Generation of 7137 non-redundant expressed sequence tags from a legume, Lotus japonicus.

For comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 22,983 5' end expressed sequence tags (ESTs) were accumulated from normalized and size-selected cDNA libraries constructed from young (2 weeks old) plants. The EST sequences were clustered into 7137 non-redundant groups. Similarity search against public non-redundant protein database indicated that 3302 groups showed similarity to genes of known function, 1143 groups to hypothetical genes, and 2692 were novel sequences. Homologues of 5 nodule-specific genes which have been reported in other legume species were contained in the collected ESTs, suggesting that the EST source generated in this study will become a useful tool for identification of genes related to legume-specific biological processes. The sequence data of individual ESTs are available at the web site: http://www.kazusa.or.jp/en/plant/lotus/EST/.     

Molecular linkage map for an intraspecific recombinant inbred population of durum wheat (Triticum turgidum L. var. durum)

Durum wheat (Triticum turgidum L. var. durum) is an economically and nutritionally important cereal crop in the Mediterranean region. To further our understanding of durum genome organization we constructed a durum linkage map using restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) known as Gatersleben wheat microsatellites (GWMs), amplified fragment length polymorphisms (AFLPs), and seed storage proteins (SSPs: gliadins and glutenins). A population of 110 F₉ recombinant inbred lines (RILs) was derived from an intraspecific cross between two durum cultivars, Jennah Khetifa and Cham 1. The two parents exhibit contrasting traits for resistance to biotic and abiotic stresses and for grain quality. In total, 306 markers have been placed on the linkage map – 138 RFLPs, 26 SSRs, 134 AFLPs, five SSPs, and three known genes (one pyruvate decarboxylase and two lipoxygenases). The map is 3598 cM long, with an average distance between markers of 11.8 cM, and 12.1% of the markers deviated significantly from the expected Mendelian ratio 1:1. The molecular markers were evenly distributed between the A and B genomes. The chromosome with the most markers is 1B (41 markers), followed by 3B and 7B, with 25 markers each. The chromosomes with the fewest markers are 2A (11 markers), 5A (12 markers), and 4B (15 markers). In general, there is a good agreement between the map obtained and the Triticeae linkage consensus maps. This intraspecific map provides a useful tool for marker-assisted selection and map-based breeding for resistance to biotic and abiotic stresses and for improvement of grain quality. Received: 14 February 2000 / Accepted: 28 April 2000     

Genome and Chromosome Dimensions of Lotus japonicus

Lotus Japonicus , Miyakojima MG-20 and Gifu B-129. The genome sizes of Miyakojima and Gifu were determined as 472.1 and 442.8 Mbp, respectively. Both the accessions were diploid (2n=12) and six chromosomes were identified and characterized based on the condensation patterns and the locations of rDNA loci. The obvious polymorphism observed in the genome size and the chromosome morphology between the two accessions, revealed specific accumulation of heterochromatin in Miyakojima or elimination in Gifu. The chromosomes L. japonicus were numbered according to their length. A quantitative chromosome map was also developed by the imaging methods using the digital data of the condensation pattern. 45S rDNA loci were localized on chromosomes A and F, and 5S rDNA locus was localized on chromosome A by fluorescence in situ hybridization (FISH). Identification of the chromosome and genome sizes and development of the quantitative chromosome map represent significant contribution to the L. japonicus genome project as the basic information. Received 29 August 2000/ Accepted in revised form 17 October 2000     

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.     

Development of an integrated genetic map of a sugarcane (Saccharum spp.) commercial cross, based on a maximum-likelihood approach for estimation of linkage and linkage phases

Sugarcane (Saccharum spp.) is a clonally propagated outcrossing polyploid crop of great importance in tropical agriculture. Up to now, all sugarcane genetic maps had been developed using either full-sib progenies derived from interspecific crosses or from selfing, both approaches not directly adopted in conventional breeding. We have developed a single integrated genetic map using a population derived from a cross between two pre-commercial cultivars (‘SP80-180’ × ‘SP80-4966’) using a novel approach based on the simultaneous maximum-likelihood estimation of linkage and linkage phases method specially designed for outcrossing species. From a total of 1,118 single-dose markers (RFLP, SSR and AFLP) identified, 39% derived from a testcross configuration between the parents segregating in a 1:1 fashion, while 61% segregated 3:1, representing heterozygous markers in both parents with the same genotypes. The markers segregating 3:1 were used to establish linkage between the testcross markers. The final map comprised of 357 linked markers, including 57 RFLPs, 64 SSRs and 236 AFLPs that were assigned to 131 co-segregation groups, considering a LOD score of 5, and a recombination fraction of 37.5 cM with map distances estimated by Kosambi function. The co-segregation groups represented a total map length of 2,602.4 cM, with a marker density of 7.3 cM. When the same data were analyzed using JoinMap software, only 217 linked markers were assigned to 98 co-segregation groups, spanning 1,340 cM, with a marker density of 6.2 cM. The maximum-likelihood approach reduced the number of unlinked markers to 761 (68.0%), compared to 901 (80.5%) using JoinMap. All the co-segregation groups obtained using JoinMap were present in the map constructed based on the maximum-likelihood method. Differences on the marker order within the co-segregation groups were observed between the two maps. Based on RFLP and SSR markers, 42 of the 131 co-segregation groups were assembled into 12 putative homology groups. Overall, the simultaneous maximum-likelihood estimation of linkage and linkage phases was more efficient than the method used by JoinMap to generate an integrated genetic map of sugarcane. E.A. Kido, A.N. Meza and H.M.B. Souza contributed equally to this work.     

Ecotypes of the model legume Lotus japonicus vary in their interaction phenotypes with the root-knot nematode Meloidogyne incognita

BACKGROUND AND AIMS: Knowledge of host factors affecting plant-nematode interactions is scarce. Here, relevant interaction phenotypes between a nodulating model host, Lotus japonicus, and the endoparasitic root-knot nematode Meloidogyne incognita are assessed via a genetic screen. METHODS: Within an alpha experimental design, 4-week-old replicate plants from 60 L. japonicus ecotypes were inoculated with 1000 nematodes from a single egg mass population, and evaluated for galling and nematode egg masses 6 weeks after inoculation. KEY RESULTS: Statistical analysis of data for 57 ecotypes showed that ecotype susceptibilities ranged from 3.5 to 406 galls per root, and correlated strongly (r = 0.8, P < 0.001, log scale) with nematode reproduction (ranging from 0.6 to 34.5 egg masses per root). Some ecotypes, however, showed a significant discrepancy between disease severity and nematode reproduction. Necrosis and developmental malformations were observed in other infected ecotypes. CONCLUSIONS: The first evidence is provided of significant variability in the interactions between L. japonicus and root-knot nematodes that may have further implications for the genetic dissection and characterization of host pathways involved in nematode parasitism and, possibly, in microbial symbiosis.     

The construction of a genetic linkage map of non-heading Chinese cabbage （Brassica campestris ssp. chinensis Makino）

Non-heading Chinese cabbage （Brassica carnpestris ssp. chinensis Makino） is one of the most important vegetables in eastern China. A genetic linkage map was constructed using 127 doubled haploid （DH） lines, and the DH population was derived from a commercial hybrid ＂Hanxiao＂ （lines SW-13 x L-118）. Out of the 614 polyrnorphic markers, 43.49% were not assigned to any of the linkage groups （LGs）. Chi-square tests showed that 42.67% markers were distorted from expected Mendelian segregation ratios, and the direction of distorted segregation was mainly toward the paternal parent L-118. After sequentially removing the markers that had an interval distance smaller than 1 cM from the upper marker, the overall quality of the linkage map was increased. Two hundred and sixty-eight molecular markers were mapped into 10 LGs, which were anchored to the corresponding chromosome of the B. rapa reference map based on com- mon simple sequence repeat （SSR） markers. The map covers 973.38 cM of the genome and the average interval distance between markers was 3.63 cM. The number of markers on each LG ranged from 18 （R08） to 64 （R07）, with an average interval distance within a single LG from 1.70 cM （R07） to 6.71 cM （R06）. Among these mapped markers, 169 were sequence-related amplified polymorphism （SRAP） molecular markers, 50 were SSR markers and 49 were random amplification polymorphic DNA （RAPD） markers. With further saturation to the LG9 the current map offers a genetic tool for loci analysis for important agronomic traits.     

AFLP linkage map of the Japanese quail Coturnix japonica

The quail is a valuable farm and laboratory animal. Yet molecular information about this species remains scarce. We present here the first genetic linkage map of the Japanese quail. This comprehensive map is based solely on amplified fragment length polymorphism (AFLP) markers. These markers were developed and genotyped in an F2 progeny from a cross between two lines of quail differing in stress reactivity. A total of 432 polymorphic AFLP markers were detected with 24 TaqI/EcoRI primer combinations. On average, 18 markers were produced per primer combination. Two hundred and fifty eight of the polymorphic markers were assigned to 39 autosomal linkage groups plus the ZW sex chromosome linkage groups. The linkage groups range from 2 to 28 markers and from 0.0 to 195.5 cM. The AFLP map covers a total length of 1516 cM, with an average genetic distance between two consecutive markers of 7.6 cM. This AFLP map can be enriched with other marker types, especially mapped chicken genes that will enable to link the maps of both species and make use of the powerful comparative mapping approach. This AFLP map of the Japanese quail already provides an efficient tool for quantitative trait loci (QTL) mapping.     

Structural analysis of a Lotus japonicus genome. IV. Sequence features and mapping of seventy-three TAC clones which cover the 7.5 mb regions of the genome.

Using the sequence information of expressed sequences tags (ESTs), cDNAs and genes from Lotus japonicus and other legumes, 73 TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of L. japonicus accession MG-20, and their nucleotide sequences were determined. The length of the DNA sequenced in this study was 7,455,959 bp, and the total length of the DNA regions sequenced so far is 26,167,443 bp together with the nucleotide sequences of 183 TAC clones previously reported. By similarity searches against the sequences in protein and EST databases and prediction by computer programs, a total of 699 potential protein-encoding genes with known or predicted functions, 163 gene segments and 267 pseudogenes were assigned to the newly sequenced regions. Based oil the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was located onto the linkage map of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Structural analysis of a Lotus japonicus genome. III. Sequence features and mapping of sixty-two TAC clones which cover the 6.7 Mb regions of the genome.

A total of sixty-two clones were selected from a TAC (transformation-competent artificial chromosome) genomic library of the Lotus japonicus accession MG-20 based on the sequence information of expressed sequence tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined. The length of the sequenced regions in this study is 6,682,189 bp, and the total length of the regions sequenced so far is 18,711,484 bp together with the nucleotide sequences of 121 TAC clones previously reported. By comparison with the sequences in protein and EST databases and analysis with computer programs for gene modeling, a total of 573 potential protein-coding genes with known or predicted functions, 91 gene segments and 272 pseudogenes were identified in the newly sequenced regions. Each of the sequenced clones was localized onto the linkage map of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20, using simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers generated based on the nucleotide sequences of the clones. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Structural analysis of a Lotus japonicus genome. I. Sequence features and mapping of fifty-six TAC clones which cover the 5.4 mb regions of the genome.

A total of 56 TAC clones with an average insert size of 100 kb were isolated from a TAC library of the Lotus japonicus genome based on the expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined according to the shot-gun based strategy. The total length of the sequenced regions is 5,473,195 bp. By comparison with the sequences in protein and EST databases and analysis with computer programs for gene modeling, a total of 605 potential protein-encoding genes with known or predicted functions, 69 gene segments, and 172 pseudogenes were identified. The average density of the genes assigned so far is 1 gene/8120 bp. Introns were identified in approximately 78% of the potential genes. There was an average of 3.8 introns per gene and the average length of the introns was 375 bp. DNA markers were generated based on the nucleotide sequences obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of L. japonicus Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Structural analysis of a Lotus japonicus genome. V. Sequence features and mapping of sixty-four TAC clones which cover the 6.4 mb regions of the genome.

We determined the nucleotide sequences of 64 TAC (transformation-competent artificial chromosome) clones selected from genomic libraries of Lotus japonicus accession Miyakojima MG-20 based on the sequence information of expressed sequence tags (ESTs), cDNAs, genes and DNA markers from L. japonicus and other legumes. The length of the DNA regions sequenced in this study was 6,370,255 bp, and the total length of the L. japonicus genome sequenced so far is 32,537,698 bp together with the nucleotide sequences of 256 TAC clones previously reported. Five hundred forty-eight potential protein-encoding genes with known or predicted functions, 127 gene segments and 224 pseudogenes were assigned to the newly sequenced regions by computer prediction and similarity searches against the sequences in protein and EST databases. Based on the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was genetically localized onto the linkage map of two accessions of L. japonicus, MG-20 and Gifu B-129. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Structural analysis of a Lotus japonicus genome. II. Sequence features and mapping of sixty-five TAC clones which cover the 6.5-mb regions of the genome.

Sixty-five TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of Lotus japonicus accession MG-20 based on the sequence information of expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined. The average insert size of the TAC clone was approximately 100 kb, and the total length of the sequenced regions in this study is 6,556,100 bp. Together with the nucleotide sequences of 56 TAC clones previously reported, the regions sequenced so far total 12,029,295 bp. By comparison with the sequences in protein and EST databases and by analysis with computer programs for gene modeling, a total of 711 potential protein-encoding genes with known or predicted functions, 239 gene segments and 90 pseudogenes were identified in the newly sequenced regions. The average gene density assigned so far was 1 gene/9140 bp. The average length of the assigned genes was 2.6 kb, which is considerably larger than that assigned in the Arabidopsis thaliana genome (1.9 kb for 6451 genes). Introns were identified in approximately 73% of the potential genes, and the average number and length of the introns per gene were 3.4 and 377 bp, respectively. Simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated based on the nucleotide sequences of the genomic clones obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

A genetic linkage map of tef [Eragrostis tef (Zucc.) Trotter] based on amplified fragment length polymorphism

A genetic linkage map of tef was constructed with amplified fragment length polymorphism (AFLP) markers using F₅ recombinant inbred lines (RILs) derived by single seed descent from the intraspecific cross of ’Kaye Murri’×’Fesho’. A total of 192 EcoRI/MseI primer combinations were screened for parental polymorphism. Around three polymorphic fragments per primer combination were detected, indicating a low polymorphism level in tef. Fifty primer combinations were selected to assay the mapping population, and 226 loci segregated among 85 F₅ RILs. Most AFLP loci behaved as dominant markers (presence or absence of a band), but about 15% of the loci were codominant. Significant deviations from the expected Mendelian segregation ratio were observed for 26 loci. The genetic linkage map comprised 211 markers assembled into 25 linkage groups and covered 2,149 cM of genome. AFLP is an efficient marker system for mapping plant species with low polymorphism such as tef. This is the first genetic linkage map constructed for tef. It will facilitate the mapping of genes controlling agronomically important traits and cultivar improvement in tef. Received: 27 April 1998 / Accepted: 4 January 1999     

Construction of a genetic linkage map for silver carp (Hypophthalmichthys molitrix)

For silver carp (Hypophthalmichthys molitrix), a combined microsatellite (or simple sequence repeat) and amplified fragment length polymorphism (AFLP) sex average linkage map was constructed. A total of 483 markers (245 microsatellites and 238 AFLPs) were assigned to 33 linkage groups. The map spanned 1352.2 cM, covering 86.4% of the estimated genome size of silver carp. The maximum and average spaces between 420 loci were 21.5 cM and 3.2 cM, respectively. The length of linkage groups ranged from 3.6 cM to 98.5 cM with an average of 41.0 cM. The number of markers per group varied from 2 to 44 with an average of 14.6. The AFLP markers significantly improved the integrity of microsatellite-based linkage groups and increased the genome coverage and marker evenness. A genome-wide recombination suppression was observed in male. In an extreme case, six microsatellites co-segregated in male, but spanned a 45.1 cM region in female.     

百脉根嫁接方法的优化及其在结瘤自调控研究中的应用

嫁接试验是研究结瘤自调控的有效方法。本文优化了百脉根的嫁接方法,并应用该方法初步研究了百脉根突变体rel3根瘤数目减少的机理。结果表明,rel3突变体根瘤数目减少的表型是由来自于茎的信号决定。     

Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences

Marker-assisted selection (MAS) offers quick and reliable prediction of the phenotypes of seedlings in large populations and thus opens new approaches for selection to breeders of apple (Malus x domestica Borkh.). The development of framework maps enables the discovery of genetic markers linked to desired traits. Although genetic maps have been reported for apple scion cultivars, none has previously been constructed for apple rootstocks. We report the construction of framework genetic maps in a cross between ‘M.9’ (‘Malling 9’) and ‘R.5’ (‘Robusta 5’) apple rootstocks. The maps comprise 224 simple sequence repeat (SSR) markers, 18 sequence-characterised amplified regions, 14 single nucleotide polymorphisms and 42 random amplified polymorphic DNAs. A new set of 47 polymorphic SSRs was developed from apple EST sequences and used for construction of this rootstock map. All 17 linkage groups have been identified and aligned to existing apple genetic maps. The maps span 1,175.7 cM (‘M.9’) and 1,086.7 cM (‘R.5’). To improve the efficiency of mapping markers to this framework map, we developed a bin mapping set. Applications of these new genetic maps include the elucidation of the genetic basis of the dwarfing effect of the apple rootstock ‘M.9’ and the analysis of disease and insect resistance traits such as fire blight (Erwinia amylovora), apple scab (Venturia inaequalis) and woolly apple aphid (Eriosoma lanigerum). Markers for traits mapped in this population will be of direct use to apple breeders for MAS and for identification of causative genes by map-based cloning.     

Split-root study of autoregulation of nodulation in the model legume Lotus japonicus

We used a split-root system to determine the timing for induction of the autoregulation of nodulation (AUT) in Lotus japonicus (Regel) Larsen after inoculation with Mesorhizobium loti. The signal took at least five days for full induction of AUT and inhibition of infection thread formation. Strain ML108 (able to nodulate but unable to fix nitrogen) induced full AUT, but ML101 (unable to nodulate or to fix nitrogen) did not induce autoregulation. These results indicate that Nod factor-producing strains induce AUT, but that the nitrogen fixed by rhizobia and supplied to the plant as ammonia does not elicit the AUT in L. japonicus.     

Lotus burttii takes a position of the third corner in the lotus molecular genetics triangle.

In order to consolidate molecular genetic system in Lotus japonicus and to further access the biological diversity in Lotea, we introduce here Lotus burttii B-303 derived from West Pakistan as the third crossing partner of the Gifu ecotype (B-129-S9) for a genetic analysis. L. burttii is a relatively small and early flowering plant with non-shattering behavior. The general chromosome morphology is very similar to Gifu, and fluorescence in situ hybridization (FISH) analysis revealed that the short arm of chromosome 1 in L. burttii is comparable to that of Gifu, indicating that the translocation event involving chromosomes 1 and 2, which was observed in L. japonicus Miyakojima MG-20, is not present in L. burttii. In addition L. burttii has a higher level of DNA polymorphism compared to Gifu and MG-20 enabling design of codominant markers such as SSR, CAPS and dCAPS. Using an F2 population from a cross between Gifu and L. burttii, codominant makers that co-segregated at the translocation site could be expanded. In order to normalize the genetic background, L. burttii was inbred for nine generations and the germplasm L. burttii B-303-S9 was established.