Identification and characterization of NBS-encoding disease resistance genes in Lotus japonicus

Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.     

A Genome-Wide Compilation of the Two-Component Systems in Lotus japonicus

The two-component systems (TCS), or histidine-to-aspartate phosphorelays, are evolutionarily conserved common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes including plants. Among higher plants, legumes including Lotus japonicus have a unique ability to engage in beneficial symbiosis with nitrogen-fixing bacteria. We previously presented a genome-wide compiled list of TCS-associated components of Mesorhizobium loti, which is a symbiont specific to L. japonicus (Hagiwara et al. 2004, DNA Res., 11, 57–65). To gain both general and specific insights into TCS of this currently attractive model legume, here we compiled TCS-associated components as many as possible from a genome-wide viewpoint by taking advantage that the efforts of whole genome sequencing of L. japonicus are almost at final stage. In the current database (http://www.kazusa.or.jp/lotus/index.html), it was found that L. japonicus has, at least, 14 genes each encoding a histidine kinase, 7 histidine-containing phosphotransmitter-related genes, 7 type-A response regulator (RR)-related genes, 11 type-B RR-related genes, and also 5 circadian clock-associated pseudo-RR genes. These results suggested that most of the L. japonicus TCS-associated genes have already been uncovered in this genome-wide analysis, if not all. Here, characteristics of these TCS-associated components of L. japonicus were inspected, one by one, in comparison with those of Arabidopsis thaliana. In addition, some critical experiments were also done to gain further insights into the functions of L. japonicus TCS-associated genes with special reference to cytokinin-mediated signal transduction and circadian clock.     

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus,Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.     

Genome Duplication and Gene Loss Affect the Evolution of Heat Shock Transcription Factor Genes in Legumes

Whole-genome duplication events (polyploidy events) and gene loss events have played important roles in the evolution of legumes. Here we show that the vast majority of Hsf gene duplications resulted from whole genome duplication events rather than tandem duplication, and significant differences in gene retention exist between species. By searching for intraspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found that genome duplications accounted for 42 of 46 Hsf-containing segments in Glycine max, while paired segments were rarely identified in Lotus japonicas, Medicago truncatula and Cajanus cajan. However, by comparing interspecies microsynteny, we determined that the great majority of Hsf-containing segments in Lotus japonicas, Medicago truncatula and Cajanus cajan show extensive conservation with the duplicated regions of Glycine max. These segments formed 17 groups of orthologous segments. These results suggest that these regions shared ancient genome duplication with Hsf genes in Glycine max, but more than half of the copies of these genes were lost. On the other hand, the Glycine max Hsf gene family retained approximately 75% and 84% of duplicated genes produced from the ancient genome duplication and recent Glycine-specific genome duplication, respectively. Continuous purifying selection has played a key role in the maintenance of Hsf genes in Glycine max. Expression analysis of the Hsf genes in Lotus japonicus revealed their putative involvement in multiple tissue-/developmental stages and responses to various abiotic stimuli. This study traces the evolution of Hsf genes in legume species and demonstrates that the rates of gene gain and loss are far from equilibrium in different species.     

Association of genetic variants in enamel-formation genes with dental caries: A meta- and gene-cluster analysis

Previous studies have reported the association between multiple genetic variants in the enamel-formation genes and the risk of dental caries with inconsistent results. We performed a systematic literature search of the PubMed, Cochrane Library, HuGE and Google Scholar databases for studies published before March 21, 2020 and conducted meta-, gene-based and gene-cluster analysis on the association between genetic variants in the enamel-formation genes and the risk of dental caries. We identified 21 relevant publications including a total of 24 studies for analysis. The genetic variant rs17878486 in AMELX was significantly associated with dental caries risk (OR = 1.40, 95% CI: 1.02–1.93, P = 0.037). We found no significant association between the risk of dental caries with rs12640848 in ENAM (OR = 1.15, 95% CI: 0.88–1.52, P = 0.310), rs1784418 in MMP20 (OR = 1.07, 95% CI: 0.76–1.49, P = 0.702) and rs3796704 in ENAM (OR = 1.06, 95% CI: 0.96–1.17, P = 0.228). Gene-based analysis indicated that multiple genetic variants in AMELX showed joint association with the risk of dental caries (6 variants; P < 10⁻⁵), so did genetic variants in MMP13 (3 variants; P = 0.004), MMP2 (3 variants; P < 10⁻⁵), MMP20 (2 variants; P < 10⁻⁵) and MMP3 (2 variants; P < 10⁻⁵). The gene-cluster analysis indicated a significant association between the genetic variants in this enamel-formation gene cluster and the risk of dental caries (P < 10⁻⁵). The present meta-analysis revealed that genetic variant rs17878486 in AMELX was associated with dental caries, and multiple genetic variants in the enamel-formation genes jointly contributed to the risk of dental caries, supporting the role of genetic variants in the enamel-formation genes in the etiology of dental caries.     

The IL-33-ST2L Pathway Is Associated with Coronary Artery Disease in a Chinese Han Population

The effects of interleukin-33 (IL-33) on the immune system have been clearly demonstrated; however, in cardiovascular diseases, especially in coronary artery disease (CAD), these effects have not yet been clarified. In this study, we investigate the genetic role of the IL-33-ST2L pathway in CAD. We performed three-stage case-control association analyses on a total of 4,521 individuals with CAD and 4,809 controls via tag SNPs in the genes encoding IL-33 and ST2L-IL-1RL1. One tag SNP in each gene was significantly associated with CAD (rs7025417^T in IL33, p_adj = 1.19 × 10⁻²⁸, OR = 1.39, 95% CI: 1.31–1.47; rs11685424^G in IL1RL1, p_adj = 6.93 × 10⁻³⁰, OR = 1.40, 95% CI: 1.32–1.48). Combining significant variants in two genes, the risk for CAD increased nearly 5-fold (p_adj = 8.90 × 10⁻²¹, OR = 4.98, 95% CI: 3.56–6.97). Traditional risk factors for CAD were adjusted for the association studies by SPSS with logistic regression analysis. With the two variants above, both located within the gene promoter regions, reporter gene analysis indicated that the rs7025417 C>T and rs11685424 A>G changes resulted in altered regulation of IL33 and IL1RL1 gene expression, respectively (p < 0.005). Further studies revealed that the rs7025417 genotype was significantly associated with plasma IL-33 levels in the detectable subjects (n = 227, R² = 0.276, p = 1.77 × 10⁻¹⁷): the level of IL-33 protein increased with the number of rs7025417 risk (T) alleles. Based on genetic evidence in humans, the IL-33-ST2L pathway appears to have a causal role in the development of CAD, highlighting this pathway as a valuable target for the prevention and treatment of CAD.     

Optimization of linkage mapping strategy and construction of a high-density American lotus linkage map

Background

Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification.

Results

We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F₁ population derived from a cross between Nelumbo nucifera ‘China Antique’ and N. lutea ‘AL1’. A total of 4,098 RADseq markers were used to construct the American lotus ‘AL1’ genetic map, and 147 markers were used to construct the Chinese lotus ‘China Antique’ genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera ‘China Antique’ draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome.

Conclusions

Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.     

Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation

Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.     

Genome Analysis of Lotus japonicus

Lotus japonicus , a model legume plant, was reviewed and compared with Medicago truncatula and soybean. Several mutant libraries are being analyzed, focusing on the nodulation mechanism. The first plant nodulation gene nin was cloned by Ac-transposon tagging. Soybean remains as the most studied legume, especially in relation to the disease resistance genes. However, Lotus japonicus offers several advantages for molecular genetics, and the remained lackings were recently filled up, namely 1) an appropriate crossing partner for Gifu, accession Miyakojima, was proposed for its 4% polymorphism and smooth recombining ability; 2) a genome library with long inserts, average of 140 kb, and 8.2 genome equivalents of library size, has been established; and 3) the rather low polymorphic rate between Gifu and Miyakojima can be overcome with the HEGS (High Efficiency Genome Scanning). With this infrastructure, positional cloning of the causative genes of several mutant libraries will be accomplished in a short term. Genome sizes of L. japonicus acc. Gifu and Miyakojima were determined with high accuracy, to be 494±0 MB and 512±1 MB, respectively. The feasibility of constructing a physical map of the entire genome, for functional genomics, was discussed. Received 5 September 2000/ Accepted in revised form 11 October 2000     

Large-Scale Development of Cost-Effective Single-Nucleotide Polymorphism Marker Assays for Genetic Mapping in Pigeonpea and Comparative Mapping in Legumes

Single-nucleotide polymorphisms (SNPs, >2000) were discovered by using RNA-seq and allele-specific sequencing approaches in pigeonpea (Cajanus cajan). For making the SNP genotyping cost-effective, successful competitive allele-specific polymerase chain reaction (KASPar) assays were developed for 1616 SNPs and referred to as PKAMs (pigeonpea KASPar assay markers). Screening of PKAMs on 24 genotypes [23 from cultivated species and 1 wild species (Cajanus scarabaeoides)] defined a set of 1154 polymorphic markers (77.4%) with a polymorphism information content (PIC) value from 0.04 to 0.38. One thousand and ninety-four PKAMs showed polymorphisms between parental lines of the reference mapping population (C. cajan ICP 28 × C. scarabaeoides ICPW 94). By using high-quality marker genotyping data on 167 F₂ lines from the population, a comprehensive genetic map comprising 875 PKAMs with an average inter-marker distance of 1.11 cM was developed. Previously mapped 35 simple sequence repeat markers were integrated into the PKAM map and an integrated genetic map of 996.21 cM was constructed. Mapped PKAMs showed a higher degree of synteny with the genome of Glycine max followed by Medicago truncatula and Lotus japonicus and least with Vigna unguiculata. These PKAMs will be useful for genetics research and breeding applications in pigeonpea and for utilizing genome information from other legume species.     

Identification of the centromere-specific histone H3 variant in Lotus japonicus

The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japonicus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofluorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species.     

LjABCB1, an ATP-binding cassette protein specifically induced in uninfected cells of Lotus japonicus nodules

Legume plants develop root nodules through symbiosis with rhizobia, and fix atmospheric nitrogen in this symbiotic organ. Development of root nodules is regulated by many metabolites including phytohormones. Previously, we reported that auxin is strongly involved in the development of the nodule vascular bundle and lenticel formation on the nodules of Lotus japonicus. Here we show that an ATP-binding cassette (ABC) protein, LjABCB1, which is a homologue of Arabidopsis auxin transporter AtABCB4, is specifically expressed during nodulation of L. japonicus. A reporter gene analysis indicated that the expression of LjABCB1 was restricted to uninfected cells adjacent to infected cells in the nodule, while no expression was observed in shoot apical meristems or root tips, in which most auxin transporter genes are expressed. The auxin transport activity of LjABCB1 was confirmed using a heterologous expression system.