Scab Resistance Gene Vf Research Articles

Identification of apple scab and powdery mildew resistance genes in Czech apple (Malus × domestica) genetic resources by PCR molecular markers

The presence of genes for resistance to scab (Venturia inaequalis) and powdery mildew (Podosphaera leucotricha) was studied using molecular markers in a sample of 279 apple cultivars from the Czech collection of apple genetic resources. The sample comprised 37 cultivars supposed to have the Vf gene for scab resistance, 97 reference world cultivars and 145 old and local cultivars. Six PCR molecular markers for the scab resistance genes Vf, Vm, Vbj, Vr and Vh and three PCR molecular markers for the powdery mildew resistance genes Pl-w, Pl-1 and Pl-d were used. The marker for the major scab resistance gene Vf was detected in all cultivars supposed to have Vf, except in Romus 1, and in the three small-fruited cultivars Malus Evereste, Golden Gem and Hilleri. The markers of the Vr and Vh scab resistance genes were detected in 22 cultivars in combination with the marker for Vf, in 56 reference world cultivars and in 82 old and local apple cultivars. PCR molecular markers for one or two of the powdery mildew resistance genes were detected in the small-fruited cultivars Malus Evereste, Golden Gem, prof. Sprengeri and Hilleri; and in the larger fruited cultivars Hagloe Crab, Borovinka and Tita Zetei. We did not find markers for the scab resistance genes Vm and Vbj in any of the studied cultivars. They are absent also in the remaining part of the Czech collection of apple genetic resources. PCR molecular markers are useful tools for the identification of resistance genes within apple germplasm collections and can be used to increase the number of sources for disease resistance in breeding programmes.

CISGENESIS IS A PROMISING APPROACH FOR FAST, ACCEPTABLE AND SAFE BREEDING OF PIP FRUIT

Introgression of traits from wild germplasm into pip fruit cultivars by means of classical breeding is painstakingly slow. Introgression of e.g., the apple scab resistance gene Vf from Malus floribunda 821 into marketable high quality apple cultivars took approximately 50 years. In the mean time the Vf resistance is being broken down in Europe. For durable resistance, different resistance genes should be accumulated. However, this may take another series of decades. This slow tempo is caused mainly by the long juvenile period of apple and the phenomenon that not only the allele of interest is inherited by the progeny, but also hundreds of unwanted alleles. The process would be much faster if only the allele of interest was inserted, without unwanted alleles. This can be achieved by cisgenesis. We defined cisgenesis as genetic modification of plants, inserting alleles of the plant itself or from crossable relatives. The allele should contain its native introns and should be flanked by its native promoter and terminator in sense-orientation. If the plant is equipped with foreign genes from outside the gene pool of the conventional breeder, the plant is named transgenic. Inquiries indicate that cisgenic plants are more acceptable to consumers than transgenic plants. As the phenotypic result of cisgenesis can, in principle, also be obtained by means of conventional breeding or translocation breeding, cisgenic plants are as safe as plants from conventional breeding or mutation breeding. Therefore we have proposed to treat cisgenic plants like conventionally bred plants, by exempting cisgenic plants from the GMO regulation. The number of isolated, functionally analysed genes and their alleles from fruit tree crops is increasing. Also technologies are available for introduction of these alleles without leaving selection genes behind. Cisgenesis is combining the knowledge of native alleles with marker free technologies. Cisgenesis is a promising path for utilizing the wealth of knowledge on plant genes to the benefit of the society in a fast, safe, and acceptable way.

Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab‐resistant accessions in Malus germplasm*

AbstractA polymerase chain reaction (PCR)‐based method was developed to isolate microsatellite markers from large‐insert genomic DNA clones of bacterial artificial chromosome (BAC) libraries. The method is fast and economic since it does not require subcloning. It was applied to isolate a microsatellite marker from a BAC clone of the chromosomal region containing the apple scab resistance gene Vf. The Vf gene of Malus floribunda 821 is the most widely used source of scab resistance in apple breeding. A second microsatellite was found on the extremity of a BAC clone flanking the Vf locus. The two microsatellites allowed the identification of the presence of the Vf gene in the scab‐resistant accessions M. micromalus SA573‐3, ‘Golden Gem’, M. prunifolia 19651 and MA 16 not previously known to carry this gene. They were also used to verify the correctness of the published genealogical tree of the Vf cultivar ‘Florina’, in which a probable mistake was identified. This analysis shows the importance of genotyping the Vf locus when choosing scab‐resistant germplasm as parents in breeding programmes.

Phenotypic Reaction and Genetic Analysis Using AFLP‐derived SCARs for Resistance to Apple Scab

AbstractSix sequence‐characterized amplified region (SCAR) markers linked to the apple scab resistance gene Vf were evaluated for their utility in marker‐assisted selection (MAS) in apple breeding. Of the six SCARs used in this study, ACS‐6 was located left of the Vf gene, ACS‐7 and ACS‐9 co‐segregated with Vf, and ACS‐8, ACS‐4, ACS‐5 were located right of the Vf gene. Three families derived from crosses between scab‐resistant and scab‐susceptible cultivars, including ‘Liberty’ × ‘Deljub’, ‘Liberty’ × ‘Delcorf’, and ‘Florina’ בDelcorf’, previously screened for scab resistance following greenhouse inoculation with the fungal pathogen Venturia inaequalis, were genotyped and compared with phenotypic reactions to scab infection in the field. For each family, a subset progeny of 30 seedlings (propagated onto Malling 9 rootstock and of 7 years old) was selected based on fungal sporulation according to the following scheme. Ten seedlings with no visible scab sporulation on leaves were given phenotypic scores of 0 (deemed resistant); 10 seedlings with moderate scab sporulation were given phenotypic scores of 1.0 (deemed moderately resistant); and 10 seedlings with heavy sporulation were given phenotypic scores of 2.0 (deemed susceptible). DNA was isolated from leaf tissue collected from all 90 seedlings, parents and Malus floribunda 821, the original source of the Vf gene, and screened with all six SCARs. All six SCARs were present in the two scab‐resistant parents, ‘Liberty’ and ‘Florina’, and M. floribunda 821; while, the two scab‐susceptible parents, ‘Deljub’ and ‘Delcorf’, lacked all SCARs. All SCARs were either present or absent in varying numbers of seedlings in each progeny with phenotypic ratings of either 0 (resistant) or 1.0 (moderately resistant); while all seedlings with phenotypic ratings of 2.0 (susceptible) lacked all SCARs. The inconsistencies between phenotypic scab ratings and SCAR marker data are discussed.

Quantitative Trait Loci (QTL) Analysis Reveals Both Broad-Spectrum and Isolate-Specific QTL for Scab Resistance in an Apple Progeny Challenged with Eight Isolates of Venturia inaequalis

ABSTRACT The major scab resistance gene Vf, extensively used in apple breeding programs, was recently overcome by the new races 6 and 7 of the fungal pathogen Venturia inaequalis. New, more durable, scab resistance genes are needed in apple breeding programs. F(1) progeny derived from the cross between partially resistant apple cv. Discovery and apple hybrid 'TN10-8' were inoculated in the greenhouse with eight isolates of V. inaequalis, including isolates able to overcome Vf. One major resistance gene, Vg, and seven quantitative trait loci (QTL) were identified for resistance to these isolates. Three QTL on linkage group (LG)12, LG13, and LG15 were clearly isolate-specific. Another QTL on LG5 was detected with two isolates. Three QTL on LG1, LG2, and LG17 were identified with most isolates tested, but not with every isolate. The QTL on LG2 displayed alleles conferring different specificities. This QTL co-localized with the major scab resistance genes Vr and Vh8, whereas the QTL on LG1 colocalized with Vf. These results contribute to a better understanding of the genetic basis of the V. inaequalis-Malus x domestica interaction.

A bacterial artificial chromosome (BAC) library ofMalus floribunda821 and contig construction for positional cloning of the apple scab resistance geneVf

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5× haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.Key words: apple, resistance Vf gene, BAC library, sequence-characterized amplified regions (SCARs), fiber fluorescence in situ hybridization (fiber-FISH), positional cloning.

A bacterial artificial chromosome (BAC) library of <i>Malus floribunda </i>821 and contig construction for positional cloning of the apple scab resistance gene <i>Vf</i>

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5x haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.

Saturation mapping of the apple scab resistance gene Vf using AFLP markers

Using the amplified fragment length polymorphism (AFLP) technique combined with a ”narrow-down” bulk segregant strategy enabled us to quickly identify 15 tightly linked AFLP markers to the Vf gene that confers resistance to the apple scab disease. High-resolution mapping placed all 15 AFLP markers within an interval of 0.6 cM around the Vf region; 7 of them were found to be inseparable from the Vf gene, 1 was localized left of, and the remaining 7 located right of the Vf gene. In addition, eight previously identified RAPD markers were also mapped, but only three, including M18, AM19, and AL07, were localized within this short interval, and none co-segregated with the Vf gene. The saturation of the Vf region with AFLP markers will accelerate both marker-assisted selection and map-based cloning. The advantages of this ”narrow-down” strategy, estimation of physical distances among AFLP markers, and their potential application are also discussed.

The use of a modified bulk segregant analysis to identify a molecular marker linked to a scab resistance gene in apple

Almost two-hundred random sequence decamer-primers were used to screen a pair of bulked samples and the donor parent Malus floribunda clone 821 for markers linked to the Vf gene conferring resistance to apple scab (Venturia inaequalis (Cke.) Wint.). A single primer was identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars confirmed that this marker was linked to the scab-resistance gene Vf with a recombination frequency of 4.3%. OPK16/1300 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were synthesized, and used in turn to screen M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. A pair of sequence-specific primers of clone OPK16/1300 amplified a distinct single band of the same size as the RAPD clone. Thus, a sequence characterized amplified region (SCAR) marker was developed which can be used to identify polymorphisms of OPK16/1300 based on the presence or absence of a single band.

A Randomly Amplified Polymorphic DNA (RAPD) Marker Tightly Linked to the Scab-resistance Gene Vf in Apple

Almost 200 random sequence decamer primers were used to screen a pair of bulked samples of apple (Malus ×domestica Borkh.) DNA and that of the donor parent Malus floribunda Sieb. clone 821 for molecular markers linked to the Vf gene conferring resistance to apple scab [Venturia inaequalis (Cke.) Wint.]. Identified was a single primer that generated a polymerase chain-reaction (PCR) fragment, OPAR4/1400, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent-parent bulk. Cosegregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant selections/cultivars confirmed that this marker was linked to the scab-resistance gene Vf OPAR4/1400 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were developed and used to screen further M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Purdue, Rutgers, and Univ. of Illinois apple breeding program selections. The sequence-specific primers identified polymorphisms of OPAR4/1400 based on the presence or absence of a single band. This molecular marker is at a distance of about 3.6 cM from the Vf gene.

A Molecular Marker Linked to a Scab Resistance Gene (Vf) in Apple

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene Vf. Our major objective is to identify RAPD markers linked to the Vf gene. The approach in this paper is based on the introgression of the Vf gene from M. floribunda into commercial cultivars. Almost 200 random sequence decamer-primers have been used to screen a pair of bulked samples and the donor parent M. floribunda clone 821 for markers linked to the Vf gene conferring resistance to apple scab. A single primer has been identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars have confirmed that this marker is linked to the scab-resistance gene Vf. OPK16/1300 has since been cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker have been synthesized and used to screen further M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. The sequence-specific primers have identified polymorphisms of OPK16/1300 based on the presence or absence of a single band.

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.