Codominant Sequence Characterized Amplified Region Marker Research Articles

Development of a diagnostic DNA marker for the geographic origin of Shorea leprosula

Abstract The development of sequence characterized amplified region (SCAR) markers derived from amplified fragment length polymorphisms (AFLPs) is described for Shorea leprosula. An AFLP fragment that showed nearly complete differentiation between Borneo and Sumatra was gel-extracted, sequenced, and converted into a SCAR marker using the inverse polymerase chain reaction (PCR) technique. The single nucleotide polymorphism (SNP) that originally caused the AFLP was found in the MseI restriction site. Differentiation between islands was detected either as size variation of the codominant SCAR marker or after digestion of the PCR products with the restriction enzyme MseI (PCR-RFLP). Size variation was due to insertions/deletions found within the sequenced region that flanked the original AFLP fragment. After genotyping 151 samples of S. leprosula from 14 populations in Sumatra and Borneo, all but one sample from Sumatra were homozygous for one size variant (427 bp), while S. leprosula populations from Borneo showed different genotypes than Sumatra populations and variation not only among populations but also within populations. Complete differentiation and fixation on alternative variants was found for the geographic regions of Sumatra and Borneo by the PCR-RFLP method. The SCAR marker did not amplify in Shorea parvifolia and thus can also be used to distinguish between S. leprosula and S. parvifolia. The marker was successfully amplified from wood DNA extracts suggesting its applicability to track the geographic origin of timber.

Mapping of Pi, a gene conferring pink leaf in ornamental kale (Brassica oleracea L. var. acephala DC)

We previously found that pink leaf is conferred by a single semi-dominant gene in ornamental kale. To map this gene, we constructed an F2 segregating population containing 184 individuals by crossing W02–7, a white-leaved inbred line, with P02–9, a pink-leaved inbred line. We screened 297 simple sequence repeat (SSR) and 437 sequence-related amplified polymorphism (SRAP) markers using bulked segregant analysis. We identified one SSR and seven SRAP markers that linked tightly to the pink-leaf (Pi) gene. To obtain more specific genomic markers, we converted six SRAPs to sequence-characterized amplified region (SCAR) markers. We constructed a genetic linkage map of the Pi locus using one SSR and five SCAR markers, spanning a total interval of 15.0 cM. One SSR marker, Ni2C12, and one co-dominant SCAR marker, Boac04, flanked Pi on either side at distances of 0.6 and 2.4 cM, respectively. Based on the reference genome sequence of Brassica oleracea, we positioned Pi on the top of chromosome C3. We also detected that pink hypocotyl co-segregated with pink inner leaf. These markers provide the basis for fine-scale mapping and cloning of Pi, and may be used for marker-assisted selection in ornamental kale breeding programs.

Development of a SCAR marker linked to bacterial wilt (Ralstonia solanacearum) resistance in tomato line Hawaii 7996 using bulked-segregant analysis

We report the development of a codominant sequence characterized amplified region (SCAR) marker linked to bacterial wilt resistance in tomato line Hawaii 7996. Bulked segregant analysis was employed for rapid identification of RAPD markers linked to resistance genes. Genomic DNA from six resistant F9 recombinant inbred lines (RILs) and six susceptible F9 RILs, which derived from a cross between S. lycopersicum Hawaii 7996 (resistant parent) and S. pimpinellifolium WVa 700 (susceptible parent) were pooled in to an R-pool and an S-pool, respectively. A total of 800 RAPD primers were screened and only six primers (UBC#176, 205, 287, 317, 350, and 676) showed polymorphism between R- and S- pools. Of these, only two markers UBC#176 and 317 revealed a 100% linkage in the individual plants comprising the contrasting bulks. Of these, the marker UBC#176 was converted into a codominant SCAR marker and designated as SCU176-534. The marker SCU176-534 was confirmed by genotyping the individual of the R- and S- pools and gave the same result as UBC#176. When the marker SCU176-534 was further validated for association with resistance and its potential for maker-assisted selection (MAS) in 92 tomato lines and cultivars, the results showed that none of these carries the resistance gene. Thus, SCAR marker SCU176-534 can be used in early selection of resistant lines when Hawaii 7996 is used as a parent in a breeding program.

RAPD and SCAR Markers Linked to Tea-Mosquito Resistance in Tea

Tea mosquito (Helopeltis theivora) is a serious pest in tea (Camellia sinensis) growing regions, causing severe crop loss. Identification of markers associated with tea-mosquito resistance is necessary to develop cultivars tolerant to tea mosquito from the available germplasm. This study was conducted to identify random amplified polymorphic DNA (RAPD) markers associated with tolerance against tea mosquito. The RAPD markers were then sequenced and converted into sequence-characterized amplified region (SCAR) markers. A total of 138 decamer primers were used to screen genomic DNA from four susceptible and four relatively tolerant tea cultivars. Only three RAPD markers (OPW-03725, OPT-01625, and OPG-11750) were linked to tea-mosquito resistance. These markers were converted into co-dominant SCAR markers, Rht1, Rht2, and Rht3. This is the first study reporting the development of markers for tea-mosquito resistance. These markers can be used as an important tool to screen tea cultivars for tea-mosquito resistance/susceptibility.

Identification and mapping of a SCAR marker linked to a locus involved in shell pigmentation of the Pacific oyster (Crassostrea gigas)

The Pacific oyster (Crassostrea gigas) is one of the most important farmed oysters worldwide. To improve breeding progress, research was done to identify molecular markers linked to shell pigmentation, which has been viewed as a key factor for product value. An F1-segregating population exhibiting a bimodal distribution of shell pigmentation intensity was obtained by crossing two wild oysters with opposite shell pigmentation. Genomic DNA from nine individuals with lightest shell pigmentation and nine individuals with darkest shell pigmentation were equally pooled for amplified fragment length polymorphism (AFLP) screening. In bulked segregant analysis, six out of 225 selective primer pair combinations produced seven polymorphic fragments tightly associated with shell pigmentation across the segregating population. The seven AFLP markers were mapped onto a single linkage group, and 80% of phenotypic variance could be explained by this locus. In conversion of the seven fragments into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a co-dominant SCAR marker, named SP-170. The SCAR marker was integrated into a previously constructed linkage map. The SCAR marker obtained in this study will be useful for marker-assisted selection of the Pacific oyster.

Development of a co-dominant SCAR marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato (Solanum lycopersicum)

Random amplified polymorphism DNA (RAPD) and bulk segregant analysis (BSA) approaches were used to characterize the molecular marker linked to the Phytophthora infestans resistance gene Ph-3 in tomato. A total of 800 RAPD primers were screened. One RAPD marker UBC#602 was identified to be tightly linked to the Ph-3 gene. The marker was successfully converted into a co-dominant sequence characterized amplified region (SCAR) marker. The SCAR marker SCU602 was used to ana- lyze 96 F2 progenies and fitted the expected 1:2:1 Mendelian segregation ratio. Forty one tomato inbred lines were screened using the SCAR marker in com- parison with a reference marker linked to the Ph-3 gene and both markers gave the same results. SCU602 was further validated for association to resis- tance and its potential in MAS in 72 tomato lines and cultivars. The marker identified three genotypes har- bouring the resistance allele. This SCAR marker can be used in breeding programs for the selection of the Ph-3 gene for Phytophthora infestans resistance.

Identification and development of molecular markers linked to Phytophthora root rot resistance in pepper (Capsicum annuum L.)

Phytopthora root rot in pepper (C. annuum) is caused by Phytophthora capsici L., which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a number of quantitative trait loci. Pyramiding resistance alleles is desirable and could be simplified by the use of molecular markers tightly linked to the resistance genes. The purpose of this study was development of molecular markers linked to Phytophthora root rot resistance. An F8 recombinant inbred line (RIL) population derived from a cross between YCM334 and a susceptible cultivar ‘Tean’ was used in combination with bulk segregant analysis utilizing RAPD and conversion of AFLP markers linked to Phytophtora root rot resistance into sequence-characterized amplified region (SCAR) markers. In conversion: one marker was successfully converted into a co-dominant SCAR marker SA133_4 linked to the trait. In bulked segregant analysis (BSA): three RAPD primers (UBC484, 504, and 553) produced polymorphisms between DNA pools among 400 primers screened. Genetic linkage analysis showed that the SCAR and RAPD markers were located on chromosome 5 of pepper. Quantitative trait locus (QTL) analysis showed that the SA133_4 and UBC553 were linked to Phytophtora root rot resistance. These markers were correctly identified as resistant or susceptible in nine promising commercial pepper varieties. These markers will be beneficial for marker-assisted selection in pepper breeding.

English

  Fusarium crown and root rot of tomato (Solanum lycopersicum) is the disease caused by the fungal pathogen Fusarium oxysporum f.sp. radicis-lycopersici(FORL). The most effective way to control this disease is to plant resistant varieties. Markers tightly linked to Fusarium crown and root rot could be used in breeding programs to introgress crown rot resistance into new varieties. In this study, we converted the random amplified polymorphic DNA (RAPD) marker UBC#116, linked to the Fusarium crown and root rot resistance gene (Frl), into a co-dominant sequence characterized amplified region (SCAR) marker. A fragment of about 480 bp, linked to the Frl gene, was polymerase chain reaction (PCR) amplified with the use of the UBC#116 primers, cloned and sequenced. A pair of primers were designed and PCR amplification was performed to develop a new SCAR marker for the Frl gene. The new marker was applied for the analysis of 96 tomato genotypes. The RAPD marker UBC#116 was also used and it revealed that the markers were equivalent to each other. However, the development of the new co-dominant SCAR marker has made marker-assisted selection (MAS) more practical, rapid and efficient.   Key words: Fusarium oxysporum f. sp. radicis-lycopersicum (FORL), marker-assisted selection (MAS), Solanum lycopersicum, breeding.

Uso prático de marcadores moleculares para seleção assistida no melhoramento de uvas de mesa apirênicas

A hipótese mais bem aceita atualmente para explicar a genética complexa da estenoespermocarpia, observada na videira, indica que a expressão deste fenótipo é controlada por três genes recessivos, independentemente herdados e controlados por um gene regulador dominante (sdI). Em estudo anterior, Lahogue et al. (1998) identificaram um marcador RAPD (Random Amplified Polymorphic DNA) ligado ao gene sdI e utilizaram-no para desenvolver um SCAR (Sequence Characterized Amplified Region) co-dominante denominado SCC8, que pode distinguir, em uma progênie, indivíduos com semente como também selecionar indivíduos apirênicos. Neste trabalho, são apresentados resultados da avaliação do potencial de aplicação do marcador molecular SCAR SCC8 para seleção assistida do caráter da apirenia no melhoramento de uvas de mesa sem sementes. A utilização deste marcador na seleção assistida para apirenia em uvas de mesa mostrou-se viável, e as conseqüências da sua utilização no programa de melhoramento da Embrapa Uva e Vinho são discutidas.

SCAR markers linked to the common bean rust resistance gene Ur-13

Rust in common bean (Phaseolus vulgaris L.) is caused by Uromyces appendiculatus Pers.:Pers. (Unger) which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a considerable number of genes. Pyramiding resistance genes is desirable and could be simplified by the use of molecular markers closely linked to the genes. The resistance gene Ur-13, present in the South African large seeded cultivar Kranskop, has been used extensively in the local breeding program. The purpose of this study was the development of a molecular marker linked to Ur-13. An F(2) population derived from a cross between Kranskop and a susceptible (South African) cultivar Bonus was used in combination with bulked segregant analysis utilizing the amplified fragment length polymorphism (AFLP) technique. Seven AFLP fragments linked significantly to the rust resistance and five were successfully converted to sequence characterized amplified region (SCAR) markers. The co-dominant SCAR markers derived from a 405 bp EAACMACC fragment, KB 126, was located 1.6 cM from the gene. Two additional SCAR markers and one cleaved amplified polymorphic sequence marker were located further from the gene. The gene was mapped to linkage group B8 on the BAT 93/Jalo EEP 558 core map (chromosome 3).

(260) Development of SCAR and CAPS Markers Linked to Tomato Begomovirus Resistance Genes Introgressed from Lycopersicon chilense

Resistance to begomoviruses tomato mottle virus (ToMoV) and tomato yellow leaf curl virus (TYLCV) has been introgressed to tomato (Lycopersicon esculentum) from L. chilense accessions LA 1932, LA 2779, and LA 1938. Resistance genes have been mapped to three regions on chromosome 6 using randomly amplified polymorphic DNA (RAPD) markers. We call these regions 1, 2, and 3. To facilitate breeding by marker assisted selection, advanced breeding lines with resistance from the above sources were assayed for the presence of RAPD markers to determine which were most tightly linked to begomovirus resistance. The best RAPD markers were then converted to sequence characterized amplified region (SCAR) markers or cleaved amplified polymorphic sequence (CAPS) markers. In addition, selected restriction fragment length polymorphism (RFLP) markers near the three regions were converted into CAPS markers, which were tested for association with the advanced breeding lines. Only LA 2779 derivatives have the L. chilense introgression in region 1, which is near the location of the Ty-1 gene and spans across CAPS markers 32.5Cla and TG118. Two region 1 RAPD markers UBC197 and UBC621 were converted co-dominant SCAR or CAPS markers, which were present in all 16 resistant breeding lines tested. Derivatives from all three accessions have introgressions in region 2. Further assays with more markers in this region are under way to determine the lengths and locations of the introgressions. No tightly linked RAPD markers have been found for the resistance gene from LA 1932 in region 3. RFLP and CAPS markers are being used to more precisely locate the region 3 gene.

共優性ＳＣＡＲマーカーによるハイブリッドアカシア（Ａｃａｃｉａ ｍａｎｇｉｕｍ × Ａ．ａｕｒｉｃｕｌｉｆｏｒｍｉｓ）の雑種鑑定

共優性ＳＣＡＲマーカーによるハイブリッドアカシア（Ａｃａｃｉａ ｍａｎｇｉｕｍ × Ａ．ａｕｒｉｃｕｌｉｆｏｒｍｉｓ）の雑種鑑定

Development of SCAR Markers Linked to L3 gene in Capsicum

Random amplified polymorphic DNA (RAPD) markers linked to the L3 locus were developed by applying the bulked segregant analysis method to two doubled haploid (DH) populations in Capsicum. The codominant RAPD markers, E18272 and E18286, were converted into sequence-characterized amplified region (SCAR) markers by molecular cloning and nucleotide sequencing. A PCR analysis using DH (n = 176) and backcross (n = 190) populations revealed that all the SCAR markers, PMFR11269, PMFR11283 and PMFR21200, co-segregated with the original RAPD markers, and were mapped at a distance of 4.0 cM from the L3 locus. Furthermore, after confirmation of their validity in 18 accessions of Capsicum spp. using the codominant SCAR marker pair, PMFR11269 and PMFR11283, it was suggested that the SCAR markers developed here could become effective in marker-assisted selection programs for the introduction of the L3 gene derived from PI159236 (C. chinense) into sweet pepper for breeding purposes.

Variation in the response of melon genotypes to Fusarium oxysporum f.sp. melonis race 1 determined by inoculation tests and molecular markers

Screening of genotypes of melon (Cucumis melo) for resistance to wilt caused by Fusarium oxysporum f.sp. melonis is often characterized by wide variability in their responses to inoculation, even under carefully controlled conditions. The variability at the seedling stage of 17 genotypes susceptible to race 1 was examined in growth‐chamber experiments. Disease incidence varied from 0 to 100% in a genotype‐dependent manner. Using four combinations of light (60 and 90 µE m−2 s−1) and temperatures of (27 and 31°C), only light intensity showed a statistically significant effect. Marker‐assisted selection for fusarium resistance breeding using cleaved amplified polymorphic sequence (CAPS) and sequence‐characterized amplified region (SCAR) markers were compared using a single set of genotypes that included 24 melon accessions and breeding lines whose genotype regarding the Fom‐2 gene was well characterized. The practical value of the markers for discriminating a range of genotypes and clarifying the scoring of phenotypes was also tested using a segregating breeding population which showed codominant SCAR markers to be useful in marker‐assisted selection.

Development of SCAR markers distinguishing pure seedlings of the endangered species Morus boninensis from M. boninensis × M. acidosa hybrids for conservation in Bonin (Ogasawara) Islands

To support the recovery of Morusboninensis Koidz. (Moraceae), a criticallyendangered species distributed in the BoninIslands of Japan, we need to distinguishbetween pure M. boninensis and hybrid(M. boninensis × M. acidosa Griff.)seedlings within populations derived from M. boninensis mother trees. Therefore, wedeveloped three co-dominant SCAR (SequenceCharacterized Amplified Region) markers todistinguish between M. boninensis, M. acidosa and their hybrids. All threemarkers could clearly identify the species andthe hybrids, although we observed one case of aputative null allele or a backcross at onelocus (Mos0251-1). Hence, we suggest that pureand hybrid seedlings could be unambiguouslyidentified by the simultaneous use of the threemarkers. When we investigated a seedlingpopulation produced by a single mother treegrowing in the Iguma-bay population ofHaha-jima Island, the proportion of embryosformed by pollination from M. acidosapollen was about 13%. All hybrid seedlingsidentified according to the molecular markersshowed incisions in their leaves, amorphological character that has never beenobserved in pure M. boninensisindividuals. Our results suggest that M.boninensis can potentially be multiply byexcluding hybrids from the seedling pools,using incisions on the leaves as morphologicalmarkers to identify them.

Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection

A Brassica juncea mapping population was generated and scored for seed coat colour. A combination of bulked segregant analysis and AFLP methodology was employed to identify markers linked to seed coat colour in B. juncea. AFLP analysis using 16 primer combinations revealed seven AFLP markers polymorphic between the parents and the bulks. Individual plants from the segregating population were analysed, and three AFLP markers were identified as being tightly linked to the seed coat colour trait and specific for brown-seeded individuals. Since AFLP markers are not adapted for large-scale application in plant breeding, our objective was to develop a fast, cheap and reliable PCR-based assay. Towards this goal, we employed PCR-walking technology to isolate sequences adjacent to the linked AFLP marker. Based on the sequence information of the cloned flanking sequence of marker AFLP8, primers were designed. Amplification using the locus-specific primers generated bands at 0.5 kb and 1.2 kb with the yellow-seeded parent and a 1.1-kb band with the brown-seeded parent. Thus, the dominant AFLP marker (AFLP8) was converted into a simple codominant SCAR (Sequence Characterized Amplified Region) marker and designated as SCM08. Scoring of this marker in a segregating population easily distinguished yellow- and brown-seeded B. juncea and also differentiated between homozygous (BB) and heterozygous (Bb) brown-seeded individuals. Thus, this marker will be useful for the development of yellow seed B. juncea cultivars and facilitate the map-based cloning of genes responsible for seed coat colour trait.

Development of a sequence characterized amplified region (SCAR) based indirect selection method for a dominant blast-resistance gene in rice.

Two randomly amplified polymorphic DNA (RAPD) markers, OPF6(2700) and OPH18(2400), tightly linked to Pi-10, a dominant blast-resistance gene conferring complete resistance to isolate 106 (international race IB46) of the blast fungus were identified. To derive sequence characterized amplified regions (SCARs) from OPF6(2700) and OPH18(2400), these amplified RAPD products were cloned and sequenced. Nucleotide sequence information, obtained for each end of the two linked RAPD markers, was used to design 24-mer oligonucleotide primers for PCR amplification of the respective SCARs. Polymorphisms appearing as differences in the length of the SCAR's alternate alleles were considered for the indirect selection of Pi-10. Such polymorphisms converted the linked dominant RAPD loci into codominant SCAR markers and also facilitated the indirect scoring of the blast-resistant and blast-susceptible genotypes. The development of length variant codominant SCAR markers linked to a major gene for blast resistance in rice is described. The codominant SCARs will facilitate marker-assisted selection of the Pi-10 locus in rice breeding programs and will also be useful as genetic markers for high resolution mapping of the Pi-10 region.

Rapid mapping of two genes for resistance to downy mildew from Lactuca serriola to existing clusters of resistance genes

Two resistances to downy mildew derived from Lactuca serriola were characterized genetically and mapped using molecular markers. Classical genetic analysis suggested monogenic inheritance; however, the presence of multiple, tightly-linked genes in each case could not be eliminated. Therefore, they were designated resistance factors R17 and R18. Analysis with molecular markers known to be linked to clusters of resistance genes quickly revealed linkage of R18 to the major cluster of resistance genes and provided six linked markers, three RAPD (Random Amplified Polymorphic DNA) markers and three codominant SCAR (Sequence Characterized Amplified Region) markers. The mapping of R17 required the screening of arbitrary RAPD markers using bulked segregant analysis; this provided five linked markers, three of which segregated in the basic mapping population. This demonstrated loose linkage to a second cluster of resistance genes and provided additional linked markers. Two RAPD markers linked to R17 were converted into SCARs. The identification of reliable PCR-based markers flanking each gene will aid in selection and in combining these resistance genes with others.