A route to de novo domestication of wild allotetraploid rice.

Cultivated rice varieties are all diploid, and polyploidization of rice has long been desired because of its advantages in genome buffering, vigorousness, and environmental robustness. However, a workable route remains elusive. Here, we describe a practical strategy, namely de novo domestication of wild allotetraploid rice. By screening allotetraploid wild rice inventory, we identified one genotype of Oryza alta (CCDD), polyploid rice 1 (PPR1), and established two important resources for its de novo domestication: (1) an efficient tissue culture, transformation, and genome editing system and (2) a high-quality genome assembly discriminated into two subgenomes of 12 chromosomes apiece. With these resources, we show that six agronomically important traits could be rapidly improved by editing O. alta homologs of the genes controlling these traits in diploid rice. Our results demonstrate the possibility that de novo domesticated allotetraploid rice can be developed into a new staple cereal to strengthen world food security.

Pan-Genome of Wild and Cultivated Soybeans.

Soybean is one of the most important vegetable oil and protein feed crops. To capture the entire genomic diversity, it is needed to construct a complete high-quality pan-genome from diverse soybean accessions. In this study, we performed individual de novo genome assemblies for 26 representative soybeans that were selected from 2,898 deeply sequenced accessions. Using these assembled genomes together with three previously reported genomes, we constructed a graph-based genome and performed pan-genome analysis, which identified numerous genetic variations that cannot be detected by direct mapping of short sequence reads onto a single reference genome. The structural variations from the 2,898 accessions that were genotyped based on the graph-based genome and the RNA sequencing (RNA-seq) data from the representative 26 accessions helped to link genetic variations to candidate genes that are responsible for important traits. This pan-genome resource will promote evolutionary and functional genomics studies in soybean.

Pan-centromere reveals widespread centromere repositioning of soybean genomes.

Centromere repositioning refers to a de novo centromere formation at another chromosomal position without sequence rearrangement. This phenomenon was frequently encountered in both mammalian and plant species and has been implicated in genome evolution and speciation. To understand the dynamic of centromeres on soybean genome, we performed the pan-centromere analysis using CENH3-ChIP-seq data from 27 soybean accessions, including 3 wild soybeans, 9 landraces, and 15 cultivars. Building upon the previous discovery of three centromere satellites in soybean, we have identified two additional centromere satellites that specifically associate with chromosome 1. These satellites reveal significant rearrangements in the centromere structures of chromosome 1 across different accessions, consequently impacting the localization of CENH3. By comparative analysis, we reported a high frequency of centromere repositioning on 14 out of 20 chromosomes. Most newly emerging centromeres formed in close proximity to the native centromeres and some newly emerging centromeres were apparently shared in distantly related accessions, suggesting their emergence is independent. Furthermore, we crossed two accessions with mismatched centromeres to investigate how centromere positions would be influenced in hybrid genetic backgrounds. We found that a significant proportion of centromeres in the S9 generation undergo changes in size and position compared to their parental counterparts. Centromeres preferred to locate at satellites to maintain a stable state, highlighting a significant role of centromere satellites in centromere organization. Taken together, these results revealed extensive centromere repositioning in soybean genome and highlighted how important centromere satellites are in constraining centromere positions and supporting centromere function.

Chromosome-level reference genome and resequencing of 322 accessions reveal evolution, genomic imprint and key agronomic traits in adzuki bean.

Adzuki bean (Vigna angularis) is an important legume crop cultivated in over 30 countries worldwide. We developed a high-quality chromosome-level reference genome of adzuki bean cultivar Jingnong6 by combining PacBio Sequel long-read sequencing with short-read and Hi-C technologies. The assembled genome covers 97.8% of the adzuki bean genome with a contig N50 of approximately 16 Mb and a total of 32 738 protein-coding genes. We also generated a comprehensive genome variation map of adzuki bean by whole-genome resequencing (WGRS) of 322 diverse adzuki beans accessions including both wild and cultivated. Furthermore, we have conducted comparative genomics and a genome-wide association study (GWAS) on key agricultural traits to investigate the evolution and domestication. GWAS identified several candidate genes, including VaCycA3;1, VaHB15, VaANR1 and VaBm, that exhibited significant associations with domestication traits. Furthermore, we conducted functional analyses on the roles of VaANR1 and VaBm in regulating seed coat colour. We provided evidence for the highest genetic diversity of wild adzuki (Vigna angularis var. nipponensis) in China with the presence of the most original wild adzuki bean, and the occurrence of domestication process facilitating transition from wild to cultigen. The present study elucidates the genetic basis of adzuki bean domestication traits and provides crucial genomic resources to support future breeding efforts in adzuki bean.

The MADS-box transcription factor GmFULc promotes GmZTL4 gene transcription to modulate maturity in soybean.

Flowering time and maturity are crucial agronomic traits that affect the regional adaptability of soybean plants. The development of soybean cultivars with early maturity adapted to longer days and colder climates of high latitudes is very important for ensuring normal ripening before frost begins. FUL belongs to the MADS-box transcription factor family and has several duplicated members in soybeans. In this study, we observed that overexpression of GmFULc in the Dongnong 50 cultivar promoted soybean maturity, while GmFULc knockout mutants exhibited late maturity. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that GmFULc could bind to the CArG, bHLH and homeobox motifs. Further investigation revealed that GmFULc could directly bind to the CArG motif in the promoters of the GmZTL3 and GmZTL4 genes. Overexpression of GmZTL4 promoted soybean maturity, whereas the ztl4 mutants exhibited delayed maturity. Moreover, we found that the cis element box 4 motif of the GmZTL4 promoter, a motif of light response elements, played an important role in controlling the growth period. Deletion of this motif shortened the growth period by increasing the expression levels of GmZTL4. Functional investigations revealed that short-day treatment promoted the binding of GmFULc to the promoter of GmZTL4 and inhibited the expression of E1 and E1Lb, ultimately resulting in the promotion of flowering and early maturation. Taken together, these findings suggest a novel photoperiod regulatory pathway in which GmFULc directly activates GmZTL4 to promote earlier maturity in soybean.

Duplication and sub-functionalization of flavonoid biosynthesis genes plays important role in Leguminosae root nodule symbiosis evolution.

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N2-fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae-gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon-intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae-gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

Frontiers of soybean pan-genome studies.

Artificial domestication provided the original motivation to the blooming of agriculture, following with the dramatic change of the genetic background of crops and livestock. According to theory and technology upgradation that contributing to the omics, we appreciate using the pan-genome instead of single reference genome for crop study. By comparison and integration of multiple genomes under the guidance of pan-genome theory, we can estimate the genomic information range of a species, leading to a global understanding of its genetic diversity. Combining pan-genome with large size chromosomal structural variations, high throughput population resequencing, and multi-omics data, we can profoundly study the genetic basis behind species traits we focus on. Soybean is one of the most important commercial crops over the world. It is also essential to our food security. Dissecting the formation of genetic diversity and the causal loci of key agricultural traits of soybean will make the modern soybean breeding more efficiently. In this review, we summarize the core idea of pan-genome and clarified the characteristics of construction strategies of pan-genome such as de novo/mapping assembly, iterative assembly and graph-based genome. Then we used the soybean pan-genome work as a case study to introduce the general way to study pan-genome. We highlighted the contribution of structural variation (SV) to the evolution/domestication of soybean and its value in understanding the genetic bases of agronomy traits. By those, we approved the value of graph-based pan-genome for data integration and SV calculation. Future research directions are also discussed for crop genomics and data science.

Global dissection of the recombination landscape in soybean using a high-density 600K SoySNP array.

Recombination is crucial for crop breeding because it can break linkage drag and generate novel allele combinations. However, the high-resolution recombination landscape and its driving forces in soybean are largely unknown. Here, we constructed eight recombinant inbred line (RIL) populations and genotyped individual lines using the high-density 600K SoySNP array, which yielded a high-resolution recombination map with 5636 recombination sites at a resolution of 1.37 kb. The recombination rate was negatively correlated with transposable element density and GC content but positively correlated with gene density. Interestingly, we found that meiotic recombination was enriched at the promoters of active genes. Further investigations revealed that chromatin accessibility and active epigenetic modifications promoted recombination. Our findings provide important insights into the control of homologous recombination and thus will increase our ability to accelerate soybean breeding by manipulating meiotic recombination rate.

Toward cis-regulation in soybean: a 3D genome scope.

In eukaryotic cells, 3D genome plays an important role in the regulation of gene spatiotemporal expression, which is essential for the biological and developmental processes in a life cycle. In the past decade, the development of high-throughput technologies greatly enhances our ability to map the 3D genome organization, identifies multiple 3D genome structures, and investigates the functional role of 3D genome organization in gene regulation, which facilitates our understandings of cis-regulatory landscape and biological development. Comparing with the comprehensive analyses of 3D genome in mammals and model plants, the progress in soybean is much less. Future development and application of tools to precisely manipulate 3D genome structure at different levels will significantly strengthen the functional genome study and molecular breeding in soybean. Here, we review the recent progresses in 3D genome study and discuss future directions, which may help to improve soybean 3D functional genome study and molecular breeding.

A recently evolved BAHD acetyltransferase, responsible for bitter soyasaponin A production, is indispensable for soybean seed germination.

Soyasaponins are major small molecules that accumulate in soybean (Glycine max) seeds. Among them, type-A soyasaponins, fully acetylated at the terminal sugar of their C22 sugar chain, are responsible for the bitter taste of soybean-derived foods. However, the molecular basis for the acetylation of type-A soyasaponins remains unclear. Here, we identify and characterize GmSSAcT1, encoding a BADH-type soyasaponin acetyltransferase that catalyzes three or four consecutive acetylations on type-A soyasaponins in vitro and in planta. Phylogenetic analysis and biochemical assays suggest that GmSSAcT1 likely evolved from acyltransferases present in leguminous plants involved in isoflavonoid acylation. Loss-of-function mutants of GmSSAcT1 exhibited impaired seed germination, which attribute to the excessive accumulation of null-acetylated type-A soyasaponins. We conclude that GmSSAcT1 not only functions as a detoxification gene for high accumulation of type-A soyasaponins in soybean seeds but is also a promising target for breeding new soybean varieties with lower bitter soyasaponin content.

Designing future crops: challenges and strategies for sustainable agriculture.

Crop production is facing unprecedented challenges. Despite the fact that the food supply has significantly increased over the past half-century, ~8.9 and 14.3% people are still suffering from hunger and malnutrition, respectively. Agricultural environments are continuously threatened by a booming world population, a shortage of arable land, and rapid changes in climate. To ensure food and ecosystem security, there is a need to design future crops for sustainable agriculture development by maximizing net production and minimalizing undesirable effects on the environment. The future crops design projects, recently launched by the National Natural Science Foundation of China and Chinese Academy of Sciences (CAS), aim to develop a roadmap for rapid design of customized future crops using cutting-edge technologies in the Breeding 4.0 era. In this perspective, we first introduce the background and missions of these projects. We then outline strategies to design future crops, such as improvement of current well-cultivated crops, de novo domestication of wild species and redomestication of current cultivated crops. We further discuss how these ambitious goals can be achieved by the recent development of new integrative omics tools, advanced genome-editing tools and synthetic biology approaches. Finally, we summarize related opportunities and challenges in these projects.

Natural allelic variation of GmST05 controlling seed size and quality in soybean.

Seed size is one of the most important agronomic traits determining the yield of crops. Cloning the key genes controlling seed size and pyramiding their elite alleles will facilitate yield improvement. To date, few genes controlling seed size have been identified in soybean, a major crop that provides half of the plant oil and one quarter of the plant protein globally. Here, through a genome-wide association study of over 1800 soybean accessions, we determined that natural allelic variation at GmST05 (Seed Thickness 05) predominantly controlled seed thickness and size in soybean germplasm. Further analyses suggested that the two major haplotypes of GmST05 differed significantly at the transcriptional level. Transgenic experiments demonstrated that GmST05 positively regulated seed size and influenced oil and protein contents, possibly by regulating the transcription of GmSWEET10a. Population genetic diversity analysis suggested that allelic variations of GmST05 were selected during geographical differentiation but have not been fixed. In summary, natural variation in GmST05 determines transcription levels and influences seed size and quality in soybean, making it an important gene resource for soybean molecular breeding.

Progress in soybean functional genomics over the past decade.

Soybean is one of the most important oilseed and fodder crops. Benefiting from the efforts of soybean breeders and the development of breeding technology, large number of germplasm has been generated over the last 100 years. Nevertheless, soybean breeding needs to be accelerated to meet the needs of a growing world population, to promote sustainable agriculture and to address future environmental changes. The acceleration is highly reliant on the discoveries in gene functional studies. The release of the reference soybean genome in 2010 has significantly facilitated the advance in soybean functional genomics. Here, we review the research progress in soybean omics (genomics, transcriptomics, epigenomics and proteomics), germplasm development (germplasm resources and databases), gene discovery (genes that are responsible for important soybean traits including yield, flowering and maturity, seed quality, stress resistance, nodulation and domestication) and transformation technology during the past decade. At the end, we also briefly discuss current challenges and future directions.

Downregulation of a gibberellin 3ß-hydroxylase enhances photosynthesis and increases seed yield in soybean.

Seed yield, determined mainly by seed numbers and seed weight, is the primary target of soybean breeding. Identifying the genes underlying yield-related traits is of great significance. Through joint linkage mapping and a genome-wide association study for 100-seed weight, we cloned GmGA3ox1, a gene encoding gibberellin 3ß-hydroxylase, which is the key enzyme in the gibberellin synthesis pathway. Genome resequencing identified a beneficial GmGA3ox1 haplotype contributing to high seed weight, which was further confirmed by soybean transformants. CRISPR/Cas9-generated gmga3ox1 mutants showed lower seed weight, but promoted seed yield by increasing seed numbers. The gmga3ox1 mutants reduced gibberellin biosynthesis while enhancing photosynthesis. Knockout of GmGA3ox1 resulted in the upregulation of numerous photosynthesis-related genes, particularly the GmRCA family encoding ribulose-1,5-bispho-sphate carboxylase-oxygenase (Rubisco) activases. The basic leucine zipper transcription factors GmbZIP97 and GmbZIP159, which were both upregulated in the gmga3ox1 mutants and induced by the gibberellin synthesis inhibitor uniconazole, could bind to the promoter of GmRCAß and activate its expression. Analysis of genomic sequences with over 2700 soybean accessions suggested that GmGA3ox1 is being gradually utilized in modern breeding. Our results elucidated the important role of GmGA3ox1 in soybean yield. These findings reveal important clues for future high-yield breeding in soybean and other crops.

The antagonistic MYB paralogs RH1 and RH2 govern anthocyanin leaf markings in Medicago truncatula.

Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear. Here, using Medicago truncatula leaf marking as a model, we show that the classic M. truncatula leaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2. RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C-terminal activation motif. RH1 physically interacts with the M. truncatula bHLH protein MtTT8 and the WDR family member MtWD40-1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C-terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40-1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2-mediated competition can repress RH1 expression. Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings in M. truncatula, providing a multidimensional paralogous-antagonistic regulatory paradigm for fine-tuning patterned pigmentation.

Fine mapping QTL and mining genes for protein content in soybean by the combination of linkage and association analysis.

Soybean is one main source of dietary protein; therefore, improving protein content is an important objective in breeding programs. There is a significant negative correlation between protein and oil content, which influenced mapping quantitative trait locus (QTL) and quantitative trait nucleotides for these two traits. In this study, a linkage map was created with 2232 single-nucleotide polymorphism markers for the four-way recombinant inbred line (FW-RIL) population derived from the cross (Kenfeng 14 × Kenfeng 15) × (Heinong 48 × Kenfeng 19), and then conditional and unconditional QTL analyses were carried out by inclusive complete interval mapping based on the phenotypic data of protein and oil content collected in 10 different environments. As shown in the results of linkage analysis, a total of 85 QTL have been detected. We have performed association analysis using 109,676 markers after quality filtering for FW-RIL, and the results have shown that a total of 60 QTNs were detected. We have performed association analysis using 63,306 markers after quality filtering for resource population, and the results have shown that a total of 123 QTNs were detected. We have combined linkage and association analysis, and there are six QTNs verified by FW-RIL and resource population. We have performed pathway analysis on the genes in these six QTN attenuation regions, and the result shows that a total of four candidate genes are related to the synthesis or metabolism of soybean protein. These findings will facilitate marker-assisted selection and molecular breeding of soybean.

Pollen grain size associated with pollinator feeding strategy.

Angiosperm pollen grain diameter varies greatly from a few microns to over 100, but the selective forces driving the interspecific variation in pollen size remain unclear. Although both pre- and post-pollination hypotheses have been proposed, empirical evidence remains scarce. Here we propose that visits by pollen-foraging pollinators have selected against large pollen grains. An association between pollinator behaviour and pollen grain size was confirmed by field studies of 80 flowering species in natural communities, showing that pollinators positively collected pollen in those species with relatively smaller pollen grains but rarely did so in species with larger ones. Allowing for the confounding effects of pollinator type, flower size or style length and pollen grain number, we found a significant effect of pollen-foraging behaviour on variation in pollen grain size, particularly in bee-pollinated plants. While these results suggest that many plant species whose pollen is collected or consumed by pollinators produce small pollen grains, it remains unclear whether pollen grain size is directly affected by pollinator foraging habit or indirectly mediated by pollen number trade-offs.

De novo assembly of a wild pear (Pyrus betuleafolia) genome.

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia-Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi-C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein-coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide-binding site (NBS)-type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR-NBS-LRR (TNL)-type genes, which represented the greatest number of TNL-type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism-related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high-quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.

An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean.

Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean [Glycine max (L.) Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Via a genome-wide association study (GWAS), we identified 28 single nucleotide polymorphisms (SNPs) that are significantly associated with isoflavone concentrations in soybean. One of these 28 SNPs was located in the 5'-untranslated region (5'-UTR) of an R2R3-type MYB transcription factor, GmMYB29, and this gene was thus selected as a candidate gene for further analyses. A subcellular localization study confirmed that GmMYB29 was located in the nucleus. Transient reporter gene assays demonstrated that GmMYB29 activated the IFS2 (isoflavone synthase 2) and CHS8 (chalcone synthase 8) gene promoters. Overexpression and RNAi-mediated silencing of GmMYB29 in soybean hairy roots resulted in increased and decreased isoflavone content, respectively. Moreover, a candidate-gene association analysis revealed that 11 natural GmMYB29 polymorphisms were significantly associated with isoflavone contents, and regulation of GmMYB29 expression could partially contribute to the observed phenotypic variation. Taken together, these results provide important genetic insights into the molecular mechanisms underlying isoflavone biosynthesis in soybean.

Mutation of YL Results in a Yellow Leaf with Chloroplast RNA Editing Defect in Soybean.

RNA editing plays a key role in organelle gene expression. Little is known about how RNA editing factors influence soybean plant development. Here, we report the isolation and characterization of a soybean yl (yellow leaf) mutant. The yl plants showed decreased chlorophyll accumulation, lower PS II activity, an impaired net photosynthesis rate, and an altered chloroplast ultrastructure. Fine mapping of YL uncovered a point mutation in Glyma.20G187000, which encodes a chloroplast-localized protein homologous to Arabidopsis thaliana (Arabidopsis) ORRM1. YL is mainly expressed in trifoliate leaves, and its deficiency affects the editing of multiple chloroplast RNA sites, leading to inferior photosynthesis in soybean. Taken together, these results demonstrate the importance of the soybean YL protein in chloroplast RNA editing and photosynthesis.