High-integrity Pueraria montana var. lobata genome and population analysis revealed the genetic diversity of Pueraria genus.

Pueraria montana var. lobata (P. lobata) is a traditional medicinal plant belonging to the Pueraria genus of Fabaceae family. Pueraria montana var. thomsonii (P. thomsonii) and Pueraria montana var. montana (P. montana) are its related species. However, evolutionary history of the Pueraria genus is still largely unknown. Here, a high-integrity, chromosome-level genome of P. lobata and an improved genome of P. thomsonii were reported. It found evidence for an ancient whole-genome triplication and a recent whole-genome duplication shared with Fabaceae in three Pueraria species. Population genomics of 121 Pueraria accessions demonstrated that P. lobata populations had substantially higher genetic diversity, and P. thomsonii was probably derived from P. lobata by domestication as a subspecies. Selection sweep analysis identified candidate genes in P. thomsonii populations associated with the synthesis of auxin and gibberellin, which potentially play a role in the expansion and starch accumulation of tubers in P. thomsonii. Overall, the findings provide new insights into the evolutionary and domestication history of the Pueraria genome and offer a valuable genomic resource for the genetic improvement of these species.

Telomere-to-telomere assembly of cassava genome reveals the evolution of cassava and divergence of allelic expression.

Cassava is a crucial crop that makes a significant contribution to ensuring human food security. However, high-quality telomere-to-telomere cassava genomes have not been available up to now, which has restricted the progress of haploid molecular breeding for cassava. In this study, we constructed two nearly complete haploid resolved genomes and an integrated, telomere-to-telomere gap-free reference genome of an excellent cassava variety, 'Xinxuan 048', thereby providing a new high-quality genomic resource. Furthermore, the evolutionary history of several species within the Euphorbiaceae family was revealed. Through comparative analysis of haploid genomes, it was found that two haploid genomes had extensive differences in linear structure, transcriptome features, and epigenetic characteristics. Genes located within the highly divergent regions and differentially expressed alleles are enriched in the functions of auxin response and the starch synthesis pathway. The high heterozygosity of cassava 'Xinxuan 048' leads to rapid trait segregation in the first selfed generation. This study provides a theoretical basis and genomic resource for molecular breeding of cassava haploids.

Genome-wide identification and expression pattern analysis of R2R3-MYB transcription factor gene family involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii.

BACKGROUND: R2R3-MYB transcription factors regulate secondary metabolism, stress responses and development in various plants. Puerarin is a bioactive ingredient and most abundant secondary metabolite isolated from Pueraria lobata. The biosynthesis of puerarin proceeds via the phenylpropanoid pathway and isoflavonoids pathway, in which 9 key enzymes are involved. The expression of these structural genes is under control of specific PtR2R3-MYB genes in different plant tissues. However, how PtR2R3-MYB genes regulates structural genes in puerarin biosynthesis remains elusive. This study mined the PtR2R3-MYB genes involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii. RESULTS: A total of 209 PtR2R3-MYB proteins were identified, in which classified into 34 subgroups based on the phylogenetic topology and the classification of the R2R3-MYB superfamily in Arabidopsis thaliana. Furtherly physical and chemical characteristics, gene structure, and conserved motif analysis were also used to further analyze PtR2R3-MYBs. Combining puerarin content and RNA-seq data, speculated on the regulated puerarin biosynthesis of PtR2R3-MYB genes and structural genes, thus 21 PtR2R3-MYB genes and 25 structural genes were selected for validation gene expression and further explore its response to MeJA and GSH treatment by using qRT-PCR analysis technique. Correlation analysis and cis-acting element analysis revealed that 6 PtR2R3-MYB genes (PtMYB039, PtMYB057, PtMYB080, PtMYB109, PtMYB115 and PtMYB138) and 7 structural genes (PtHID2, PtHID9, PtIFS3, PtUGT069, PtUGT188, PtUGT286 and PtUGT297) were directly or indirectly regulation of puerarin biosynthesis in ZG11. It is worth noting that after MeJA and GSH treatment for 12-24 h, the expression changes of most candidate genes were consistent with the correlation of puerarin biosynthesis, which also shows that MeJA and GSH have the potential to mediate puerarin biosynthesis by regulating gene expression in ZG11. CONCLUSIONS: Overall, this study provides a comprehensive understanding of the PtR2R3-MYB and will paves the way to reveal the transcriptional regulation of puerarin biosynthesis and response to phytohormone of PtR2R3-MYB genes in Pueraria lobata var. thomsonii.

Chromosome-level and graphic genomes provide insights into metabolism of bioactive metabolites and cold-adaption of Pueraria lobata var. montana.

Pueraria lobata var. montana (P. montana) belongs to the genus Pueraria and originated in Asia. Compared with its sister P. thomsonii, P. montana has stronger growth vigour and cold-adaption but contains less bioactive metabolites such as puerarin. To promote the investigation of metabolic regulation and genetic improvement of Pueraria, the present study reports a chromosome-level genome of P. montana with length of 978.59 Mb and scaffold N50 of 80.18 Mb. Comparative genomics analysis showed that P. montana possesses smaller genome size than that of P. thomsonii owing to less repeat sequences and duplicated genes. A total of 6,548 and 4,675 variety-specific gene families were identified in P. montana and P. thomsonii, respectively. The identified variety-specific and expanded/contracted gene families related to biosynthesis of bioactive metabolites and microtubules are likely the causes for the different characteristics of metabolism and cold-adaption of P. montana and P. thomsonii. Moreover, a graphic genome was constructed based on 11 P. montana accessions. Total 92 structural variants were identified and most of which are related to stimulus-response. In conclusion, the chromosome-level and graphic genomes of P. montana will not only facilitate the studies of evolution and metabolic regulation, but also promote the breeding of Pueraria.

DNA Methylome and LncRNAome Analysis Provide Insights Into Mechanisms of Genome-Dosage Effects in Autotetraploid Cassava.

Whole genome duplication (WGD) increases the dosage of all coding and non-coding genes, yet the molecular implications of genome-dosage effects remain elusive. In this study, we generated integrated maps of the methylomes and lncRNAomes for diploid and artificially generated autotetraploid cassava (Manihot esculenta Crantz). We found that transposable elements (TEs) suppressed adjacent protein coding gene (PCG)-expression levels, while TEs activated the expression of nearby long non-coding RNAs (lncRNAs) in the cassava genome. The hypermethylation of DNA transposons in mCG and mCHH sites may be an effective way to suppress the expression of nearby PCGs in autotetraploid cassava, resulting in similar expression levels for most of PCGs between autotetraploid and diploid cassava. In the autotetraploid, decreased methylation levels of retrotransposons at mCHG and mCHH sites contributed to reduced methylation of Gypsy-neighboring long intergenic non-coding RNAs, potentially preserving diploid-like expression patterns in the major of lncRNAs. Collectively, our study highlighted that WGD-induced DNA methylation variation in DNA transposons and retrotransposons may be as direct adaptive responses to dosage of all coding-genes and lncRNAs, respectively.

Chromosomal-level genome and multi-omics dataset of Pueraria lobata var. thomsonii provide new insights into legume family and the isoflavone and puerarin biosynthesis pathways.

Pueraria lobata var. thomsonii (hereinafter abbreviated as Podalirius thomsonii), a member of legumes, is one of the important traditional Chinese herbal medicines, and its puerarin extraction is widely used in health and pharmaceutical industry. Here, we assembled a high-quality genome of P. thomsonii using long-read single-molecule sequencing and Hi-C technologies. The genome assembly is approximately 1.37 Gb in size and consists of 5145 contigs with a contig N50 of 593.70 Kb, further clustered into 11 pseudochromosomes. The genome structural annotation resulted in about 869.33 Mb (about 62.70% of the genome) repeat regions and 45 270 protein-coding genes. Genome evolution analysis revealed that P. thomsonii is most closely related to soybean and underwent two ancient whole-genome duplication events, one was in the common ancestor shared by legume species, the other occurred independently at around 7.2 million years ago after its specification. A total of 2373 gene families were found unique in P. thomsonii comparing to five other legume species. Genes and metabolites related to puerarin content in tuberous tissues were characterized. A total of 572 genes upregulated in the puerarin biosynthesis pathway were identified, and 235 candidate genes were further enriched by omics data. Furthermore, we identified 6 8-C-glucosyltransferase (8-C-GT) candidate genes significantly involved in puerarin metabolism. Our study filled in a key genomic gap in legume family, and provided valuable multi-omic resources for the genetic improvement of P. thomsonii.

Identification of Nutritional Ingredients and Medicinal Components of Pueraria lobata and Its Varieties Using UPLC-MS/MS-Based Metabolomics.

Pueraria lobata and its variety P. lobata var. thomsonii are both traditional Chinese medicines that have high nutritional and medical value; whereas another variety, P. lobata var. montana has low nutritional and medicinal value and can cause ecological disasters. The material basis of different nutritional and medicinal values, which are caused by metabolite differences among these varieties, remains to be further clarified. Here, we performed ultra performance liquid chromatography-tandem mass spectrometry based widely targeted metabolome analysis on Pueraria lobata, P. lobata var. thomsonii, and P. lobata var. montana. Among them, a total of 614 metabolites were identified, and distinguished from each other using orthogonal partial least squares discriminant analysis. Our results suggest that the nutritional differences between P. lobata and its varieties can be explained by variations in the abundance of amino acids, nucleotides, saccharides, and lipids; differences in flavonoids, isoflavones, phenolic acids, organic acids, and coumarins contents caused the differences in the medicinal quality of P. lobata and its varieties. Additionally, the key metabolites responsible for the classification of the three Pueraria varieties were identified. This study provides new insights into the underlying metabolic causes of nutritional and medicinal variation in P. lobata and its varieties.

Metabolomic and transcriptomic profiling reveals distinct nutritional properties of cassavas with different flesh colors.

Cassava is a significant food security crop in several developing countries. Metabolites in cassava roots provide numerous nutrients essential for human health. Exploiting the diversity of nutritional ingredients present in cassavas is vital for improving its nutritional value. To address this problem, root metabolomes of three cassava cultivars with white-flesh, light-yellow-flesh and yellow-flesh were comprehensively measured, respectively. A total of 508 metabolites were detected in cassava roots, including 300 primary metabolites and 185 secondary metabolites. There were 22.6% to 34.1% metabolites exhibiting significant variations among the three cassava cultivars. The light-yellow-flesh cassava contained higher contents of secondary metabolites, especially flavone, phenylpropanoids and alkaloids, and lower contents of primary metabolites except lipids, alcohols, vitamins and derivatives. Compared with light-yellow-flesh cassava, the yellow-flesh cassava contained higher contents of amino acid and derivatives, but lower contents of phenylpropanoids, nucleotide and derivates. White-flesh cassava contained higher contents of primary metabolites, especially amino acid and derivatives, but lower contents of secondary metabolites except flavonoid and indole derivatives. Transcriptome analyses were parallelly performed to decipher the potential mechanisms regulating the accumulations of related metabolites. Several pathways were both enriched by differentially expressed genes and differentially accumulated metabolites, supporting that metabolisms of these metabolites were regulated at transcriptional level. These results expand the knowledge on metabolite compositions in cassava roots and provide substantial information for genetic improvement of cassavas with high nutritional values.

Comparative physiology and transcriptome analysis allows for identification of lncRNAs imparting tolerance to drought stress in autotetraploid cassava.

BACKGROUND: Polyploidization, pervasive among higher plant species, enhances adaptation to water deficit, but the physiological and molecular advantages need to be investigated widely. Long non-coding RNAs (lncRNAs) are involved in drought tolerance in various crops. RESULTS: Herein, we demonstrate that tetraploidy potentiates tolerance to drought stress in cassava (Manihot esculenta Crantz). Autotetraploidy reduces transpiration by lesser extent increasing of stomatal density, smaller stomatal aperture size, or greater stomatal closure, and reducing accumulation of H2O2 under drought stress. Transcriptome analysis of autotetraploid samples revealed down-regulation of genes involved in photosynthesis under drought stress, and less down-regulation of subtilisin-like proteases involved in increasing stomatal density. UDP-glucosyltransferases were increased more or reduced less in dehydrated leaves of autotetraploids compared with controls. Strand-specific RNA-seq data (validated by quantitative real time PCR) identified 2372 lncRNAs, and 86 autotetraploid-specific lncRNAs were differentially expressed in stressed leaves. The co-expressed network analysis indicated that LNC_001148 and LNC_000160 in autotetraploid dehydrated leaves regulated six genes encoding subtilisin-like protease above mentioned, thereby result in increasing the stomatal density to a lesser extent in autotetraploid cassava. Trans-regulatory network analysis suggested that autotetraploid-specific differentially expressed lncRNAs were associated with galactose metabolism, pentose phosphate pathway and brassinosteroid biosynthesis, etc. CONCLUSION: Tetraploidy potentiates tolerance to drought stress in cassava, and LNC_001148 and LNC_000160 mediate drought tolerance by regulating stomatal density in autotetraploid cassava.