In 2017, I joined Tian’s group as a PhD student. We discussed the project on cloning and functional characterization of the gene that controlling branch number in soybean. I realized that it is a very study-worthy scientific question, because branch number is one of the most profound traits that determine the final yield of soybean. It is also a big challenge because the branch number is affected not only by internal genetic factors but also by external environment condition. Nevertheless, few major genes controlling branch number had been reported in soybean, which make it more study-worthy. Thereafter, a challenging journey opened. The first tough task is to phenotype the natural population in the field. To make the phenotyping accuracy, I spent three months in each growing season, which is not an easy time to spend. We need to endure loneliness and keep our original intention. We need to cheer ourself up every day. We overcame various problems and finally phenotyped the branch number of 2,409 soybean accessions in two years (Fig. 1).
Figure 1. a) Soybean natural population planted in field in 2017 on Wuqing Farm, Tianjin City, China. b) Soybean natural population planted in field in 2018 on Shunyi Farm, Beijing City, China.
The good thing is that our team has accumulated genotyping data for these accessions1-3. When we got the two years’ phenotypic data, we can’t wait to carried out the GWAS with trepidation and begging for a good signal. And the exciting thing was a stable association signal on the chromosome 18 within 40 kb interval was found. However, upon careful analysis, we determined that a known star gene Dt2 might be the candidate gene, which made me frustrated again.
In 2014, Dt2 was identified to be one of the key genes to regulate stem growth in soybean4. Thereafter, Dt2 was further functional characterized as a post-domestication mutation modulating multiple agronomic traits5,6.However, whether Dt2 could modulate branch number had not been investigated in the previous study. Therefore, we wondered that a further dig of this “old” gene may shed light on its new function.
To check if Dt2 control branch number, we knocked out this gene using CRISPR-Cas9 technology and over-expressed it driven by 35S promoter. In 2020, we investigated the field phenotype with trepidation and found the Dt2CR lines exhibited increased branch number compared with the wild type DN50. We also found that the Dt2CR lines showed significantly delayed flowering, increased plant height, increased higher 100 seed weight and higher grain weight per plant, resulting in significantly increased yield per plot. In contrast to the results from the Dt2CR lines, the Dt2OE lines exhibited decreased branch number, promoted flowering time and maturity, decreased plant height, thus exhibiting decreased yield per plot (Fig 2).
Figure 2. Transgenic lines of Dt2 phenotype identification in filed in 2020. The 1-8 represent the DN50, Dt2-OE-1, Dt2-OE-2, Dt2-OE-3, Dt2-CR-1, Dt2-CR-2, Dt2-CR-3, Dt2-CR-4 respectively.
Next, we made some basic experiments to figure out how Dt2 affect branching. Transcriptional profiling and in situ hybridization assay show that Dt2 mainly expressed in the shoot apical meristem and axillary meristem. In view of the function that Dt2 affect the transition from the vegetative to the reproductive phase, we wanted to know which genes were regulated by Dt2 in this process. So we made RNA-seq analysis with lateral buds in leaf axils, and many of these regulated genes were found to be related to flowering, in which three GmAp1 gene family members were significantly up-regulated in the Dt2OE lines and down-regulated in the Dt2CR line. In soybean, GmAP1 family genes are the characteristic genes of inflorescence meristem development7. Nevertheless, none of these flowering bases had ever been studied for branching number. So we constructed the transgenic lines and investigated the field phenotype. And the exciting thing was that GmAp1 genes, their phenotypes had a significant effect on branching number. And the subsequent biochemistry experiments showed that Dt2 functioned as a transcriptional activator to promote the transcription of GmAp1a and GmAp1d.
Next, we wanted to explore the gene function in protein level. Protein structure domain assay showed that Dt2 had four domains and the K-box domain was considered as the interaction binding region with other protein4,5, so we inferred that Dt2 protein may exert the function by interacting with other protein. Then we conducted a yeast two hybrid (Y2H) experiment and found GmAgl22, a gene belong to MADS-box gene family and related to the flower development, was identified and further verified by the basic protein interaction biochemistry assays. A previous study reported that Dt2 could interact with GmSoc1a to affect the soybean growth habit5, so we suspected that the three protein could interact each other and subsequent was verified by the Y2H, BiFC and Co-IP assay. We also found that the GmSoc1aCR line showed significantly increased branch number, and the GmAgl22OE lines showed significantly decreased branch number. These results indicated that Dt2, GmAgl22 and GmSoc1a may function by forming a complex to control branching in soybean, further biochemistry experiments found that Dt2-GmAgl22-GmSoc1a together showed stronger activity than Dt2 alone.
When we made the statistical analysis of geographical distribution of different haplotypes, we were surprised to find that the geographical distribution in different haplotypes show some pattern. The Dt2HapII mainly distributed in the high latitude in I ecoregion, while Dt2HapI mainly distributed in middle and low latitudes. The branch number of accessions increased gradually from high latitude to low latitude. Further we wanted to know whether this difference was domesticated or not, so we made the adaptation analysis and found that the distinct geographic distribution of Dt2 haplotypes maybe related to soybean adaptation to different latitudes.
Interestingly, modulating the Dt2HapII (such as knocked out) could enhance the latitudes adaption. Because when a soybean accession was planted from higher latitudes to lower latitudes, it usually exhibited a significant yield decrease due to the early flowering and maturity8. The geographic and genetic differentiations of Dt2 inspired us that modification of Dt2 may improve the adaptation of soybean. Therefore, we tried to plant Dt2CR lines in different ecoregions in China, from Heilongjiang province, a region located at high latitudes to Beijing (in the middle of China) and Hainan province (in the southern China). The Dt2CR lines showed significantly higher yields than DN50 (Fig 3). These results suggested that modulating Dt2HapII could break the restriction of ecoregions, enhance soybean adaptation ability and will provide more insight for soybean improvement.
Figure 3. Phenotype identification between Dt2-CR liens and DN50 in different ecoregions. a-c represent the location of Haerbin of Heilong jiang province, Beijing and Sanya of Hainan province, respectively.
Therefore, after a long challenge journey, we shed light on that the previously reported “old” gene, Dt2, is the key gene controlling branch in soybean.
- Zhou, Z. et al. Resequencing 302 wild and cultivated accessions identifies genes related to domestication and improvement in soybean. Nature Biotechnology 33, 408-414 (2015).
- Fang, C. et al. Genome-wide association studies dissect the genetic networks underlying agronomical traits in soybean. Genome Biol. 18, 161 (2017).
- Liu, Y. et al. Pan-genome of wild and cultivated soybeans. Cell 182, 162-176 (2020).
- Ping, J. et al. Dt2 is a gain-of-function MADS-domain factor gene that specifies semideterminacy in soybean. Plant Cell 26, 2831-2842 (2014).
- Liu, Y. et al. Innovation of a regulatory mechanism modulating semi-determinate stem growth through artificial selection in soybean. PLoS Genet. 12, e1005818 (2016).
- Zhang, D. et al. A post-domestication mutation, Dt2, triggers systemic modification of divergent and convergent pathways modulating multiple agronomic traits in soybean. Mol. Plant 12, 1366-1382 (2019).
- Chen, L. et al. Soybean AP1 homologs control flowering time and plant height. J. Integr. Plant Biol. 62, 1868-1879 (2020).
- Lu, S. et al. Natural variation at the soybean J locus improves adaptation to the tropics and enhances yield. Nat. Genet. 49, 773-779 (2017).