nucleotide polymorphism (SNP) markers and phenotyped for the two traits at three different developmental stages over two locations in order to identify their quantitative trait nucleotides (QTNs) using compressed mixed linear model (CMLM) and multi-locus random-SNP-effect mixed linear model (mrMLM) approaches. As a result, 11 and 13 QTNs were found by CMLM to be associated with PH and NN, respectively. Among these QTNs, 8, 3, and 4 for PH and 6, 6, and 8 for NN were found at the three stages, and 3 and 6 were repeatedly detected for PH and NN. In addition, 34 and 30 QTNs were found by mrMLM to be associated with PH and NN, respectively. Among these QTNs, 11, 13, and 16 for PH and 11, 15, and 8 for NN were found at the three stages. A majority of these QTNs overlapped with the previously reported loci. Moreover, one QTN within the known E2 locus for flowering time was detected for the two traits at all three stages, and another that overlapped with the Dt1 locus for stem growth habit was also identified for the two traits at the mature stage. This may explain the highly significant correlation between the two traits. Our findings provide evidence for mixed major plus polygenes inheritance for dynamic traits and an extended understanding of their genetic architecture for molecular dissection and breeding utilization in soybeans.

The colour of plant organs is a useful trait in crop breeding. The pod colours of soybeans primarily include black, brown and tan types, which are controlled by two classical genetic loci, L1 and L2. Most wild soybeans have black pods, which reflect a possible role in adaptation to the natural environment. Here, an improved chromosome segment substitution line (CSSL) population SojaCSSLP3 was established to identify the L1 gene. The segment on the 19th chromosome represented by the SSR marker Satt313 was found to link with locus L1. The region was further delimited three times with increased SSR and InDel markers using a population derived from a heterozygous plant of CSSL124 from SojaCSSLP3. The L1 gene was finally located in a 184.43-kb region between SSR_19p09 and Indel_19P7. Thirteen putative genes in this region were analysed with qRT-PCR. The expression level of Glyma19 g27460, which is a member of the SANT superfamily with a MYB DNA-binding domain, was significantly upregulated in black pods and was recognized to be the most likely candidate for the L1 gene.

Annual wild soybean characterized with more number of branches and pods may contain favourable exotic genes/alleles for improving the yield potential of cultivated soybeans. To evaluate the wild alleles/segments, the chromosome segment substitution line population SojaCSSLP3 comprising 158 lines with N24852 (wild) as the donor and NN1138-2 (cultivated) as the recurrent parent was tested under three environments. The phenotypic data along with 198 simple sequence repeat markers were analysed for qualitative trait loci (QTL)/segments associated with the number of branches on the main stem (BN) and number of pods per plant (PN) using the inclusive composite interval mapping procedure (RSTEP-LRT-ADD model) of ICIM version 3.0. The analysis was carried out for individual environments due to a significant G × E interaction. A total of eight QTL/segments associated with BN and eight QTL/segments associated with PN were detected under the three environments, with all the wild segments having positive effects. Among these, two QTL/segments for each of the two traits could be detected under two or three environments and three QTL/segments could be detected for both traits. Four QTL/segments associated with BN and one QTL/segment associated with PN were identified only in SojaCSSLP3, not reported for cultivated crosses in the literature. The detected wild segments may provide materials for further characterization, cloning and pyramiding of the alleles conferring the two traits.

Annual wild soybean characterized by low 100-seed weight (100SW), high protein content (PRC) and low oil content (OIC) may have favourable exotic genes/alleles for broadening the genetic base of the cultivated soybean. To evaluate the wild alleles/segments, a chromosome segment substitution line population comprising 151 lines with N24852 (wild) as the donor and NN1138-2 (cultivated) as the recurrent parent was analysed using single-marker analysis, interval mapping, inclusive composite interval mapping and mixed linear composite interval mapping. On 14 segments of ten chromosomes, 17 quantitative trait loci (QTL) were identified, with two segments each containing two QTL for 100SW and OIC and one segment containing two QTL for PRC and OIC, respectively. All the seven wild alleles/segments for 100SW were associated with negative effects and three were associated with positive effects, but one was associated with a negative effect for PRC, and five were associated with negative effects, but one was associated with a positive effect for OIC. Except Satt216 and Sat_224 for 100SW, the identified QTL/segments have been reported from cultivated soybean mapping populations. The detected wild segments may provide materials for further characterization, cloning and pyramiding of the alleles conferring the seed-quality traits.

Chromosome segment substitution line (CSSL) population is potential in precisely detecting and pyramiding genes/QTL/segments due to the genetic background noise removed. To exploit and utilize the favorable wild alleles, a CSSL population with 151 lines (SojaCSSLP1) was generated using a wild soybean (Glycine soja Sieb. et Zucc.) N24852 as donor parent and the elite cultivar NN1138-2 as its genetic background. An improved CSSL construction strategy was used, i.e. continuous backcross after initial crossing followed with alternation of backcross and selfing combined with marker-assisted selection based on pedigree DNA pools and phenotypic differences among pedigrees. The SojaCSSLP1 with an average recovery ratio of 95.7 % of the NN1138-2 genome could cover the entire genome of wild soybean. Four wild alleles/segments for each of the two wild characteristics, longer plant height (PH) and more number of nodes on main stem (NN), in a total of six segments, were detected with additive effects all positive. Among them, Satt243 on Chr.10 and Sat_286 on Chr.19 associated with both PH and NN while Satt338 and SOYGPATR on Chr.4 and Satt314 neighboring with Satt192 on Chr.12 had the former and latter on each chromosome associated with PH and NN, respectively. That could explain the high positive correlation between the two traits (r = 0.88). Compared with those in the literature, three QTL/segments for PH and one for NN were detected also among cultivated soybeans, indicating allele differentiation happened not only between wild and cultivated but also among cultivated soybeans. Therefore these QTL/segments might be the key ones to explain the domestication and evolution of soybean. In addition, SojaCSSLP1 should be also potential in studies for multiple wild traits due to its broad variation.