花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展, 初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记, 根据花生EST序列开发EST-SSR标记, 根据豆科植物序 列信息和SSR标记开发花生SSR标记, 将SSR标记与其它分子标记结合开发新的DNA标记, 以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

花生微卫星标记的研究进展

近年来花生微卫星标记的开发取得了一定的进展,初步揭示了花生在DNA水平上的遗传多样性。花生微卫星标记的开发途径主要包括通过构建小片段基因组文库开发基因组SSR标记,根据花生EST序列开发EST-SSR标记,根据豆科植物序列信息和SSR标记开发花生SSR标记,将SSR标记与其它分子标记结合开发新的DNA标记,以及基于SSR核心序列开发ISSR标记。花生微卫星标记主要应用于遗传多样性研究、遗传图谱与品种指纹图谱构建以及分子标记辅助育种等领域。本文综述了花生SSR标记开发研究的进展及应用。     

Integration Efficiency in DNA-Induced Transformation of PNEUMOCOCCUS. II. Genetic Studies of Mutant Integrating All the Markers with a High Efficiency

Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl(3). The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl(3). However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl(3) by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.-The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.-Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.     

Identifying subjects who benefit from additional information for better prediction of the outcome variables

Summary .  Suppose that we are interested in using new bio- or clinical markers, in addition to the conventional markers, to improve prediction or diagnosis of the patient's clinical outcome. The incremental value from the new markers is typically assessed by averaging across patients in the entire study population. However, when measuring the new markers is costly or invasive, an overall improvement does not justify measuring the new markers in all patients. A more practical strategy is to utilize the patient's conventional markers to decide whether the new markers are needed for improving prediction of his/her health outcomes. In this article, we propose inference procedures for the incremental values of new markers across various subgroups of patients classified by the conventional markers. The resulting point and interval estimates can be quite useful for medical decision makers seeking to balance the predictive or diagnostic value of new markers against their associated cost and risk. Our proposals are theoretically justified and illustrated empirically with two real examples.     

Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat.

Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.     

Assessment of AFLP marker behaviour in enriching STS radiation hybrid maps

Radiation hybrid (RH) mapping provides a powerful tool to build high-resolution maps of genomes. Here, we demonstrate the use of the AFLP^® technique for high-throughput typing of RH cell lines. Cattle were used as the model species because an RH panel was available to investigate the behaviour of AFLP markers within the microsatellite- and STS-based maps of this species. A total of 747 AFLP markers were typed on the TM112 RH radiation panel and 651 of these were assigned by two-point analysis to the 29 bovine autosomes and sex chromosomes. AFLP markers were added to the 1222 microsatellite and STS markers that were included in earlier RH maps. Multipoint maps were constructed for seven example chromosomes, which retained 248 microsatellite and STS markers, and added 123 AFLP markers at LOD 4. The addition of the AFLP markers increased the number of markers by 42.1% and the map length by 10.4%. The AFLP markers showed lower retention frequency (RF) values than the STS markers. The comparison of RF values in AFLP markers and their corresponding AFLP-derived STSs demonstrated that the lower RF values were due to the lower detection sensitivity of the AFLP technique. Despite these differences, AFLP and AFLP-derived STS markers mapped to identical or similar positions. These results demonstrate that it is possible to merge AFLP and microsatellite markers in the same map. The application of AFLP technology could permit the rapid construction of RH maps in species for which extensive genome information and large numbers of SNP and microsatellite markers are not available.     

Deletion-based physical mapping of barley chromosome 7H

Chromosomal mutations in barley (Hordeum vulgare, 2n=2x=14, HH) chromosome 7H added to the common wheat (Triticum aestivum, 2n=6x=42, AABBDD) cultivar Chinese Spring were induced genetically by the gametocidal activity of certain alien chromosomes derived from wild species of the genus Aegilops. The rearranged barley chromosomes were characterized by C-banding, FISH and GISH. Twenty two deletion or translocation chromosomes in a hemizygous condition were selected for deletion mapping of 17 AFLP and 28 STS markers that are specific to 7H. Of the 22 breakpoints in chromosome 7H, seven involved the short arm (7HS), 12 the long arm (7HL) and three were in the centromeric region. The seven 7HS breakpoints separated all four 7HS-specific AFLP markers and split the 21 STS markers into six groups. One breakpoint occurred between two STS markers formerly occupying the same position in the genetic map. All seven 7HS breakpoints were separated from each other by either the AFLP or STS markers. The 12 breakpoints in 7HL divided the 13 7HL-specific AFLP markers into seven groups, and the seven STS markers into three groups. On the other hand, the 12 breakpoints in 7HL were divided into six groups by the AFLP markers and into two groups by the STS markers. This deletion-based map was in accordance with previously published genetic and physical maps using the same STS markers. The breakpoints, AFLP markers and STS markers were arrayed in a consistent order. Received: 5 February 2001 / Accepted: 19 February 2001     

Development and application of RAPD-SCAR markers to identify intra-species hybrids of industrial Saccharomyces cerevisiae

To overcome the drawbacks of protoplast fusion in industrial breeding, strain-specific molecular markers were applied to select hybrids of industrial Saccharomyces cerevisiae strains. Random Amplified Polymorphic DNA (RAPD) analysis was used to generate strain-specific RAPD markers for two industrial yeast strains, Z8 and Z9. For industrial and technical controls, two RAPD markers with non-coding regions were converted into stable Sequence Characterized Amplified Region (SCAR) markers. Hybrids of Z8 and Z9 were obtained by protoplast fusion in combination with SCAR markers and were found to increase ethanol production by 4.3–8.1%. Results suggested that protoplast fusion could be combined with RAPD-SCAR molecular markers and applied in industrial breeding instead of auxotrophic markers.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Barley chromosome addition lines of wheat for screening of AFLP markers on barley chromosomes.

We conducted AFLP (Amplified Fragment Length Polymorphism) analysis with the six wheat-barley chromosome addition lines of common wheat cultivar Chinese Spring. We analyzed the AFLP fingerprints generated by 36 combinations of selective-amplification primers to find 103 markers specific to the barley chromosomes (2.9 markers per combination on average). The numbers of AFLP markers mapped to the barley chromosomes varied (one to 16) depending of the primer combinations. Each barley chromosome had 10 to 27 AFLP markers (17.2 markers on average). We identified the chromosome arms in which these markers are located using the barley telocentric addition lines (one to 20 markers per chromosome arm). The AFLP markers were not distributed evenly among chromosomes and chromosome arms. We could not determine the chromosome-arm locations for some of the barley-specific markers, either because such markers were found in both the short- and long-arm telocentric lines, or in neither line.     

Assessment of genetic diversity in Indian rice germplasm (Oryza sativa L.): use of random versus trait-linked microsatellite markers

Assessment of genetic diversity in a crop germplasm is a vital part of plant breeding. DNA markers such as microsatellite or simple sequence repeat markers have been widely used to estimate the genetic diversity in rice. The present study was carried out to decipher the pattern of genetic diversity in terms of both phenotypic and genotypic variability, and to assess the efficiency of random vis-à-vis QTL linked/gene based simple sequence repeat markers in diversity estimation. A set of 88 rice accessions that included landraces, farmer’s varieties and popular Basmati lines were evaluated for agronomic traits and molecular diversity. The random set of SSR markers included 50 diversity panel markers developed under IRRI’s Generation Challenge Programme (GCP) and the trait-linked/gene based markers comprised of 50 SSR markers reportedly linked to yield and related components. For agronomic traits, significant variability was observed, ranging between the maximum for grains/panicle and the minimum for panicle length. The molecular diversity based grouping indicated that varieties from a common centre were genetically similar, with few exceptions. The trait-linked markers gave an average genetic dissimilarity of 0.45 as against that of 0.37 by random markers, along with an average polymorphic information constant value of 0.48 and 0.41 respectively. The correlation between the kinship matrix generated by trait-linked markers and the phenotype based distance matrix (0.29) was higher than that of random markers (0.19). This establishes the robustness of trait-linked markers over random markers in estimating genetic diversity of rice germplasm.     

编码区和非编码区SSR标记对水稻类群的比较研究

设计14对水稻编码区SSR引物和选取已公布的非编码区SSR引物12对、编码区SSR引物3对,采用SSR技术,对29个标记在60个水稻材料中的多态性进行分析。结果表明,编码区SSR标记平均检测到3.59个多态性位点,多态信息量PIC(polymorphism information conten)在0.032～P0.853之间,平均值为0.447;非编码区SSR标记平均检测到3.92个多态性位点,PIC在0.063～P0.795之间,平均值为0.521。聚类分析显示,非编码区SSR标记能更加精确地区分来自不同地区的水稻类群,编码区SSR标记也具有良好的多态性,同样可以用于分析水稻的亲缘关系。     

Using AFLP markers and the Geneland program for the inference of population genetic structure

The use of dominant markers such as amplified fragment length polymorphism (AFLP) for population genetics analyses is often impeded by the lack of appropriate computer programs and rarely motivated by objective considerations. The point of the present note is twofold: (i) we describe how the computer program Geneland designed to infer population structure has been adapted to deal with dominant markers; and (ii) we use Geneland for numerical comparison of dominant and codominant markers to perform clustering. AFLP markers lead to less accurate results than bi-allelic codominant markers such as single nucleotide polymorphisms (SNP) markers but this difference becomes negligible for data sets of common size (number of individuals n≥100, number of markers L≥200). The latest Geneland version (3.2.1) handling dominant markers is freely available as an R package with a fully clickable graphical interface. Installation instructions and documentation can be found on http://www2.imm.dtu.dk/~gigu/Geneland.     

Cloning of AFLP markers detected by fluorescence capillary electrophoresis

The available methods to isolate specific amplified fragment length polymorphism (AFLP) markers can be used only if markers are detected by radioactive labeling, silver staining, or ethidium bromide staining; these methods are useless if modern and automated genetic analyzers are used to detect AFLP markers by fluorescent labeling. We have developed a method that allows for isolation and cloning of specific AFLP markers obtained with a laser-induced fluorescence capillary electrophoresis system. This procedure has been tested on 5Arabidopsis thaliana polymorphic AFLP markers, and the nucleotide sequences obtained from these cloned markers were identified and located in theArabidopsis genome.     

An integrated genetic linkage map with 1,137 markers constructed from five F2 crosses of autoimmune disease-prone and -resistant inbred rat strains

The rat (Rattus norvegicus) is an important experimental model for many human diseases including arthritis, diabetes, and other autoimmune and chronic inflammatory diseases. The rat genetic linkage map, however, is less well developed than those of mouse and human. Integrated rat genetic linkage maps have been previously reported by Pravenec et al. (1996, Mamm. Genome 7: 117-127) (500 markers mapped in one cross), Bihoreau et al. (1997, Genome Res. 7: 434-440) (767 markers mapped in three crosses), Wei et al. (1998, Mamm. Genome 9: 1002-1007) (562 markers mapped in two crosses), Brown et al. (1998, Mamm. Genome 9: 521-530) (678 markers mapped in four crosses), and Nordquist et al. (1999, Rat Genome 5: 15-20) (330 markers mapped in two crosses). The densest linkage map combined with a radiation hybrid map, reported by Steen et al. (1999, Genome Res. 9: AP1-AP8), includes 4736 markers mapped in two crosses. Here, we present an integrated linkage map with 1137 markers. We have constructed this map by genotyping F2 progeny of five crosses: F344/NHsd x LEW/NHsd (673 markers), DA/Bkl x F344/NHsd (531 markers), BN/SsN x LEW/N (714 markers), DA/Bkl x BN/SsNHsd (194 markers), and DA/Bkl x ACI/SegHsd (245 markers). These inbred rat strains vary in susceptibility/resistance to multiple autoimmune diseases and are used extensively for many types of investigation. The integrated map includes 360 loci mapped in three or more crosses. The map contains 196 new SSLP markers developed by our group, as well as many SSLP markers developed by other groups. Two hundred forty genes are incorporated in the map. This integrated map should allow comparison of rat genetic maps from different groups and thereby facilitate genetic studies of rat autoimmune and related disease models.     

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。     

Development of 50 gene-associated microsatellite markers using BAC clones and the construction of a linkage map of swine chromosome 4

The development of informative polymorphic markers is essential for QTL mapping. We developed 50 microsatellite markers from BAC clones containing genes that were predicted to map swine chromosome 4 (SSC4) according to comparative analysis between human and swine chromosomes, and constructed a linkage map that consisted of 37 markers including 24 markers closely linked to genes in BAC clones. Microsatellite markers were developed by direct-sequencing of BAC clones and our results demonstrated that this method was effective for developing microsatellite markers in specific regions on chromosomes. Effective development of microsatellite markers closely linked to genes can further accelerate the comparative studies of chromosomes between different species.     

Mapping of rat chromosome 5 markers generated from chromosome-sorted DNA

Nineteen markers for rat Chromosome 5 (Chr) were generated by screening chromosome-sorted DNA libraries and were subsequently mapped by linkage to known markers by use of five F₂ rat populations. Along with existing markers, these newly produced markers are potentially useful for fine mapping of certain quantitative trait loci for blood pressure and for obesity. Received: 20 January 1997 / Accepted: 17 March 1997     

Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

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.