Application of AFLP™-based molecular markers to breeding of Populus spp.

Molecular marker technologies have eased and potentiated genetic analysis of plants and have become an extremely useful tool in forest tree breeding. The information provided by molecular markers has made it possible to acquire further knowledge about the structure and organization of plant genomes as well as about the evolution of these plant genomes through phylogenetic analysis. Using Populus spp. as a model tree, this paper aims at showing and discussing the possible applications of AFLP, a high-density DNA marker technology developed by Keygene N.V. (Wageningen, The Netherlands). Applications include: (i) AFLP analysis of the disease resistance against Melampsora larici-populina using bulked-segregant analysis, (ii) AFLP fingerprinting for identification and taxonomic analysis of individual trees, and (iii) AFLP-based mapping strategies in Populus.Abbreviations AFLP amplified fragment length polymorphism - RFLP restriction fragment length polymorphism - PCR polymerase chain reaction - QTL quantitative trait loci - RAPD random amplified polymorphic DNA     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

High-resolution Mapping and Analysis of the Resistance Locus <Emphasis Type="Italic">Rpi-abpt</Emphasis> Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Introduction of more durable resistance against Phytophthora infestans causing late blight into the cultivated potato is of importance for sustainable agriculture. We identified a new monogenically inherited resistance locus that is localized on chromosome 4. The resistance is derived from an ABPT clone, which is originally a complex quadruple hybrid in which Solanum acaule, S. bulbocastanum, S. phureja and S. tuberosum were involved. Resistance data of the original resistant accessions of the wild species and analysis of mobility of AFLP markers linked to the resistance locus suggest that the resistance locus is originating from S. bulbocastanum. A population of 1383 genotypes was screened with two AFLP markers flanking the Rpi-abpt locus and 98 recombinants were identified. An accurate high-resolution map was constructed and the Rpi-abpt locus was localized in a 0.5 cM interval. One AFLP marker was found to co-segregate with the Rpi-abpt locus. Its DNA sequence was highly similar with sequences found on a tomato BAC containing several resistance gene analogues on chromosome 4 and its translated protein sequence appeared to be homologous to several disease resistance related proteins. The results indicated that the Rpi-abpt gene is a member of an R gene cluster.     

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     

A PCR-based marker tightly linked to the nematode resistance gene,Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F₂ population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.     

Genetic mapping of a fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.)

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis is one of the most devastating diseases in melon production worldwide. The most effective control measure available is the use of resistant varieties. Identifying molecular markers linked to resistance genes can serve as a valuable tool for the selection of resistant genotypes. Bulked segregant analysis was used to identify markers linked to the Fom-2 genes, which confers resistance to races 0 and 1 of the fungal pathogen. Pooled DNA from homozygous resistant or homozygous susceptible progeny of F₂ cross between MR-1 and AY was screened using 240 PstI/MseI and 200 EcoRI/MseI primer combinations to identify AFLP markers linked to Fom-2. Fifteen markers potentially linked to Fom-2 were identified, all with EcoRI/MseI primer pairs. These were mapped relative to Fom-2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3 cM, respectively. Moreover, AFLP marker AGG/CCC and the previously identified RAPD marker 596-1 cosegregated with Fom-2. These two dominant markers were converted to co-dominant markers by designing specific PCR primers that produced product length polymorphisms between the parents. A survey of 45 melon genotypes from diverse geographic origins with the co-dominant markers demonstrated a high correlation between fragment size and the resistance phenotype. These markers may therefore be useful in marker-assisted breeding programs.     

Mapping and diagnostic marker development for Soil-borne cereal mosaic virus resistance in bread wheat

Monogenically-inherited resistance to Soil-borne cereal mosaic virus (SBCMV) in hexaploid bread wheat cultivars ‘Tremie’ and ‘Claire’ was mapped on chromosome 5D. The two closest flanking markers identified in the Claire-derived mapping population, Xgwm469-5D and E37M49, are linked to the resistance locus at distances of 1 and 9 cm, respectively. Xgwm469-5D co-segregated with the SBCMV resistance in the Tremie-derived population and with the recently identified Sbm1 locus in the cv. Cadenza. This suggested that Tremie and Claire carry a resistance gene allelic to Sbm1, or one closely linked to it. The diagnostic value of Xgwm469-5D was assessed using a collection of SBCMV resistant and susceptible cultivars. Importantly, all susceptible genotypes carried a null allele of Xgwm469-5D, whereas resistant genotypes presumably related to either Claire and Tremie or Cadenza revealed a 152 or 154 bp allele of Xgwm469-5D, respectively. Therefore, Xgwm469-5D is well suited for marker assisted selection for SBCMV resistance.     

Association of AFLP markers with downy mildew resistance in autotetraploid alfalfa

The amplified fragment length polymorphism (AFLP) assay is an efficient method for the identification of molecular markers useful in the improvement of numerous crop species. The identification of AFLP markers linked to disease resistance genes has been shown in segregating populations from crosses of inbred lines. The development of inbred lines in alfalfa is not possible, but existing breeding programs have produced populations selected for resistance to a single pest. Two such populations, UC-123 and UC-143, differing only in selection for resistance to downy mildew (Peronospora trifoliorum de Bary) isolate I-8, were used in this study. Thirty-six resistant plants from UC-143, and 36 susceptible plants from UC-123 were screened for DNA polymorphisms using fourteen AFLP primer combinations. Four AFLP fragment markers, ACACTC₂₀₈, ACACTC₁₅₀, ACACAT₂₁₆ and ACACTC₄₈₆, were found to be significantly associated with disease susceptibility or resistance. Resistant and susceptible plants were crossed in a diallel scheme and the progeny were screened for resistance to P. trifoliorum isolate I8. Two of the AFLP markers, ACACTC₂₀₈ and ACACTC₄₈₆ were significantly associated with resistance in the F₁ and S₁ progeny. The utilization of two populations, comprised of 36 resistant and 36 susceptible plants, for the identification of DNA fragments associated with disease resistance proved successful. Seventy-two plants is a very manageable number and provides a starting point for further refinement of marker-trait associations.     

SSR Markers Closely Linked to the Pi-z Locus are Useful for Selection of Blast Resistance in a Broad Array of Rice Germplasm

Pi-z is a disease resistance gene that has been effectively used to combat a broad-spectrum of races of the rice blast fungus Magnaporthe grisea. Although DNA markers have been reported for selection of the Pi2(t) and Pi-z resistance genes at the Pi-z locus, markers that are more tightly linked to the Pi-z locus would benefit rapid and effective cultivar development. Analysis of the publicly available genome sequence of Nipponbare near the Pi-z locus revealed numerous SSRs that could be converted into markers. Three SSRs on rice PAC AP005659 were found to be very tightly linked to the Pi-z locus, with one marker, AP5659-3, co-segregating with the Pi-z resistance reaction. The Pi-z factor conferring resistance to two races of blast was mapped to a 57 kb region on the physical map of Nipponbare in a location where the Pi2(t) gene was physically mapped. Two SSR marker haplotypes were unique for cultivars carrying the Pi-z gene, which indicates these markers are useful for selection of resistance genes at the Pi-z locus in rice germplasm.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

Identification of AFLP markers in soybean linked to resistance to Meloidogyne javanica and conversion to Sequence Characterized Amplified Regions (SCARs)

Meloidogynejavanica is the most widely spread nematode pest on soybean in SouthAfrica. Only a few registered commercial South African cultivars are poor hostsof this nematode species and there is an urgent need for an efficient breedingprogramme for resistant cultivars of all maturity groups. However, breeding ishampered by laborious screening procedures for selection of poor host cultivarsand/or lines. The objective of this study was to develop an economically viablemolecular marker system for application in selection procedures. BothRestriction Fragment Length Polymorphism (RFLP) and Amplified Fragment LengthPolymorphism (AFLP) screening techniques identified markers linked togall-indexvariation in a segregating population of 60 F₂ progeny from a crossbetween a resistant cultivar (Gazelle) and a highly susceptible variety(Prima).A codominant RFLP marker( B212) was linked significantly to M.javanica resistance and explained 62% of the variation ingall-index.Seven AFLP markers were linked significantly to the resistance trait, of whichfour were linked in repulsion phase and three in coupling phase. All seven AFLPmarkers mapped to LG-F (Linkage Group F) on the public soybean molecular map.The major quantitative trait locus (QTL) for resistance mapped between markersE-ACC/M-CTC2(SOJA6) (linked in coupling phase), B212 and E-AAC/M-CAT1(SOJA7)(linked in repulsion phase). These two AFLP markers bracketing the majorresistance QTL were successfully converted to SCARs (Sequence CharacterizedAmplified Regions). Marker E-ACC/M-CTC2 was converted to a codominant SCARmarker SOJA6, which accounted for 41% of variation in gall-index in the mappingpopulation. Marker E-AAC/M-CAT1 was converted to a dominant SCAR marker (SOJA7)and explained 42% of gall-index variation in the mapping population. These twomarkers mapped approximately 3.8 cM and 2.4 cMrespectively from the resistance QTL. This study represents the first report ofthe development of PCR-based sequence specific markers linked to M.javanica resistance in soybean.     

Identification of a major quantitative trait locus (QTL) for yellow spot (<Emphasis Type="Italic">Mycovellosiella koepkei</Emphasis>) disease resistance in sugarcane

In this study we used amplified fragment length polymorphism (AFLP) and microsatellite (short sequence repeat or SSR) markers to identify a major quantitative trail locus (QTL) for yellow spot (Mycovellosiella koepkei) disease resistance in sugarcane. A bi-parental cross between a resistant variety, M 134/75, and a susceptible parent, R 570, generated a segregating population of 227 individuals. These clones were evaluated for yellow spot infection in replicated field trials in two locations across two consecutive years. A χ²-test (χ² at 98% confidence level) of the observed segregation pattern for yellow spot infection indicated a putative monogenic dominant inheritance for the trait with a 3 (resistant):1(susceptible) ratio. The AFLP and SSR markers identified 666 polymorphisms as being present in the resistant parent and absent in the susceptible one. A genetic map of M 134/75 was constructed using 557 single-dose polymorphisms, resulting in 95 linkage groups containing at least two markers based on linkages in coupling. QTL analysis using QTLCartographer v1.17d and MAPMAKER/QTL v1.1 identified a single major QTL located on LG87, flanked by an AFLP marker, actctc10, and an SSR marker, CIR12284. This major QTL, which was found to be linked at 14 cM to an AFLP marker, was detected with LOD 8.7, had an additive effect of −10.05% and explained 23.8% of the phenotypic variation of yellow spot resistance.     

Mapping a new nematode resistance locus in Lycopersicon peruvianum

Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.     

High-resolution genetic and physical mapping of the region containing the Bs2 resistance gene of pepper

The Bs2 resistance gene of pepper confers resistance against the bacterial pathogen Xanthomonas campestris pv. vesicatoria. As a first step toward isolation of the Bs2 gene, molecular markers tightly linked to the gene were identified by randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of near-isogenic lines. Markers flanking the locus were identified and a high-resolution linkage map of the region was developed. One AFLP marker, A2, was found to cosegregate with the locus, while two others, F1 and B3, flank the locus and are within 0.6 cM. Physical mapping of the A2 and F1 markers indicates that these markers may be within 150 kb of each other. Together, these results indicate that the Bs2 region may be cloned either by chromosome walker or landing. The linked markers were also used to characterize gamma-irradiation-induced mutants at the Bs2 locus. Received: 15 January 1999 / Accepted: 11 May 1999     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.     

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata,L. Wilczek)

Summary Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F₂ progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F₂ population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.     

DNA markers linked to Malus floribunda 821 scab resistance

Breeding resistant apple plants is an alternative way to control fungal pathogens reducing the environmental impact due to the use of pesticides. The breeding of apple cultivars resistant to Venturia inaequalis could be much improved by marker-assisted selection. A molecular marker closely linked to the resistance locus called Vf could replace selection based on infection studies. To find such molecular markers, DNA of progenies from crossings of a resistant and a susceptible apple tree was subject to bulked segregant analysis. Two markers were found with a genetic distance of 10.6% and 19.7% recombination frequency to the Vf locus.     

Phenotypic assessment and mapped markers for<Emphasis Type="Italic"> H31,</Emphasis> a new wheat gene conferring resistance to Hessian fly (Diptera: Cecidomyiidae)

A new source of resistance to the highly virulent and widespread biotype L of the Hessian fly, Mayetiola destructor (Say), was identified in an accession of tetraploid durum wheat, Triticum turgidum Desf., and was introgressed into hexaploid common wheat, Triticum aestivum L. Genetic analysis and deletion mapping revealed that the common wheat line contained a single locus for resistance, H31, residing at the terminus of chromosome 5BS. H31 is the first Hessian fly-resistance gene to be placed on 5BS, making it unique from all previously reported sources of resistance. AFLP analysis identified two markers linked to the resistance locus. These markers were converted to highly specific sequence-tagged site markers. The markers are being applied to the development of cultivars carrying multiple genes for resistance to Hessian fly biotype L in order to test gene pyramiding as a strategy for extending the durability of deployed resistance.Communicated by J. Dvorak     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.