Searching for interacting QTL in related populations of an outbreeding species

Many important crop species are outbreeding. In outbreeding species the search for genes affecting traits is complicated by the fact that in a single cross up to four alleles may be present at each locus. This paper is concerned with the search for interacting quantitative trait loci (QTL) in populations which have been obtained by crossing a number of parents. It will be assumed that the parents are unrelated, but the methods can be extended easily to allow a pedigree structure. The approach has two goals: (1) finding QTL that are interacting with other loci and also loci which behave additively; (2) finding parents which segregate at two or more interacting QTL. Large populations obtained by crossing these parents can be used to study interactions in detail. QTL analysis is carried out by means of regression on predictions of QTL genotypes

The Vf gene for scrab resistance in apple is linked to sub-lethal genes

V f is the most widely used resistance gene in the breeding for scab resistant apple cultivars. Distorted segregation ratios for V f -resistance have frequently been reported. Here we revealed that sub-lethal genes caused the distorted segregation. The inheritance of V f was examined in six progenies by testing linked molecular markers. Three progenies showed distorted segregations that could be explained by three sub-lethal genes (sl1, sl2 and sl3), of which sl1, sl2 were closely linked to V f . The s11 gene was located at about 14 cM from V f and expressed itself only in the presence of another independently segregating sub-lethal gene sl3. Only the double homozygous recessive genotypes (sl1sl1 sl3sl3) were lethal, which occurred at first as dwarf and poor vigour plants during the first three months after germination. The sl2 gene was also linked to V f and its lethality was expressed prior to seed germination and also required the homozygous recessive presence of sl3. The map position of sl3 has not yet been identified. The linkage of V f to sub-lethal genes usually results in a shortage of V f -resistant progenies. But in some exceptional crosses, it will lead to abundance of resistant seedlin

Identification and mapping of the novel apple scab resistance gene Vd3

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing in temperate zones with humid springs and summers. Breeding programs around the world have been able to identify several sources of resistance, the Vf from Malus floribunda 821 being the most frequently used. The appearance of two new races of V. inaequalis (races 6 and 7) in several European countries that are able to overcome the resistance of the Vf gene put in evidence the necessity of the combination of different resistance genes in the same genotype (pyramiding). Here, we report the identification and mapping of a new apple scab resistance gene (Vd3) from the resistant selection “1980-015-25” of the apple breeding program at Plant Research International, The Netherlands. This selection contains also the Vf gene and the novel V25 gene for apple scab resistance. We mapped Vd3 on linkage group 1, 1 cM to the south of Vf in repulsion phase to it. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder “D3.” This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis capable of overcoming the resistance from Vf and Vg

Construction of an integrated consensus map of the Apple genome based on four mapping populations

An integrated consensus genetic map for apple was constructed on the basis of segregation data from four genetically connected crosses (C1¿=¿Discovery × TN10-8, C2¿=¿Fiesta × Discovery, C3¿=¿Discovery × Prima, C4¿=¿Durello di Forli × Fiesta) with a total of 676 individuals using CarthaGene® software. First, integrated female¿male maps were built for each population using common female¿male simple sequence repeat markers (SSRs). Then, common SSRs over populations were used for the consensus map integration. The integrated consensus map consists of 1,046 markers, of which 159 are SSR markers, distributed over 17 linkage groups reflecting the basic chromosome number of apple. The total length of the integrated consensus map was 1,032 cM with a mean distance between adjacent loci of 1.1 cM. Markers were proportionally distributed over the 17 linkage groups (¿ 2¿=¿16.53, df¿=¿16, p¿=¿0.41). A non-uniform marker distribution was observed within all of the linkage groups (LGs). Clustering of markers at the same position (within a 1-cM window) was observed throughout LGs and consisted predominantly of only two to three linked markers. The four integrated female¿male maps showed a very good colinearity in marker order for their common markers, except for only two (CH01h01, CH05g03) and three (CH05a02z, NZ02b01, Lap-1) markers on LG17 and LG15, respectively. This integrated consensus map provides a framework for performing quantitative trait locus (QTL) detection in a multi-population design and evaluating the genetic background effect on QTL expression

High throughput marker development and application in horticultural crops

In this paper we present an overview of current developments in sequencing that offer the possibility to generate large numbers of markers in ornamental crops. The prospects of this new sequence technology for the application of markers in breeding of outcrossing and/or polyploid crops are discussed using examples in rose and lily

QTL analysis of the genetic architecture determining resistance to fire blight in an apple progeny

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is one of the most destructive diseases of apple (Malus x domestica). In order to analyse the genetic determinism of resistance to fire blight in apple, a quantitative trait analysis (QTL) approach was used. A F1 progeny of 164 individuals derived from a cross between the apple cultivars `Prima¿ and `Fiesta¿ was inoculated in greenhouse conditions. Seven copies per genotype were used. The length of the necrosis observed on shoots was scored 7 and 14 days after inoculation. The MapQTL software was used for QTL analyses, using two previously built maps of the parents, and the symptoms scored on shoots. Digenic interactions between all pairwise combinations of genetic markers were tested using a two-way ANOVA model with the SAS software. QTL were detected at the same locations both 7 and 14 days after inoculation. Two weak effect QTL deriving from `Prima¿ were detected on linkage groups (LG) 3 and LG16. One strong effect QTL deriving from `Fiesta¿ was detected on LG7 that explained 46.6% of the phenotypic variation observed in the progeny. Two additional significant (

An assessment of the durability and susceptibility of scab resistance in apple cultivars

Durable resistance to scab (Venturia inaequalis) of newly introduced cultivars is needed to achieve a considerable reduction of pesticide use. However, the assessment in the field on the level and durability of scab resistance is time and space consuming. A proper and fast indoor test should be a convenient tool to discard cultivars with a limited level of resistance at an early stage. As a result, field tests can include only the better cultivars. This approach has been tried out by comparing field data with data from a glasshouse test, comprising twelve new and seven well-known susceptible and resistant (with and without Vf) cultivars which have been tested with six isolates of scab from different origins, virulence and aggressiveness, including race 6 and 7. Young trees on M.9 rootstock were infected and after a 17 day period at high humidity, sporulation and chlorosis have been observed. If the set of isolates is broad and well-chosen it seems possible to assess susceptibility and possibly also durability. To optimize the choice of isolates, it is desirable to know the pedigree of the cultivars that are tested. In addition, this procedure allows the use of highly virulent and aggressive races, that should be avoided in field tests for the risk of spreadin

Development and validation of a 20K Single Nucleotide Polymorphism (SNP) whole genome genotyping array for apple (Malus × domestica Borkh)

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8 K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20 K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ~3.7 K validated SNPs from the IRSC 8 K array. The array has already been used in other studies where ~15.8 K SNP markers were mapped with an average of ~6.8 K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs

Pedigree genotyping: a new pedigree-based approach of QTL identification and allele mining

To date, molecular markers are available for many economically important traits. Unfortunately, lack of knowledge of the allelic variation of the related genes hampers their full exploitation in commercial breeding programs. These markers have usually been identified in one single cross. Consequently, only one or two favourable alleles of the related QTL are identified and exploitable for marker-assisted breeding (MAB), whereas a breeding program may include several alleles. Selection for just these alleles means that many favourable genotypes are ignored, which decreases efficiency and leads to genetic erosion. A new approach, called Pedigree Genotyping, allows the identification and exploitation of most alleles present in an ongoing breeding program. This is achieved by including breeding material itself in QTL detection, thus covering multiple generations and linking many crosses through their common ancestors in the pedigree. The principle of Identity by Descent (IBD) is utilised to express the identity of an allele of a modern selection in terms of alleles of founding cultivars. These founder alleles are used as factors in statistical analyses. Co-dominant markers like SSR (microsatellite) markers are essential in this approach since they are able to connect cultivars, breeding selections and progenies at the molecular marker level by monitoring specific chromosomal segments along family trees. Additional advantages of the use of breeding genetic material are (1) a major reduction in experimental costs since plant material is already available and phenotyped by default (2) continuity over generations within breeding programs with regard to marker research (3) the testing of QTL-alleles against a wide range of genetic backgrounds, making results generally applicable, (4) intra- as well as inter-QTL interactions can be explored. Fruit firmness in apple will be used as an example to illustrate the principles of this powerful approach to detect QTLs and estimate their allelic variatio

A qRT-PCR assay for the expression of all Mal d 1 isoallergen genes

Background - A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars 'Florina' and 'Gala'. Results - We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent. Conclusion - The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy