Molecular characterization of the SCAR markers tightly linked to the Tm-2 locus of the genus Lycopersicon

The Tm-2 gene and its alleles conferring tomato mosaic virus resistance in tomato originate from Lycopersicon peruvianum, a wild relative of tomato. DNA fragments of several RAPD markers tightly linked with the Tm-2 locus in tomato were successfully cloned and sequenced. Subsequently, the 24-mer oligonucleotide primer pairs of the SCAR markers corresponding to the RAPD markers were designed based on the 5’-endmost sequences. A fragment of the same size as that of a SCAR marker was amplified in the ToMV-susceptible tomato line with no Tm-2, but the digests of the PCR fragments by AccI exhibited polymorphism in fragment length between the two lines. We chose three SCAR markers and three RAPD markers tightly linked with the Tm-2 locus, and examined whether the same-sized fragments corresponding to these markers were also present in three other lines carrying Tm-2a or one of the other Tm-2 alleles. The fragments corresponding to the three SCAR markers were present in all of the three lines, but the other markers (three RAPDs ) were absent in one or two lines, suggesting that the three SCAR markers are closer to Tm-2 than the other markers. Comparison of the nucleotide sequences of these fragments revealed that they are all homologous to the corresponding SCAR markers. Received: 8 November 1999 / Accepted: 15 November 1999     

Identification of RAPD markers linked to the Tm-2 locus in tomato

Tm-2 and Tm-2a are genes conferring resistance to tomato mosaic virus in Lycopersicon esculentum. They are allelic and originated from different lines of L. peruvianum, a wild relative of tomato. In this study, random amplified polymorphic DNA (RAPD) markers linked to these genes were screened in nearly isogenic lines (NILs). To detect RAPDs differentiating NILs, 220 different 10-base oligonucleotide primers were examined by the polymerase chain reaction (PCR), and 43 of them generated 53 consistent polymorphic fragments among the NILs. Out of these 53 fragments, 13 were arbitrarily chosen and examined in respect of whether they were linked to the netted virescent (nv) gene, since nv is tightly linked to the Tm-2 locus and its phenotype is more easily distinguishable. As a result, all 13 markers were shown to be linked to nv, and hence to the Tm-2 locus. Among them, two fragments specific to the NIL carrying Tm-2 three specific to the NIL carrying Tm-2a, and four specific to both of these NILs were closely linked to nv.     

Identification and mapping on chromosome 9 of RAPD markers linked to Sw-5 in tomato by bulked segregant analysis

Bulked segregant analysis was used to identify random amplified polymorphic DNA (RAPD) markers linked to the Sw-5 gene for resistance to tomato spotted wilt virus (TSWV) in tomato. Using two pools of phenotyped individuals from one segregating population, we identified four RAPD markers linked to the gene of interest. Two of these appeared tightly linked to Sw-5, whereas another, linked in repulsion phase, enabled the identification of heterozygous and susceptible plants. After linkage analysis of an F₂ population, the RAPD markers were shown to be linked to Sw-5 within a distance of 10.5 cM. One of the RAPD markers close to Sw-5 was used to develop a SCAR (sequence characterized amplified region) marker. Another RAPD marker was stabilized into a pseudo-SCAR marker by enhancing the specificity of its primer sequence without cloning and sequencing. RAPD markers were mapped to chromosome 9 on the RFLP tomato map developed by Tanksley et al. (1992). The analysis of 13 F₃ families and eight BC₂ populations segregating for resistance to TSWV confirmed the linkage of the RAPD markers found. These markers are presently being used in marker-assisted plant breeding.     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

Construction of a high-resolution genetic map and YAC-contigs in the tomato Tm-2a region

With the ultimate goal of cloning the Tobacco Mosaic Virus (TMV) resistance gene Tm-2a from tomato by means of positional cloning, a high-resolution map of a 4.3-cM region surrounding the Tm-2a gene has been constructed. In total, 13 RFLP and RAPD markers were mapped in close proximity to Tm-2a using 2112 individuals from an intraspecific Lycopersicon peruvianum backcross. The closest flanking markers were separated from Tm-2a by 0.05 cM on each side. Only one marker, the cDNA clone R12, co-segregated with Tm-2a. In order to physically cover the Tm-2a region, R12 and the flanking DNA marker TG207 were used to select homologous YAC clones. To-date, two YAC-contigs spanning approximately 340 kb and 360 kb have been constructed. The data obtained from these experiments indicate that recombination around the centromere of chromosome 9 is extremely suppressed.     

Identification of 27 <Emphasis Type="Italic">Porphyra</Emphasis> lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

Identification ofporphyra lines (rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-sevenPorphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27Porphyra lines has its unique AFLP fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germ-plasm identification-AFLP) was designed for identification of the 27Porphyra lines. In addition, 21 specific AFLP markers from 15Porphyra lines were identified; 6 AFLP markers from 4Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of thePorphyra lines.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

DNA markers for sex: Molecular evidence for gender dimorphism in dioecious Mercurialis annua L.

Male specific Random Amplified Polymorphic DNA (RAPD) markers, OPB01-1562 and OPC07-303, were identified and sequenced in dioecious Mercurialis annua. Sequence Characterized Amplified Region (SCAR) primers were designed. Several internal segments of OPB01-1562 were amplified as male specific SCAR markers. These markers were PCR amplified from strong, intermediate and weak male subtypes selected according to their resistance to feminization by cytokinin. Nucleotide sequence of OPB01-1562 isolated from three male subtypes were near identical. The OPB01-1562 and derived SCAR markers were absent in females as well as hexaploid Mercurialis male and monoecious individuals. The gender relationship of the markers was maintained in all ecotypes tested. There were 2 internal fragments of OPB01-1562, which were PCR amplified from all genotypes of diploid and hexaploid Mercurialis. It is argued that identification of gender specific DNA suggests a dimorphic differentiation of the genome of dioecious Mercurialis annua.     

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.     

Molecular Identification of Tropical Tasar Silkworm (Antheraea mylitta) Ecoraces with RAPD and SCAR Markers

The tropical tasar silkworm, Antheraea mylitta, has several ecoraces, 10 of which are commercially exploited for the production of tasar silk. These ecoraces are identified by morphological markers that are greatly influenced by photoperiod, humidity, altitude, and host plants. The DNA markers, random amplification of polymorphic DNA (RAPD), and sequence-characterized amplified region (SCAR) are identified to complement the existing morphological markers. Seven RAPD bands are selected that identify 8 of the 10 ecoraces. These identified RAPD fragments are sequenced and primers are designed for SCAR markers. Of the seven sets of primers, a single primer pair produced polymorphic SCAR bands that diagnose 5 of the 10 ecoraces. All 10 ecoraces are identified by the use of RAPD and SCAR markers together.     

烟草SCAR标记技术的建立

目的:通过烟草随机扩增多态性DNA(RAPD)标记技术建立烟草特征序列扩增区域(SCAR)标记技术,用于烟草品种鉴定。方法:对12个烟草品种的复烤叶片DNA进行RAPD分析,得到2个RAPD特异片段S1和S2,通过切胶回收,连接pUCm-T载体克隆转化,片段测序,设计特异性引物S1-1/S1-2和S2-1/S2-2,对SCAR-PCR扩增退火温度进行优化。结果:2个RAPD标记成功地转化为稳定快捷的SCAR标记,可将红花大金元和NC102等2个品种从12个烟草品种中快捷准确地鉴别出来。结论:SCAR标记可作为准确稳定的DNA水平的烟草品种鉴定方法,可对种植、复烤和配方品种的烟叶或叶片进行鉴别。     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale     

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in China

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.