Development and characterization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata.

Twelve new dominant randomly amplified polymorphic DNA (RAPD) fragments associated with a single dominant gene for resistance to citrus tristeza virus (CTV) were identified using bulked segregant analysis of an intergeneric backcross family. These and eight previously reported RAPDs were mapped in the resistance gene (Ctv) region; the resulting localized linkage map spans about 32 cM, with nine close flanking markers within 2.5 cM of Ctv. Seven of 20 RAPD fragments linked with the resistance gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence-absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.     

Linkage mapping of the Mediterranean cypress, Cupressus sempervirens, based on molecular and morphological markers

Gene mapping for a Cupressus species is presented for the first time. Two linkage maps for the Mediterranean cypress (Cupressus sempervirens) varieties, C. sempervirens var. horizontalis and C. sempervirens var. pyramidalis, were constructed following the pseudo-testcross mapping strategy and employing RAPD, SCAR and morphological markers. A total of 427 loci (425 RAPDs, two SCARs) representing parents and F(1) progeny were screened for polymorphism with 32 random decamer and two SCAR primers. A morphological marker defined as "crown form" was also included. Of 274 polymorphic loci, the 188 that presented Mendelian inheritance formed the mapping dataset. Of these loci, 30% were mapped into seven linkage groups for the horizontalis (maternal) and four linkage groups for the pyramidalis (paternal) map. The putative "crown form" locus was included in a linkage group of both maps. The horizontalis and the pyramidalis maps covered 160.1 and 144.5 cM, respectively, while genome length was estimated to be 1696 cM for the former variety and 1373 cM for the latter. The four RAPD markers most tightly linked to crown form were cloned and converted to SCARs. Each of the cloned RAPD markers yielded two to three different sequences behaving as co-migrating fragments. Two SCAR markers, SC-D05(432) and SC-D09(667), produced amplified bands of the expected sizes and maintained linkage with the appropriate phenotype, but to a lesser extent compared to their original RAPD counterparts. These linkage maps represent a first step towards the localization of QTLs and genes controlling crown form and other polygenic traits in cypress.     

Use of a subset of doubled-haploid lines for RAPD interval mapping in barley.

Molecular markers have been used in barley to locate genes and quantitative trait loci. Only a few RAPD markers have been located on barley marker maps. The objectives of this study were (i) to place RAPD markers in specific intervals on the barley linkage map developed from the cross Steptoe (S) x Morex (M), (ii) to examine the distribution of RAPD markers, and (iii) to compare markers amplified by Taq DNA polymerase with those amplified by the Stoffel fragment of Taq DNA polymerase. Screening of DNA from S and M with 362 decamer primers identified 85 that amplified 127 reliable RAPDs. A subset of 15 doubled-haploid (DH) lines from the 150 DH line mapping population was used to place these RAPD markers in intervals on the SM map. This subset can be used for rapid placement of any new markers on the SM linkage map. Most of the RAPD markers were dominant but four codominant RAPDs were identified. The RAPDs were not evenly distributed, with many clustered around the centromeric region of each chromosome. Two of these clusters were located in intervals larger than 15 cM. Testing of 38 to 42 additional DH lines provided more precise placement of eight of the markers in these clusters. Reliable RAPDs were detected with 44% of the primers tested with the Stoffel fragment, but with only 17% of the primers tested with Taq DNA polymerase. These RAPDs provide additional markers for use in barley improvement.     

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo

BACKGROUND AND AIMS: The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. METHODS: Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. KEY RESULTS: As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. CONCLUSION: These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.     

Identification and mapping of chi115 gene and DNA markers linked to it in pea (Pisum sativum L.)

Chlorophyll mutant Chi115 was induced by ethylmethane sulfonate (EMS) treatment of seeds of genotype Torsdag in Moscow State University and is characterized by lighter plant color. The monogenic nature of the mutant was determined by analyzing the F2 population from a cross between two P. sativum genotypes, WL1238 and Chi115. To establish a local map around the chi115 gene, the RAPD and ISSR techniques were used with 45 RAPD and 10 ISSR primers in combination with bulked segregant analysis (BSA). Linkage of 12 RAPDs and 2 ISSRs to the chi115 locus was observed in analysis of F2 single plants. Two RAPD markers that were closely associated with the chi115 gene were converted into the sequence characterized amplified region (SCAR) markers. By lowering the LOD score to 2, the linkage group containing the chi115 gene could be linked to the b gene (color of the flower) on linkage group III. Nevertheless, to prove the result obtained, three CAPS markers Sodmt, TubA1, and Rb were chosen on linkage group III. The results of linkage analysis showed that these CAPS markers were located within the linkage group including the chi115 gene.     

Construction of a linkage map of Lentinula edodes (shiitake) with the HEGS (high-efficiency genome scanning) system: use of versatile AFLP and PCR-based gene markers

An improved linkage map of Lentinula edodes (shiitake) was constructed with an HEGS (high-efficiency genome scanning) system. Two hundred twenty-one HEGS-derived amplified fragment length polymorphism (AFLP-H) markers and 21 gene markers were developed and combined with 203 previously developed sequencer-derived AFLP markers (AFLP-S markers) and 3 mating factor loci (A, Bα, and Bβ) to construct a comprehensive linkage analysis. As a result, a novel linkage map with 166 markers including 2 mating factors (A and B), 10 HEGS-derived gene markers, 72 AFLP-H markers, and 82 AFLP-S markers was obtained. Of the total 448 markers, 273 could not be located on a linear map and thus were assigned to linkage groups as accessory markers. The map covers a total length of 1398.4 centimorgans (cM) with an average marker interval distance of 8.4 cM. The map consists of 11 linkage groups (LGs) in agreement with our previous map, and 7 LGs among them were found to contain branched linkages, which may be the result of reciprocal translocations representing dynamic reorganization of the shiitake genome. The previously reported linkage map was improved in terms of number of markers, marker density, linear order of markers, and total map length. Contribution no. 384 of the Tottori Mycological Institute     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Construction of a genetic linkage map based on amplified fragment length polymorphism markers and development of sequence-tagged site markers for marker-assisted selection of the sporeless trait in the oyster mushroom (Pleurotus eryngii)

A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.     

Megagametophyte-derived linkage maps of white spruce ( Picea glauca) based on RAPD,SCAR and ESTP markers

We have constructed linkage maps for two parents of white spruce [ Picea glauca (Moench) Voss]. Haploid megagametophytes from 92 and 96 seeds of parents M2 and 80132, respectively, were analysed with RAPD, SCAR and ESTP markers. Fragments segregating in a 1:1 Mendelian ratio were classified and mapped using MAPMAKER, GMENDEL and JOINMAP. For M2, the analysis with JOINMAP resulted in 165 loci (152 RAPDs, 3 SCARs and 10 ESTPs) mapping to 23 linkage groups and covering 2,059.4 cM(Kosambi function, K). For 80132, the analysis resulted in 144 loci (137 RAPDs, 1 SCAR and 7 ESTPs) mapping to 19 linkage groups and covering 2,007.7 cM(K). The maps covered 87 and 73% of the entire genome of parents M2 and 80132, respectively. Similar results were obtained with MAPMAKER and GMENDEL. A comparison was made between the two individual maps and 16 loci were shared between the two maps.     

Segregating random amplified polymorphic DNAs (RAPDs) in Betula alleghaniensis

Summary Molecular markers are currently being developed for Betula alleghaniensis Britton using random amplified polymorphic DNA (RAPD). Arbitrarily designed 11-mer primers were tested on three intraspecific controlled crosses for which more than 15 full-sibs were available. Using two of these primers, we were able to genetically characterize a total of nine polymorphic RAPD markers. Segregation of these markers was consistent with a biparental diploid mode of inheritance, and all appeared dominant. RAPDs were valuable in detecting contaminants and, therefore, in assessing the validity of controlled crosses. Limitations of the technique are discussed in relation to the determination of parental genotypes and construction of linkage maps for hardwood species.     

Development of SCAR markers for the DNA-based detection of the Asian long-horned beetle,Anoplophora glabripennis (Motschulsky)

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure.     

Identification and Mapping of chi115 Gene and DNA Markers Linked to It in Pea (Pisum sativum L.)

Chlorophyll mutant Chi115 was induced by ethylmethane sulfonate (EMS) treatment of seeds of genotype Torsdag in Moscow State University and is characterized by lighter plant color. The monogenic nature of the mutant was determined by analyzing the F₂ population from a cross between two P. sativum genotypes, WL1238 and Chi115. To establish a local map around the chi115gene, the RAPD and ISSR techniques were used with 45 RAPD and 10 ISSR primers in combination with bulked segregant analysis (BSA). Linkage of 12 RAPDs and 2 ISSRs to the chi115locus was observed in analysis of F₂ single plants. Two RAPD markers that were closely associated with the chi115 gene were converted into the sequence characterized amplified region (SCAR) markers. By lowering the LOD score to 2, the linkage group containing the chi115 gene could be linked to the b gene (color of the flower) on linkage group III. Nevertheless, to prove the result obtained, three CAPS markers Sodmt, TubA1, and Rb were chosen on linkage group III. The results of linkage analysis showed that these CAPS markers were located within the linkage group including thechi115gene.     

中国白菜RAPD分子遗传图谱的构建

A molecular genetic map of Brassica campestris L. (syn. B. rapa) was constructed based on the segregation of 99 RAPDs (random amplified polymorphic DNAs) markers from eighty-four 10-base random primers using DNA samples extracted from F2 population of turnip (B． campestris L. ssp. rapifera Metzg) × Chinese cabbage (B． campestris L. ssp. pekinensis Lour. Olsson). This genetic map covered 1 632.4 cM (centiMorgan) genome (Kosambi Function) with 16.5 cM mean intervals between flanking markers and defined thirteen linkage groups, in which the longest linkage group is 267.5 cM with 20.6 cM mean interval and the shortest linkage group is 62.2 cM with 15.6 cM mean interval. The size and distribution of linkage groups in this map is similar to other RFLP maps and karyotype data in B. campestris.     

白桦RAPD遗传连锁图谱的构建

以80个来自欧洲白桦(Betula pendula Roth)×中国白桦(Betula platyphylla Suk)的F1个体为作图群体。利用2个亲本和10个F1个体对1,200个10 bp的随机寡核苷酸引物进行筛选, 确定了208个多态性引物。利用RAPD标记, 按照拟测交的作图策略, 分别构建了欧洲白桦和中国白桦的分子标记连锁图谱。对2个亲本和80个F1代作图群体进行随机扩增, 共获得了364个多态性位点。χ2检验结果表明有307个位点符合1∶1分离的拟测交分离, 26个位点符合3∶1分离, 31个位点属偏分离位点。拟测交位点中有145个位点来自欧洲白桦, 有162个位点来自中国白桦。利用2点连锁分析, 欧洲白桦中的145个连锁标记构成了14个不同的连锁群(4个以上标记), 6个三连体和6个连锁对, 37个为非连锁位点, 连锁标记覆盖的总图距为955.6 cM (centimorgan), 平均图距14.9 cM。而来自中国白桦的162个标记构成了15个连锁群(4个以上标记), 4个三连体和6个连锁对, 21个为非连锁位点, 连锁标记覆盖的总图距为1,545.8 cM (centimorgan), 平均图距15.2 cM。该图谱的建立为进一步将两个图谱整合为一个高密度图谱及重要基因的定位奠定了基础。     

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) for marker-assisted selection in plant breeding

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.     

杉木种子随体染色体SCAR标记的建立

回收、纯化由引物OPB07(5’-GGTGACGCAG-3’) OPB18(5’-CCACAGCAGT-3’)扩增而得的杉木(Cunninghamia lanceolata(Lamb.)Hook)种子随体染色体特异性RAPD(随机扩增的DNA多态性分析)片段OPB07-18907,将该片段克隆至pUCm-T载体,转化受体菌后筛选出阳性克隆,并对插入片段进行测序,根据序列特点设计两对SCAR(序列特异性扩增区)引物,PCR结果显示,这两对引物的4种组合都可以扩增出属于随体染色体的特征带,适宜退火温度为57℃。成功将特异RAPD标记OPB07-18907转化为稳定的SCAR标记。开发随体染色体SCAR标记目的是:一方面能在分子水平上鉴定微分离的杉木随体染色体,另一方面,也可以将杉木已构建的遗传图谱中连锁群与染色体进行对应。探讨了该SCAR标记对杉木核型分析的作用。     

Tagging and mapping of a rice gall midge resistance gene, <Emphasis Type="Italic">Gm8</Emphasis>, and development of SCARs for use in marker-aided selection and gene pyramiding

Using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNAs (RAPDs), we have tagged and mapped Gm8, a gene conferring resistance to the rice gall midge (Orseolia oryzae), a major insect pest of rice, onto rice chromosome 8. Using AFLPs, two fragments, AR257 and AS168, were identified that were linked to the resistant and susceptible phenotypes, respectively. Another resistant phenotype-specific marker, AP19₅₈₇, was also identified using RAPDs. SCAR primers based on the sequence of the fragments AR257 and AS168 failed to reveal polymorphism between the resistant and the susceptible parents. However, PCR using primers based on the regions flanking AR257 revealed polymorphism that was phenotype-specific. In contrast, PCR carried out using primers flanking the susceptible phenotype-associated fragment AS168 produced a monomorphic fragment. Restriction digestion of these monomorphic fragments revealed polymorphism between the susceptible and resistant parents. Nucleotide BLAST searches revealed that the three fragments show strong homology to rice PAC and BAC clones that formed a contig representing the short arm of chromosome 8. PCR amplification using the above-mentioned primers on a larger population, derived from a cross between two indica rice varieties, Jhitpiti (resistant parent) and TN1 (susceptible parent), showed that there is a tight linkage between the markers and the Gm8 locus. These markers, therefore, have potential for use in marker-aided selection and pyramiding of Gm8 along with other previously tagged gall midge resistance genes [Gm2, Gm4(t), and Gm7].The nucleotide sequence data reported here will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers AY545920–AY545923     

SCAR markers for discriminating species of two genera of medicinal plants, Liriope and Ophiopogon

The development of DNA markers that can closely discriminate between Liriope and Ophiopogon species is vital for efficient and accurate identification of these species, and to ensure the quality, safety, and efficacy of medicines made from these plants. We developed species-specific molecular markers for these two genera. Forty RAPD primers were tested to detect polymorphism; species-specific RAPD bands were gel-purified, cloned, and sequenced. Primers for sequence-characterized amplified regions (SCARs) were then designed, based on nucleotide sequences of specific RAPD primers. SCAR markers SA06 and SB05, specific to Ophiopogon japonicus, amplified 460- and 553-bp DNA fragments, respectively. The marker SA12 amplified a 485-bp fragment specific to Liriope platyphylla. This is the first report of a species-specific SCAR marker for this group. These markers will be useful for rapid identification of closely related Liriope and Ophiopogon species.     

Development of PCR-based SCAR and CAPS markers linked to beta-glucan and protein content QTL regions in oat.

A key breeding objective in oat (Avena sativa L.) is cultivars with high and low beta-glucan content. In a targeted strategy to develop PCR-based markers linked to published beta-glucan content quantitative trait loci (QTLs) regions, 15 random amplified polymorphic DNA (RAPD) fragments were cloned and their sequences used to design sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) primers. The 13 derived SCAR markers and 2 derived CAPS markers were mapped on either the 'Kanota' x 'Ogle' (KO) or the 'Terra' x 'Marion' (TM) oat reference map. In addition, 3 previously reported SCAR markers were characterized further. Ten SCAR markers and one CAPS marker were associated with beta-glucan QTL regions and many of these are also associated with QTLs for protein content or other traits. These markers have the potential to help define homologous and homologous relationships in oat and investigate the complex genetics of beta-glucan and protein content.     

The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L

Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.