Gibberellins Inhibit Flavonoid Biosynthesis and Promote Nitrogen Metabolism in Medicago truncatula

Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA₃ and GA₄) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA₃. In contrast, nitrogen metabolism was promoted by exogenous GA₃ but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.     

Genome-Wide Analysis of the Cyclin Gene Family and Their Expression Profile in Medicago truncatula

Cyclins, together with highly conserved cyclin-dependent kinases (CDKs), play an important role in the process of cell cycle in plants, but less is known about the functions of cyclins in legume plants, especially Medicago truncatula. Our genome-wide analysis identified 58, 103, and 51 cyclin members in the M. truncatula, Glycine max, and Phaseolus vulgaris genomes. Phylogenetic analysis suggested that these cyclins could be classified into 10 types, and the CycB-like types (CycBL1-BL8) were the specific subgroups in M. truncatula, which was one reason for the expansion of the B-type in M. truncatula. All putative cyclin genes were mapped onto their own chromosomes of each genome, and 9 segmental duplication gene pairs involving 20 genes were identified in M. truncatula cyclins. Determined by quantitative real-time PCR, the expression profiling suggested that 57 cyclins in M. truncatula were differentially expressed in 9 different tissues, while a few genes were expressed in some specific tissues. Using the publicly available RNAseq data, the expression of Mtcyclins in the wild-type strain A17 and three nodule mutants during rhizobial infection showed that 23 cyclins were highly upregulated in the nodulation (Nod) factor-hypersensitive mutant sickle (skl) mutant after 12 h of rhizobium inoculation. Among these cyclins, six cyclin genes were also specifically expressed in roots and nodules, which might play specific roles in the various phases of Nod factor-mediated cell cycle activation and nodule development. Our results provide information about the cyclin gene family in legume plants, serving as a guide for further functional research on plant cyclins.     

Chromosome-Level Genome Assembly of a Fragrant Japonica Rice Cultivar ‘Changxianggeng 1813’ Provides Insights into Genomic Variations between Fragrant and Non-Fragrant Japonica Rice

East Asia has an abundant resource of fragrant japonica rice that is gaining increasing interest among both consumers and producers. However, genomic resources and in particular complete genome sequences currently available for the breeding of fragrant japonica rice are still scarce. Here, integrating Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C methods, we presented a high-quality chromosome-level genome assembly (~378.78 Mb) for a new fragrant japonica cultivar ‘Changxianggeng 1813’, with 31,671 predicated protein-coding genes. Based on the annotated genome sequence, we demonstrated that it was the badh2-E2 type of deletion (a 7-bp deletion in the second exon) that caused fragrance in ‘Changxianggeng 1813’. Comparative genomic analyses revealed that multiple gene families involved in the abiotic stress response were expanded in the ‘Changxianggeng 1813’ genome, which further supported the previous finding that no generalized loss of abiotic stress tolerance associated with the fragrance phenotype. Although the ‘Changxianggeng 1813’ genome showed high genomic synteny with the genome of the non-fragrant japonica rice cultivar Nipponbare, a total of 289,970 single nucleotide polymorphisms (SNPs), 96,093 small insertion-deletion polymorphisms (InDels), and 8690 large structure variants (SVs, >1000 bp) were identified between them. Together, these genomic resources will be valuable for elucidating the mechanisms underlying economically important traits and have wide-ranging implications for genomics-assisted breeding in fragrant japonica rice.     

Genetic Mapping of a Major Resistance Gene to Pea Aphid (Acyrthosipon pisum) in the Model Legume Medicago truncatula

Resistance to the Australian pea aphid (PA; Acyrthosiphon pisum) biotype in cultivar Jester of the model legume Medicago truncatula is mediated by a single dominant gene and is phloem-mediated. The genetic map position for this resistance gene, APR (Acyrthosiphon pisum resistance), is provided and shows that APR maps 39 centiMorgans (cM) distal of the A. kondoi resistance (AKR) locus, which mediates resistance to a closely related species of the same genus bluegreen aphid (A. kondoi). The APR region on chromosome 3 is dense in classical nucleotide binding site leucine-rich repeats (NLRs) and overlaps with the region harbouring the RAP1 gene which confers resistance to a European PA biotype in the accession Jemalong A17. Further screening of a core collection of M. truncatula accessions identified seven lines with strong resistance to PA. Allelism experiments showed that the single dominant resistance to PA in M. truncatula accessions SA10481 and SA1516 are allelic to SA10733, the donor of the APR locus in cultivar Jester. While it remains unclear whether there are multiple PA resistance genes in an R-gene cluster or the resistance loci identified in the other M. truncatula accessions are allelic to APR, the introgression of APR into current M. truncatula cultivars will provide more durable resistance to PA.     

Overexpression of Mtr-miR319a Contributes to Leaf Curl and Salt Stress Adaptation in Arabidopsis thaliana and Medicago truncatula

Salt stress is a worldwide agronomic issue that limits crop yield and quality. Improving salt stress tolerance via genetic modification is the most efficient method to conquer soil salinization problems in crops. Crop miRNAs have been declared to be tightly associated with responding and adapting to salt stress and are advantageous for salt tolerance modification. However, very few studies have validated vital salt tolerance miRNAs and coupled potent target genes in Medicago species, the most economically important forage legume species. In this study, Mtr-miR319a, a miRNA that was identified from the previous next-generation sequencing assay of salt-treated Medicago truncatula, was overexpressed in M. truncatula and Arabidopsis thaliana, inducing the curly leaves and salt stress tolerance phenotypes. Combining the elevated expression level of Mtr-miR319a in the M. truncatula overexpression lines under normal and salt-treatment conditions, the regulatory roles of Mtr-miR319a in leaf development and salt stress adaptation were demonstrated. Several predicted target genes of Mtr-miR319a were also regulated by Mtr-miR319a and were associated with the aforementioned phenotypes in M. truncatula plants, most notably MtTCP4. Our study clarified the functional role of Mtr-miR319a and its target genes in regulating leaf development and defending salt stress, which can help to inform crop breeding efforts for improving salt tolerance via genetic engineering.     

Integrating Genetic and Chromosome Maps of Allium cepa: From Markers Visualization to Genome Assembly Verification

The ability to directly look into genome sequences has opened great opportunities in plant breeding. Yet, the assembly of full-length chromosomes remains one of the most difficult problems in modern genomics. Genetic maps are commonly used in de novo genome assembly and are constructed on the basis of a statistical analysis of the number of recombinations. This may affect the accuracy of the ordering and orientation of scaffolds within the chromosome, especially in the region of recombination suppression. Moreover, it is impossible to assign contigs lacking DNA markers. Here, we report the use of Tyr-FISH to determine the position of the short DNA sequence of markers and non-mapped unique copy sequence on the physical chromosomes of a large-genome onion (Allium cepa L.). In order to minimize potential background masking of the target signal, we improved our earlier developed pipeline for probe design. A total of 23 markers were located on physical chromosomes 2 and 6. The order of markers was corrected by the integration of genetic, pseudochromosome maps and cytogenetic maps. Additionally, the position of the mlh1 gene, which was not on the genetic map, was defined on physical chromosome 2. Tyr-FISH mapping showed that the order of 23.1% (chromosome 2) and 27.3% (chromosome 6) of the tested genes differed between physical chromosomes and pseudochromosomes. The results can be used for the improvement of pseudochromosome 2 and 6 assembly. The present study aims to demonstrate the value of the in situ visualization of DNA sequences in chromosome-scaffold genome assembly.     

Genome-Wide Identification and Analysis of the Polycomb Group Family in Medicago truncatula

Polycomb group (PcG) proteins, which are important epigenetic regulators, play essential roles in the regulatory networks involved in plant growth, development, and environmental stress responses. Currently, as far as we know, no comprehensive and systematic study has been carried out on the PcG family in Medicago truncatula. In the present study, we identified 64 PcG genes with distinct gene structures from the M. truncatula genome. All of the PcG genes were distributed unevenly over eight chromosomes, of which 26 genes underwent gene duplication. The prediction of protein interaction network indicated that 34 M. truncatula PcG proteins exhibited protein–protein interactions, and MtMSI1;4 and MtVRN2 had the largest number of protein–protein interactions. Based on phylogenetic analysis, we divided 375 PcG proteins from 27 species into three groups and nine subgroups. Group I and Group III were composed of five components from the PRC1 complex, and Group II was composed of four components from the PRC2 complex. Additionally, we found that seven PcG proteins in M. truncatula were closely related to the corresponding proteins of Cicer arietinum. Syntenic analysis revealed that PcG proteins had evolved more conservatively in dicots than in monocots. M. truncatula had the most collinearity relationships with Glycine max (36 genes), while collinearity with three monocots was rare (eight genes). The analysis of various types of expression data suggested that PcG genes were involved in the regulation and response process of M. truncatula in multiple developmental stages, in different tissues, and for various environmental stimuli. Meanwhile, many differentially expressed genes (DEGs) were identified in the RNA-seq data, which had potential research value in further studies on gene function verification. These findings provide novel and detailed information on the M. truncatula PcG family, and in the future it would be helpful to carry out related research on the PcG family in other legumes.     

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda)

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.     

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA⁻). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1–MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.     

Genome-Wide Identification and Analyses of Drought/Salt-Responsive Cytochrome P450 Genes in Medicago truncatula

Cytochrome P450 monooxygenases (P450s) catalyze a great number of biochemical reactions and play vital roles in plant growth, development and secondary metabolism. As yet, the genome-scale investigation on P450s is still lacking in the model legume Medicago truncatula. In particular, whether and how many MtP450s are involved in drought and salt stresses for Medicago growth, development and yield remain unclear. In this study, a total of 346 MtP450 genes were identified and classified into 10 clans containing 48 families. Among them, sixty-one MtP450 genes pairs are tandem duplication events and 10 MtP450 genes are segmental duplication events. MtP450 genes within one family exhibit high conservation and specificity in intron–exon structure. Meanwhile, many Mt450 genes displayed tissue-specific expression pattern in various tissues. Specifically, the expression pattern of 204 Mt450 genes under drought/NaCl treatments were analyzed by using the weighted correlation network analysis (WGCNA). Among them, eight genes (CYP72A59v1, CYP74B4, CYP71AU56, CYP81E9, CYP71A31, CYP704G6, CYP76Y14, and CYP78A126), and six genes (CYP83D3, CYP76F70, CYP72A66, CYP76E1, CYP74C12, and CYP94A52) were found to be hub genes under drought/NaCl treatments, respectively. The expression levels of these selected hub genes could be induced, respectively, by drought/NaCl treatments, as validated by qPCR analyses, and most of these genes are involved in the secondary metabolism and fatty acid pathways. The genome-wide identification and co-expression analyses of M. truncatula P450 superfamily genes established a gene atlas for a deep and systematic investigation of P450 genes in M. truncatula, and the selected drought-/salt-responsive genes could be utilized for further functional characterization and molecular breeding for resistance in legume crops.     

Comparison of Long-Read Methods for Sequencing and Assembly of Lepidopteran Pest Genomes

Lepidopteran species are mostly pests, causing serious annual economic losses. High-quality genome sequencing and assembly uncover the genetic foundation of pest occurrence and provide guidance for pest control measures. Long-read sequencing technology and assembly algorithm advances have improved the ability to timeously produce high-quality genomes. Lepidoptera includes a wide variety of insects with high genetic diversity and heterozygosity. Therefore, the selection of an appropriate sequencing and assembly strategy to obtain high-quality genomic information is urgently needed. This research used silkworm as a model to test genome sequencing and assembly through high-coverage datasets by de novo assemblies. We report the first nearly complete telomere-to-telomere reference genome of silkworm Bombyx mori (P50T strain) produced by Pacific Biosciences (PacBio) HiFi sequencing, and highly contiguous and complete genome assemblies of two other silkworm strains by Oxford Nanopore Technologies (ONT) or PacBio continuous long-reads (CLR) that were unrepresented in the public database. Assembly quality was evaluated by use of BUSCO, Inspector, and EagleC. It is necessary to choose an appropriate assembler for draft genome construction, especially for low-depth datasets. For PacBio CLR and ONT sequencing, NextDenovo is superior. For PacBio HiFi sequencing, hifiasm is better. Quality assessment is essential for genome assembly and can provide better and more accurate results. For chromosome-level high-quality genome construction, we recommend using 3D-DNA with EagleC evaluation. Our study references how to obtain and evaluate high-quality genome assemblies, and is a resource for biological control, comparative genomics, and evolutionary studies of Lepidopteran pests and related species.     

Genome-Wide Identification and Characterization of Short-Chain Dehydrogenase/Reductase (SDR) Gene Family in Medicago truncatula

Short-chain dehydrogenase/reductase (SDR) belongs to the NAD(P)(H)-dependent oxidoreductase superfamily. Limited investigations reveal that SDRs participate in diverse metabolisms. A genome-wide identification of the SDR gene family in M. truncatula was conducted. A total of 213 MtSDR genes were identified, and they were distributed on all chromosomes unevenly. MtSDR proteins were categorized into seven subgroups based on phylogenetic analysis and three types including ‘classic’, ‘extended’, and ‘atypical’, depending on the cofactor-binding site and active site. Analysis of the data from M. truncatula Gene Expression Atlas (MtGEA) showed that above half of MtSDRs were expressed in at least one organ, and lots of MtSDRs had a preference in a tissue-specific expression. The cis-acting element responsive to plant hormones (salicylic acid, ABA, auxin, MeJA, and gibberellin) and stresses were found in the promoter of some MtSDRs. Many genes of MtSDR7C, MtSDR65C, MtSDR110C, MtSDR114C, and MtSDR108E families were responsive to drought, salt, and cold. The study provides useful information for further investigation on biological functions of MtSDRs, especially in abiotic stress adaptation, in the future.     

Construction of Whole Genomes from Scaffolds Using Single Cell Strand-Seq Data

Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.     

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes’ organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes’ map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.     

From FISH to Hi-C: The Chromatin Architecture of the Chromosomal Region 7q36.3, Frequently Rearranged in Leukemic Cells,Is Evolutionary Conserved

Fluorescence in situ hybridization (FISH) and Hi-C methods are largely used to investigate the three-dimensional organization of the genome in the cell nucleus and are applied here to study the organization of genes (LMBR1, NOM1, MNX1, UBE3C, PTPRN2) localized in the human 7q36.3 band. This region contains the MNX1 gene, which is normally not expressed in human lymphocytes beyond embryonic development. However, this homeobox gene is frequently activated in leukemic cells and its expression is associated with an altered gene positioning in the leukemia cell nuclei. In this study, we used FISH on 3D-preserved nuclei to investigate the nuclear positioning of MNX1 in the leukemia-derived cell line K562. Of the five copies of the MNX1 gene present in K562, four alleles were positioned in the nuclear periphery and only one in the nuclear interior. Using the Juicebox’s Hi-C dataset, we identified five chromatin loops in the 7q36.3 band, with different extensions related to the size and orientation of the genes located here, and independent from their expression levels. We identified similar loops in 11 human and three mouse cell lines, showing that these loops are highly conserved in different human cell lines and during evolution. Moreover, the chromatin loop organization is well conserved also during neuronal cell differentiation, showing consistency in genomic organization of this region in development. In this report, we show that FISH and Hi-C are two different approaches that complement one another and together give complete information on the nuclear organization of specific chromosomal regions in different conditions, including cellular differentiation and genetic diseases.     

The Genome of the Mimosoid Legume Prosopis cineraria,a Desert Tree

The mimosoid legumes are a clade of ~40 genera in the Caesalpinioideae subfamily of the Fabaceae that grow in tropical and subtropical regions. Unlike the better studied Papilionoideae, there are few genomic resources within this legume group. The tree Prosopis cineraria is native to the Near East and Indian subcontinent, where it thrives in very hot desert environments. To develop a tool to better understand desert plant adaptation mechanisms, we sequenced the P. cineraria genome to near-chromosomal assembly, with a total sequence length of ~691 Mb. We predicted 77,579 gene models (76,554 CDS, 361 rRNAs and 664 tRNAs) from the assembled genome, among them 55,325 (~72%) protein-coding genes that were functionally annotated. This genome was found to consist of over 58% repeat sequences, primarily long terminal repeats (LTR-)-retrotransposons. We find an expansion of terpenoid metabolism genes in P. cineraria and its relative Prosopis alba, but not in other legumes. We also observed an amplification of NBS-LRR disease-resistance genes correlated with LTR-associated retrotransposition, and identified 410 retrogenes with an active burst of chimeric retrogene creation that approximately occurred at the same time of divergence of P. cineraria from a common lineage with P. alba~23 Mya. These retrogenes include many biotic defense responses and abiotic stress stimulus responses, as well as the early Nodulin 93 gene. Nodulin 93 gene amplification is consistent with an adaptive response of the species to the low nitrogen in arid desert soil. Consistent with these results, our differentially expressed genes show a tissue specific expression of isoprenoid pathways in shoots, but not in roots, as well as important genes involved in abiotic salt stress in both tissues. Overall, the genome sequence of P. cineraria enriches our understanding of the genomic mechanisms of its disease resistance and abiotic stress tolerance. Thus, it is a very important step in crop and legume improvement.