利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

大粒裸燕麦(Avena nuda L.)遗传连锁图谱的构建

以“元莜麦”和“555”杂交得到的281个F2单株为作图群体,利用20对AFLP引物、3对SSR引物和1个穗型性状构建了一张大粒裸燕麦遗传连锁图。该图谱全长1544.8cM,包含19个连锁群,其上分布有92个AFLP标记、3个SSR标记和1个穗型形态标记,不同连锁群标记数为2-14个,长度在23.7-276.3cM之间,平均长度为81.3cM,标记间平均距离为20.1cM。穗型标记分离比符合3：1,11个AFLP标记表现为偏分离,偏分离比为11.5%。该图谱符合遗传连锁框架图的要求,为今后大粒裸燕麦的QTL定位、分子标记辅助育种和比较基因组学等研究奠定基础。     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

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     

Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1

We established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population was composed of 2450 plant lines, from which a total of 4532 flanking sequence tags of LORE1 were recovered by pyrosequencing. The two-dimensional arrangement of the plant population, together with the use of multiple identifier sequences in the primers used to amplify the flanking regions, made it possible to trace insertions back to the original plant lines. The large-scale detection of new LORE1 insertion sites revealed a preference for genic regions, especially in exons of protein-coding genes, which is an interesting feature to consider in the interaction between host genomes and chromoviruses, to which LORE1 belongs, a class of retrotransposon widely distributed among plants. Forward screening of the symbiotic mutants from the population succeeded to identify five symbiotic mutants of known genes. These data suggest that LORE1 is robust as a genetic tool.     

家蚕AFLP连锁框架图谱的构建

利用改进的AFLP分子标记方法,对家蚕Bombyx mori回交一代BC1群体进行连锁图谱的构建。经17组AFLP引物的选择性扩增,共得到430个多态位点,卡方检验后有253个为有效位点。利用Mapmaker/Exp (Version 3.0b)软件作连锁分析,其中163个标记分属28个连锁群,连锁群标记数变化范围是2～28个,平均每个连锁群标记数为5.8个,该图覆盖的基因组长度为2.998.9cM(图距单位),连锁群长度变化范围为4.5～652.8 cM,连锁群的平均长度为107.1 cM,平均图距为4.5～36.7 cM。     

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     

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.     

A genetic linkage map of the sea cucumber, Apostichopus japonicus (Selenka), based on AFLP and microsatellite markers

We present the first genetic maps of the sea cucumber ( Apostichopus japonicus ), constructed with an F₁ pseudo-testcross strategy. The 37 amplified fragment length polymorphism (AFLP) primer combinations chosen identified 484 polymorphic markers. Of the 21 microsatellite primer pairs tested, 16 identified heterozygous loci in one or other parent, and six were fully informative, as they segregated in both parents. The female map comprised 163 loci, spread over 20 linkage groups (which equals the haploid chromosome number), and spanned 1522.0 cM, with a mean marker density of 9.3 cM. The equivalent figures for the male map were 162 loci, 21 linkage groups, 1276.9 and 7.9 cM. About 2.5% of the AFLP markers displayed segregation distortion and were not used for map construction. The estimated coverage of the genome was 84.8% for the female map and 83.4% for the male map. The maps generated will serve as a basis for the construction of a high-resolution genetic map and mapping of the functional genes and quantitative trait loci, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.     

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 linkage map in F2 population of selected parents with respect to Macrophomina phaseolina resistance trait using screened polymorphic RAPD and developed SCAR markers of jute

Two molecular markers (RAPD and simple sequence repeat (SSR)) were applied on 12 Corchorus capsularis jute samples. Two of them were Macrophomina phaseolina-resistant and the remaining eight were M. phaseolina-susceptible accessions. Eleven SSR primer combinations out of 18 gave the polymorphic results between M. phaseolina-resistant and -susceptible accessions. Five pairs of sequence characterised amplified region (SCAR) primers designated as SCP-4, SCS-3, SCS-13, SCG-10 and SCU-10 were designed based on the polymorphic loci obtained between JRC-412 and CIM-036. Only SCU-10 and SCS-13 produced polymorphic markers corresponding to OPU-10 and OPS-13 amplified from ‘CIM-036’ and JRC-412, respectively. RAPD and SCAR markers were employed for construction of a linkage map using a set of 67 F2 population of a cross between JRC-412 and CIM-036 as a mapping population. Nine markers were assigned to two linkage groups (LGs) covering a total length of 628.4 cM with an average distance of 28 cM between markers.     

利用鸡F2资源群体构建1号染色体遗传连锁图谱

在鸡1号染色体上选取23个微卫星标记,利用东北农业大学鸡F2资源群体构建了遗传连锁图谱。选用369只F2个体用于基因型测定。结果表明23个微卫星位点除MCW0058为低度多态,其他位点均为中高度多态。构建的连锁图谱覆盖1号染色体全长,总共637.9 cM。MCW0115和ROS0025标记顺序与EL图谱不同,但与WAU图谱一致。其他标记顺序与3大参考家系标记顺序一致,图谱总长和标记间距离大于3大参考家系。此连锁图谱的构建为数量性状位点（QTL）定位奠定了良好的基础。     

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.     

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.     

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.