Development and validation of CAPS and AFLP markers for white rust resistance gene in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

White rust, caused by Albugo candida, is a very serious disease in crucifers. In Indian mustard (Brassica juncea), it can cause a yield loss to the extent of 89.9%. The locus Ac2(t) controlling resistance to white rust in BEC-144, an exotic accession of mustard, was mapped using RAPD markers. In the present study, we developed: (1) a more tightly linked marker for the white rust resistance gene, using AFLP in conjunction with bulk segregant analysis, and (2) a PCR-based cleaved amplified polymorphic sequence (CAPS) marker for the closely linked RAPD marker, OPB06₁₀₀₀. The data obtained on 94 RILs revealed that the CAPS marker for OPB06₁₀₀₀ and the AFLP marker E-ACC/M-CAA₃₅₀ flank the Ac2(t) gene at 3.8 cM and 6.7 cM, respectively. Validation of the CAPS marker in two different F₂ populations of crosses Varuna × BEC-144 and Varuna × BEC-286 was also undertaken, which established its utility in marker-assisted selection (MAS) for white rust resistance. The use of both flanking markers in MAS would allow only 0.25% misclassification and thus provide greater efficiency to selection.Communicated by C. Möllers     

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.     

小麦抗白粉病基因Pm23的RAPD标记

小麦抗白粉病基因Pm23对世界上很多麦区流行的白粉病表现高抗或免疫.本研究以Pm23和Chancellor为抗感亲本,用集群分离分析法对抗性基因Pm23进行了RAPD分析,从320个十碱基随机引物中筛选到一个与Pm23紧密连锁的相引相标记OPE051100. 对F2分离群体进行RAPD分析表明,该标记与Pm23基因之间的连锁距离为10.65±3.25 cM.该标记可以有效用于小麦育种分子标记辅助选择中.     

Development of RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in pepper ( Capsicum annuum L.)

Potato Virus Y (PVY) is the only potyvirus infecting pepper ( Capsicum annuum L.) in Europe. Currently, the development of pepper varieties resistant to PVY seems to be the most-efficient method to control PVY damage. Among the sources of resistance, a monogenic dominant gene Pvr4 confers resistance against all known PVY pathotypes. In this work, bulked segregant analysis (BSA) was used to search for randomly amplified polymorphic DNA (RAPD) markers linked to the Pvr4 gene, using segregating progenies obtained by crossing a homozygous resistant ('Serrano Criollo de Morelos-334') with a homozygous susceptible ('Yolo Wonder') cultivar. Eight hundred decamer primers were screened to identify one RAPD marker (UBC19(1432)) linked in repulsion phase to Pvr4. This marker was converted into a dominant sequence characterised amplified region (SCAR) marker (SCUBC19(1423)). This marker was mapped into a dense Capsicum genetic map in a region where several genes for resistance to different diseases are located. This marker can be useful to identify PVY-resistant genotypes in segregating progenies of pepper in marker-assisted selection (MAS) breeding programs.     

Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.     

Development of a CAPS marker for the Pvr4 locus: a tool for pyramiding potyvirus resistance genes in pepper.

The Pvr4 resistance gene in pepper confers a complete resistance to the three pathotypes of potato virus Y (PVY) and to pepper mottle virus (PepMoV). In order to use this gene in a marker-assisted selection (MAS) program and to permit the pyramiding of several potyvirus resistance genes in the same cultivar, tightly linked amplified fragment length polymorphism (AFLP) markers were obtained by the bulked segregant analysis method. Eight linked AFLP markers were mapped in an interval from 2.1 +/- 0.8 to 13.8 +/- 2.9 cM around this locus. The closest codominant AFLP marker was converted into a codominant CAPS (cleaved amplified polymorphic sequence) marker using data from the alignment of the two allele sequences. We have further characterized the relevance of the CAPS marker for MAS programs in different pepper breeding lines.     

RAPD-based SCAR marker SCA 12 linked to recessive gene conferring resistance to anthracnose in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, caused by Colletotrichum graminicola, infects all aerial parts of sorghum, Sorghum bicolor (L.) Moench, plants and causes loss of as much as 70%. F₁ and F₂ plants inoculated with local isolates of C. graminicola indicated that resistance to anthracnose in sorghum accession G 73 segregated as a recessive trait in a cross with susceptible cultivar HC 136. To facilitate the use of marker-assisted selection in sorghum breeding programs, a PCR-based specific sequence characterized amplified region (SCAR) marker was developed. A total of 29 resistant and 20 susceptible recombinant inbred lines (RILs) derived from a HC 136 × G 73 cross was used for bulked segregant analysis to identify a RAPD marker closely linked to a gene for resistance to anthracnose. The polymorphism between the parents HC 136 and G 73 was evaluated using 84 random sequence decamer primers. Among these, only 24 primers generated polymorphism. On bulked segregant analysis, primer OPA 12 amplified a unique band of 383 bp only in the resistant parent G 73 and resistant bulk. Segregation analysis of individual RILs showed the marker OPA 12₃₈₃ was 6.03 cM from the locus governing resistance to anthracnose. The marker OPA 12₃₈₃ was cloned and sequenced. Based on the sequence of cloned RAPD product, a pair of SCAR markers SCA 12-1 and SCA 12-2 was designed using the MacVector program, which specifically amplified this RAPD fragment in resistant parent G 73, resistant bulk and respective RILs. Therefore, it was confirmed that SCAR marker SCA 12 is at the same locus as RAPD marker OPA 12₃₈₃ and hence, is linked to the gene for resistance to anthracnose.     

Isolation of microsatellite and RAPD markers flanking the Yr15 gene of wheat using NILs and bulked segregant analysis.

Microsatellite and random amplified polymorphic DNA (RAPD) primers were used to identify molecular markers linked to the Yr15 gene which confer resistance to stripe rust (Puccina striiformis Westend) in wheat. By using near isogenic lines (NILs) for the Yr15 gene and a F2 mapping population derived from crosses of these lines and phenotyped for resistance, we identified one microsatellite marker (GWM33) and one RAPD marker (OPA19(800)) linked to Yr15. Then, bulked segregant analysis was used in addition to the NILs to identify RAPD markers linked to the target gene. Using this approach, two RAPD markers linked to Yr15 were identified, one in coupling (UBC199(700)) and one in repulsion phase (UBC212(1200)). After MAPMAKER linkage analysis on the F2 population, the two closest markers were shown to be linked to Yr15 within a distance of about 12 cM. The recombination rates were recalculated using the maximum likelihood technique to take into account putative escaped individuals from the stripe rust resistance test and obtain unbiased distance estimates. As a result of this study, the stripe rust resistance gene Yr15 is surrounded by two flanking PCR markers, UBC199(700) and GWM33, at about 5 cM from each side.     

Inheritance of resistance to covered smut in barley and development of a tightly linked SCAR marker

Inheritance of resistance to covered smut in the barley line Q21861 was studied using a doubled-haploid population produced by crossing Q21861 with the line SM89010. Based on 3 years of screening in the field and two seasons in the greenhouse, segregation for resistance/susceptibility fits a one-gene ratio, indicating a single major gene for resistance in Q21861. Of 440 random 10-mer primers tested using bulked segregant analysis, one primer (OPJ10) resulted in a reproducible polymorphic band. RAPD marker OPJ10₄₅₀ co-segregated in repulsion with the covered smut resistance. This marker was converted to a sequence-characterized amplified region (SCAR) marker linked in coupling (5.5 cM) with the covered smut resistant gene in Q21861. The SCAR marker was amplified in the line TR640 which is also resistant to covered smut, but not in the other resistant lines. The SCAR marker will be useful for marker-assisted selection for covered smut in barley breeding programs. Received: 9 January 2001 / Accepted: 31 May 2001     

The role of RAPD markers in breeding for disease resistance in common bean

Diseases are regarded as the leading constraint to increased common bean (Phaseolus vulgaris L.) production worldwide. The range in variability and complexity among bean pathogens can be controlled with different single gene and quantitative resistance sources. Combining these resistance sources into commercial cultivars is a major challenge for bean breeders. To assist breeders, a major effort to identify RAPD markers tightly linked to different genes was undertaken. To date, 23 RAPD and five SCAR markers linked to 15 different resistance genes have been identified, in addition to QTL conditioning resistance to seven major pathogens of common bean. We review the feasibility of using marker-assisted selection (MAS) to incorporate disease resistance into common bean. Indirect selection of single resistance genes in the absence of the pathogen and the opportunity afforded breeders to pyramid these genes to improve their longevity and retain valuable hypostatic genes is discussed. The role of markers linked to the QTL controlling complex resistance and the potential to combine resistance sources using marker based selection is reviewed. Improving levels of selection efficiency using flanking markers, repulsion-phase linkages, co-dominant marker pairs, recombination-facilitated MAS and SCAR markers is demonstrated. Marker-assisted selection for disease resistance in common bean provides opportunities to breeders that were not feasible with traditional breeding methods.     

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.     

DNA markers linked to a T10 loose smut resistance gene in wheat (Triticum aestivum L.).

Screening for loose smut resistance in wheat is difficult. Selecting lines with DNA markers linked to loose smut resistance would be more reliable and less costly. Molecular markers linked to a race T10 loose smut resistance gene were identified using a F6 single seed descent segregating population. A RAPD marker and a RFLP marker were located on opposite flanks of the resistance gene and were shown to be loosely linked. The RAPD marker was converted to a user friendly polymorphic SCAR marker that represented a single genetically defined locus in hexaploid wheat. Using these two bracketing markers simultaneously, the error rate for T10 resistance selection due to crossing-over was reduced to 4%. These markers can be used for a faster and more reliable selection of T10 resistant plants than previous conventional loose smut ratings.     

Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.)

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line ’Vedrantais’ were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible (’Vedrantais’) lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F₂ individuals from crosses of ’Vedrantais’ x PI 161375 and ’Ananas Yokneam’×MR-1 respectively, and 17 families from a backcross BC₁S₁ population derived from the breeding line ’MD8654’ as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed over 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F₂ individuals and BC₁S₁ families tested. This is the first report of PCR-based CAPS markers linked to resistance/susceptibility for Fusarium wilt in melon. The RFLP markers resulting from probing with a clone-derived 1.05-kb SCG17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were co-dominant, easier to score, and more accurate and consistent in predicting the melon phenotype than the RAPD markers from which they were derived. Received: 28 July 1998 / Accepted: 7 December 1998     

Identification of RAPD markers linked to the karnal bunt resistance genes in wheat

A set of 104 wheat recombinant inbred lines (RILs) obtained from a cross between parents resistant (HD 29) and susceptible (WH 542) to karnal bunt (KB) (caused by Neovossia indica) were screened and used to identify random amplified polymorphic DNA (RAPD) markers linked with resistance to karnal bunt as these would allow indirect marker assisted selection of KB resistant genotypes. The two parents were analysed with 92 RAPD primers. A total of 65 primers proved functional by giving scorable polymerase chain reaction (PCR) products. Of these, 21 (32 %) primers detected polymorphism between the two parental genotypes. Using these primers, bulked segregant analysis was carried out on two bulk DNAs, one obtained by pooling DNA from 10 KB resistant RILs and the other similarly derived by pooling 10 KB susceptible RILs. One marker, OPM-20 showed apparent association with resistance to KB. This was confirmed following selective genotyping of individual RILs included in the bulks.     

Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses

Blackspot resistance in the tetraploid rose genotype 91/100–5 had been characterised previously as a single dominant gene in duplex configuration. In the present study a tetraploid progeny (95/3) segregating for the presence of the blackspot resistance gene Rdr1 were screened with 868 RAPD and 114 AFLP primers/primer combinations. Seven AFLP markers were found to be linked to Rdr1 at distances between 1.1 and 7.6 cM. The most closely linked AFLP marker was cloned and converted into a SCAR marker that could be screened in a larger population than the original AFLP and was linked at a distance of 0.76 cM. The cloned fragment was used as an RFLP probe to locate the marker on a chromosome map of diploid roses. This is the first report of markers linked to a resistance gene in roses, and the possibilities of using them for a marker-assisted selection for blackspot resistance as well as for map-based cloning approaches are discussed. Received: 23 December 1999 / Accepted: 25 March 2000     

Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat

 RAPD (random amplified polymorphic DNA) analysis was used to identify molecular markers linked to the Dn2 gene conferring resistance to the Russian wheat aphid (Diuraphis noxia Mordvilko). A set of near-isogenic lines (NILs) was screened with 300 RAPD primers for polymorphisms linked to the Dn2 gene. A total of 2700 RAPD loci were screened for linkage to the resistance locus. Four polymorphic RAPD fragments, two in coupling phase and two in repulsion phase, were identified as putative RAPD markers for the Dn2 gene. Segregation analysis of these markers in an F₂ population segregating for the resistance gene revealed that all four markers were closely linked to the Dn2 locus. Linkage distances ranged from 3.3 cM to 4.4 cM. Southern analysis of the RAPD products using the cloned RAPD markers as probes confirmed the homology of the RAPD amplification products. The coupling-phase marker OPB10_880c and the repulsion-phase marker OPN1_400r were converted to sequence characterized amplified region (SCAR) markers. SCAR analysis of the F₂ population and other resistant and susceptible South African wheat cultivars corroborated the observed linkage of the RAPD markers to the Dn2 resistance locus. These markers will be useful for marker-assisted selection of the Dn2 gene for resistance breeding and gene pyramiding. Received: 1 July 1997 / Accepted: 20 October 1997     

Identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to resistance against beet necrotic yellow vein virus (BNYVV) in Beta accessions

Molecular markers linked to resistance genes are useful to facilitate the introgression of one or more of these genes in breeding materials. Following the approach of bulked segregant analysis, RAPD markers linked to resistance genes against beet necrotic yellow vein virus were identified in the four Beta accessions Holly-1-4, R104, R128 and WB42. Two primers were found which generate RAPD markers tightly linked to resistance in segregating families of Holly-1-4, R104 and R128, indicating that the resistance genes in these accessions might be situated at the same locus. Other, specific, primers were identified which generate RAPD markers linked to resistance in each of these accessions. Short-range maps were established around the resistance locus in these accessions. For WB42, RAPD markers were only identified at a relatively large distance from the resistance gene. Conversion of three RAPD primers of Holly-1-4, R104 and R128 into STS primers resulted in STS markers which can be readily used for marker-assisted selection in breeding programmes. Received: 8 January 1996 / Accepted: 14 June 1996     

Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 ¹, and pvr1 ². These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.     

RAPD Marker Linked to a Gene Conferring Resistance to Race IB-9 of Pyricularia grisea in a Somaclone of the Rice Cultivar Araguaia

The gene Pi-ar confers resistance to Pyricularia grisea in a somaclone of the upland rice cultivar Araguaia developed from callus culture of immature panicles. The somaclone SC09 exhibited resistant reaction to all of the 182 P. grisea test isolates belonging to 15 different races. The study on inheritance showed that the resistance to pathotype IB-9 of P. grisea is monogenic and dominant. In order to identify marker linked to this gene, the F₂ population from a cross between the highly susceptible cultivar Lijiangxintuanheigu (LTH) and the somaclone SC09 of rice cultivar Araguaia was screened using RAPD primers. Initially, the polymorphism between parents, the cultivar LTH and somaclone SC09 was analyzed using 577 random 10-bp primers. The susceptible and resistant bulks of the F₂ population, along with DNA of the two parents were tested with 176 primers that differentiated susceptible and resistant parents. Thirty-six primers differentiated the susceptible and resistant bulks, as well as the cultivar LTH of the somaclone SC09. However, one primer OPK17 was found to be closely linked (5.3 cM) to the resistance gene of somaclone and this can be used in the marker assisted selection.     

Molecular markers linked to the Rf2 fertility restorer gene in cotton.

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.