Genomic prediction for root and yield traits of barley under a water availability gradient: a case study comparing different spatial adjustments.

BACKGROUND: In drought periods, water use efficiency depends on the capacity of roots to extract water from deep soil. A semi-field phenotyping facility (RadiMax) was used to investigate above-ground and root traits in spring barley when grown under a water availability gradient. Above-ground traits included grain yield, grain protein concentration, grain nitrogen removal, and thousand kernel weight. Root traits were obtained through digital images measuring the root length at different depths. Two nearest-neighbor adjustments (M1 and M2) to model spatial variation were used for genetic parameter estimation and genomic prediction (GP). M1 and M2 used (co)variance structures and differed in the distance function to calculate between-neighbor correlations. M2 was the most developed adjustment, as accounted by the Euclidean distance between neighbors. RESULTS: The estimated heritabilities ([Formula: see text]) ranged from low to medium for root and above-ground traits. The genetic coefficient of variation ([Formula: see text]) ranged from 3.2 to 7.0% for above-ground and 4.7 to 10.4% for root traits, indicating good breeding potential for the measured traits. The highest [Formula: see text] observed for root traits revealed that significant genetic change in root development can be achieved through selection. We studied the genotype-by-water availability interaction, but no relevant interaction effects were detected. GP was assessed using leave-one-line-out (LOO) cross-validation. The predictive ability (PA) estimated as the correlation between phenotypes corrected by fixed effects and genomic estimated breeding values ranged from 0.33 to 0.49 for above-ground and 0.15 to 0.27 for root traits, and no substantial variance inflation in predicted genetic effects was observed. Significant differences in PA were observed in favor of M2. CONCLUSIONS: The significant [Formula: see text] and the accurate prediction of breeding values for above-ground and root traits revealed that developing genetically superior barley lines with improved root systems is possible. In addition, we found significant spatial variation in the experiment, highlighting the relevance of correctly accounting for spatial effects in statistical models. In this sense, the proposed nearest-neighbor adjustments are flexible approaches in terms of assumptions that can be useful for semi-field or field experiments.

Integration of DNA Methylation and Transcriptome Data Improves Complex Trait Prediction in Hordeum vulgare.

Whole-genome multi-omics profiles contain valuable information for the characterization and prediction of complex traits in plants. In this study, we evaluate multi-omics models to predict four complex traits in barley (Hordeum vulgare); grain yield, thousand kernel weight, protein content, and nitrogen uptake. Genomic, transcriptomic, and DNA methylation data were obtained from 75 spring barley lines tested in the RadiMax semi-field phenomics facility under control and water-scarce treatment. By integrating multi-omics data at genomic, transcriptomic, and DNA methylation regulatory levels, a higher proportion of phenotypic variance was explained (0.72-0.91) than with genomic models alone (0.55-0.86). The correlation between predictions and phenotypes varied from 0.17-0.28 for control plants and 0.23-0.37 for water-scarce plants, and the increase in accuracy was significant for nitrogen uptake and protein content compared to models using genomic information alone. Adding transcriptomic and DNA methylation information to the prediction models explained more of the phenotypic variance attributed to the environment in grain yield and nitrogen uptake. It furthermore explained more of the non-additive genetic effects for thousand kernel weight and protein content. Our results show the feasibility of multi-omics prediction for complex traits in barley.

Pyramiding of scald resistance genes in four spring barley MAGIC populations.

Genome-Wide Association Studies (GWAS) of four Multi-parent Advanced Generation Inter-Cross (MAGIC) populations identified nine regions on chromosomes 1H, 3H, 4H, 5H, 6H and 7H associated with resistance against barley scald disease. Three of these regions are putatively novel resistance Quantitative Trait Loci (QTL). Barley scald is caused by Rhynchosporium commune, one of the most important barley leaf diseases that are prevalent in most barley-growing regions. Up to 40% yield losses can occur in susceptible barley cultivars. Four MAGIC populations were generated in a Nordic Public-Private Pre-breeding of spring barley project (PPP Barley) to introduce resistance to several important diseases. Here, these MAGIC populations consisting of six to eight founders each were tested for scald resistance in field trials in Finland and Iceland. Eight different model covariate combinations were compared for GWAS studies, and the models that deviated the least from the expected p-values were selected. For all QTL, candidate genes were identified that are predicted to be involved in pathogen defence. The MAGIC progenies contained new haplotypes of significant SNP-markers with high resistance levels. The lines with successfully pyramided resistance against scald and mildew and the significant markers are now distributed among Nordic plant breeders and will benefit development of disease-resistant cultivars.

You Had Me at "MAGIC"!: Four Barley MAGIC Populations Reveal Novel Resistance QTL for Powdery Mildew.

Blumeria graminis f. sp. hordei (Bgh), the causal agent of barley powdery mildew (PM), is one of the most important barley leaf diseases and is prevalent in most barley growing regions. Infection decreases grain quality and yields on average by 30%. Multi-parent advanced generation inter-cross (MAGIC) populations combine the advantages of bi-parental and association panels and offer the opportunity to incorporate exotic alleles into adapted material. Here, four barley MAGIC populations consisting of six to eight founders were tested for PM resistance in field trials in Denmark. Principle component and STRUCTURE analysis showed the populations were unstructured and genome-wide linkage disequilibrium (LD) decay varied between 14 and 38 Mbp. Genome-wide association studies (GWAS) identified 11 regions associated with PM resistance located on chromosomes 1H, 2H, 3H, 4H, 5H and 7H, of which three regions are putatively novel resistance quantitative trait locus/loci (QTL). For all regions high-confidence candidate genes were identified that are predicted to be involved in pathogen defense. Haplotype analysis of the significant SNPs revealed new allele combinations not present in the founders and associated with high resistance levels.

The cytosine methylation landscape of spring barley revealed by a new reduced representation bisulfite sequencing pipeline, WellMeth.

Patterns and level of cytosine methylation vary widely among plant species and are associated with genome size as well as the proportion of transposons and other repetitive elements in the genome. We explored epigenetic patterns and diversity in a representative proportion of the spring barley (Hordeum vulgare L.) genome across several commercial and historical cultivars. This study adapted a genotyping-by-sequencing (GBS) approach for the detection of methylated cytosines in genomic DNA. To analyze the data, we developed WellMeth, a complete pipeline for analysis of reduced representation bisulfite sequencing. WellMeth enabled quantification of context-specific DNA methylation at the single-base resolution as well as identification of differentially methylated sites (DMCs) and regions (DMRs). On average, DNA methylation levels were significantly higher than what is commonly observed in many plants species, reaching over 10-fold higher levels than those in Arabidopsis thaliana (L.) Heynh. in the CHH methylation. Preferential methylation was observed within and at the edges of long-terminal repeats (LTR) retrotransposons Gypsy and Copia. From a pairwise comparison of cultivars, numerous DMRs could be identified of which more than 5,000 were conserved within the analyzed set of barley cultivars. The subset of regions overlapping with genes showed enrichment in gene ontology (GO) categories associated with chromatin and cellular structure and organization. A significant correlation between genetic and epigenetic distances suggests that a considerable portion of methylated regions is under strict genetic control in barley. The data presented herein represents the first step in efforts toward a better understanding of genome-level structural and functional aspects of methylation in barley.

Identification of Ideal Allele Combinations for the Adaptation of Spring Barley to Northern Latitudes.

The northwards expansion of barley production requires adaptation to longer days, lower temperatures and stronger winds during the growing season. We have screened 169 lines of the current barley breeding gene pool in the Nordic region with regards to heading, maturity, height, and lodging under different environmental conditions in nineteen field trials over 3 years at eight locations in northern and central Europe. Through a genome-wide association scan we have linked phenotypic differences observed in multi-environment field trials (MET) to single nucleotide polymorphisms (SNP). We have identified an allele combination, only occurring among a few Icelandic lines, that affects heat sum to maturity and requires 214 growing degree days (GDD) less heat sum to maturity than the most common allele combination in the Nordic spring barley gene pool. This allele combination is beneficial in a cold environment, where autumn frost can destroy a late maturing harvest. Despite decades of intense breeding efforts relying heavily on the same germplasm, our results show that there still exists considerable variation within the current breeding gene pool and we identify ideal allele combinations for regional adaptation, which can facilitate the expansion of cereal cultivation even further northwards.

A Novel QTL for Powdery Mildew Resistance in Nordic Spring Barley (Hordeum vulgare L. ssp. vulgare) Revealed by Genome-Wide Association Study.

The powdery mildew fungus, Blumeria graminis f. sp. hordei is a worldwide threat to barley (Hordeum vulgare L. ssp. vulgare) production. One way to control the disease is by the development and deployment of resistant cultivars. A genome-wide association study was performed in a Nordic spring barley panel consisting of 169 genotypes, to identify marker-trait associations significant for powdery mildew. Powdery mildew was scored during three years (2012-2014) in four different locations within the Nordic region. There were strong correlations between data from all locations and years. In total four QTLs were identified, one located on chromosome 4H in the same region as the previously identified mlo locus and three on chromosome 6H. Out of these three QTLs identified on chromosome 6H, two are in the same region as previously reported QTLs for powdery mildew resistance, whereas one QTL appears to be novel. The top NCBI BLASTn hit of the SNP markers within the novel QTL predicted the responsible gene to be the 26S proteasome regulatory subunit, RPN1, which is required for innate immunity and powdery mildew-induced cell death in Arabidopsis. The results from this study have revealed SNP marker candidates that can be exploited for use in marker-assisted selection and stacking of genes for powdery mildew resistance in barley.

Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding.

Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man(3)XylFucGlcNAc(4), were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor-ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The K(d) values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes.

The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation.

The number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads to hypernodulation. We have characterised the Pisum sativum (pea) Sym28 locus involved in autoregulation and shown that it encodes a protein similar to the Arabidopsis CLAVATA2 (CLV2) protein. Inactivation of the PsClv2 gene in four independent sym28 mutant alleles, carrying premature stop codons, results in hypernodulation of the root and changes to the shoot architecture. In the reproductive phase sym28 shoots develops additional flowers, the stem fasciates, and the normal phyllotaxis is perturbed. Mutational substitution of an amino acid in one leucine rich repeat of the corresponding Lotus japonicus LjCLV2 protein results in increased nodulation. Similarly, down-regulation of the Lotus Clv2 gene by RNAi mediated reduction of the transcript level also resulted in increased nodulation. Gene expression analysis of LjClv2 and Lotus hypernodulation aberrant root formation Har1 (previously shown to regulate nodule numbers) indicated they have overlapping organ expression patterns. However, we were unable to demonstrate a direct protein-protein interaction between LjCLV2 and LjHAR1 proteins in contrast to the situation between equivalent proteins in Arabidopsis. LjHAR1 was localised to the plasma membrane using a YFP fusion whereas LjCLV2-YFP localised to the endoplasmic reticulum when transiently expressed in Nicotiana benthamiana leaves. This finding is the most likely explanation for the lack of interaction between these two proteins.

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

Dissection of symbiosis and organ development by integrated transcriptome analysis of lotus japonicus mutant and wild-type plants.

Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set.

The proteome of seed development in the model legume Lotus japonicus.

We have characterized the development of seeds in the model legume Lotus japonicus. Like soybean (Glycine max) and pea (Pisum sativum), Lotus develops straight seed pods and each pod contains approximately 20 seeds that reach maturity within 40 days. Histological sections show the characteristic three developmental phases of legume seeds and the presence of embryo, endosperm, and seed coat in desiccated seeds. Furthermore, protein, oil, starch, phytic acid, and ash contents were determined, and this indicates that the composition of mature Lotus seed is more similar to soybean than to pea. In a first attempt to determine the seed proteome, both a two-dimensional polyacrylamide gel electrophoresis approach and a gel-based liquid chromatography-mass spectrometry approach were used. Globulins were analyzed by two-dimensional polyacrylamide gel electrophoresis, and five legumins, LLP1 to LLP5, and two convicilins, LCP1 and LCP2, were identified by matrix-assisted laser desorption ionization quadrupole/time-of-flight mass spectrometry. For two distinct developmental phases, seed filling and desiccation, a gel-based liquid chromatography-mass spectrometry approach was used, and 665 and 181 unique proteins corresponding to gene accession numbers were identified for the two phases, respectively. All of the proteome data, including the experimental data and mass spectrometry spectra peaks, were collected in a database that is available to the scientific community via a Web interface (http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi). This database establishes the basis for relating physiology, biochemistry, and regulation of seed development in Lotus. Together with a new Web interface (http://bioinfoserver.rsbs.anu.edu.au/utils/PathExpress4legumes/) collecting all protein identifications for Lotus, Medicago, and soybean seed proteomes, this database is a valuable resource for comparative seed proteomics and pathway analysis within and beyond the legume family.

Defects in rhizobial cyclic glucan and lipopolysaccharide synthesis alter legume gene expression during nodule development.

cDNA array technology was used to compare transcriptome profiles of Lotus japonicus roots inoculated with a Mesorhizobium loti wild-type and two mutant strains affected in cyclic beta(1-2) glucan synthesis (cgs) and in lipopolysaccharide synthesis (lpsbeta2). Expression of genes associated with the development of a fully functional nodule was significantly affected in plants inoculated with the cgs mutant. Array results also revealed that induction of marker genes for nodule development was delayed when plants were inoculated with the lpsbeta2 mutant. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to quantify gene expression of a subset of genes involved in plant defense response, redox metabolism, or genes that encode for nodulins. The majority of the genes analyzed in this study were more highly expressed in roots inoculated with the wild type compared with those inoculated with the cgs mutant strain. Some of the genes exhibited a transient increase in transcript levels during intermediate steps of normal nodule development while others displayed induced expression during the final steps of nodule development. Ineffective nodules induced by the glucan mutant showed higher expression of phenylalanine ammonia lyase than wild-type nodules. Differences in expression pattern of genes involved in early recognition and signaling were observed in plants inoculated with the M. loti mutant strain affected in the synthesis of cyclic glucan.

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci.

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development.

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus.

Research on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic pathways in many different legume species. We utilized the tools of transcriptomics and metabolomics to obtain an unprecedented overview of the metabolic differentiation that results from nodule development in the model legume, Lotus japonicus. Using an array of more than 5000 nodule cDNA clones, representing 2500 different genes, we identified approximately 860 genes that were more highly expressed in nodules than in roots. One-third of these are involved in metabolism and transport, and over 100 encode proteins that are likely to be involved in signalling, or regulation of gene expression at the transcriptional or post-transcriptional level. Several metabolic pathways appeared to be co-ordinately upregulated in nodules, including glycolysis, CO(2) fixation, amino acid biosynthesis, and purine, haem, and redox metabolism. Insight into the physiological conditions that prevail within nodules was obtained from specific sets of induced genes. In addition to the expected signs of hypoxia, numerous indications were obtained that nodule cells also experience P-limitation and osmotic stress. Several potential regulators of these stress responses were identified. Metabolite profiling by gas chromatography coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis.

Shoot control of root development and nodulation is mediated by a receptor-like kinase.

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci.

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.