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Rochester, United Kingdom

Fernandez-Fernandez F.,East Malling Research EMR | Padmarasu S.,Research and Innovation Center | Surbanovski N.,Research and Innovation Center | Evans K.M.,Tree Fruit Research and Extension Center | And 2 more authors.
Molecular Breeding

Certain progenies of Malling apple rootstocks (Malus pumila) have been reported to segregate for a virescent trait: leaves are chlorotic at germination or bud break but turn green as the season progresses. The M432 rootstock mapping progeny, from which a linkage map has recently been elaborated with 323 simple sequence repeat (SSR) markers and 3,069 single nucleotide polymorphism (SNP) markers, also segregates for this phenotype. In this investigation, 188 seedlings were scored and, on the basis of a 3:1 segregation, virescence was attributed to the recessive gene (vir) for which the two parents, M.27 and M.116, are heterozygous. At least seven of 28 Malling rootstocks are heterozygous for this apparently deleterious trait. With the published marker data the gene was mapped to linkage group 12, tightly flanked by the SSR CH01g12 and the SNP marker 475880474, and was located in a physical interval of 2.36 Mb on the Golden Delicious genome sequence. A PCR-based marker was developed from the SNP and along with the SSR was scored in a set of Malus rootstock accessions. The screening of this collection demonstrated that those accessions known to be heterozygous at the vir locus all carried the 152 allele of the SSR and the G allele of the SNP, whilst a virescent accession was homozygous for the alleles. The results we present here could help predict the genotype of apple rootstocks at the vir locus, assist in the fine mapping of the vir locus to identify potential candidate genes for the trait and also aid rootstock breeding. © 2013 Springer Science+Business Media Dordrecht. Source

Ward J.A.,Cornell University | Bhangoo J.,Sector 18 | Fernandez-Fernandez F.,East Malling Research EMR | Moore P.,Washington State University | And 6 more authors.
BMC Genomics

Background: Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker density, but result in some genotype errors and a large number of missing genotype values. Imputation can reduce the number of missing values and can correct genotyping errors, but current methods of imputation require a reference genome and thus are not an option for most species.Results: Genotyping by Sequencing (GBS) was used to produce highly saturated maps for a R. idaeus pseudo-testcross progeny. While low coverage and high variance in sequencing resulted in a large number of missing values for some individuals, a novel method of imputation based on maximum likelihood marker ordering from initial marker segregation overcame the challenge of missing values, and made map construction computationally tractable. The two resulting parental maps contained 4521 and 2391 molecular markers spanning 462.7 and 376.6 cM respectively over seven linkage groups. Detection of precise genomic regions with segregation distortion was possible because of map saturation. Microsatellites (SSRs) linked these results to published maps for cross-validation and map comparison.Conclusions: GBS together with genome-independent imputation provides a rapid method for genetic map construction in any pseudo-testcross progeny. Our method of imputation estimates the correct genotype call of missing values and corrects genotyping errors that lead to inflated map size and reduced precision in marker placement. Comparison of SSRs to published R. idaeus maps showed that the linkage maps constructed with GBS and our method of imputation were robust, and marker positioning reliable. The high marker density allowed identification of genomic regions with segregation distortion in R. idaeus, which may help to identify deleterious alleles that are the basis of inbreeding depression in the species. © 2013 Ward et al.; licensee BioMed Central Ltd. Source

Fernandez-Fernandez F.,East Malling Research EMR | Antanaviciute L.,East Malling Research EMR | van Dyk M.M.,University of Pretoria | Tobutt K.R.,East Malling Research EMR | And 5 more authors.
Tree Genetics and Genomes

An apple rootstock progeny raised from the cross between the very dwarfing 'M. 27' and the more vigorous 'M. 116' ('M. M. 106' × 'M. 27') was used for the construction of a linkage map comprising a total of 324 loci: 252 previously mapped SSRs, 71 newly characterised or previously unmapped SSR loci (including 36 amplified by 33 out of the 35 novel markers reported here), and the self-incompatibility locus. The map spanned the 17 linkage groups (LG) expected for apple covering a genetic distance of 1,229.5 cM, an estimated 91% of the Malus genome. Linkage groups were well populated and, although marker density ranged from 2.3 to 6.2 cM/SSR, just 15 gaps of more than 15 cM were observed. Moreover, only 17.5% of markers displayed segregation distortion and, unsurprisingly in a semi-compatible backcross, distortion was particularly pronounced surrounding the self-incompatibility locus (S) at the bottom of LG17. DNA sequences of 273 SSR markers and the S locus, representing a total of 314 loci in this investigation, were used to anchor to the 'Golden Delicious' genome sequence. More than 260 of these loci were located on the expected pseudo-chromosome on the 'Golden Delicious' genome or on its homeologous pseudo-chromosome. In total, 282.4 Mbp of sequence from 142 genome sequence scaffolds of the Malus genome were anchored to the 'M. 27' × 'M. 116' map, providing an interface between the marker data and the underlying genome sequence. This will be exploited for the identification of genes responsible for traits of agronomic importance such as dwarfing and water use efficiency. © 2012 Springer-Verlag. Source

Bai T.,Cornell University | Bai T.,Henan Agricultural University | Zhu Y.,Cornell University | Fernandez-Fernandez F.,East Malling Research EMR | And 3 more authors.
Molecular Genetics and Genomics

Tree architecture is an important, complex and dynamic trait affected by diverse genetic, ontogenetic and environmental factors. 'Wijcik McIntosh', a columnar (reduced branching) sport of 'McIntosh' and a valuable genetic resource, has been used intensively in apple-breeding programs for genetic improvement of tree architecture. The columnar growth habit is primarily controlled by the dominant allele of gene Co (columnar) on linkage group-10. But the Co locus is not well mapped and the Co gene remains unknown. To preciselymap the Co locus and to identify candidate genes of Co, a sequence-based approach using both peach and apple genomes was used to develop new markers linked more tightly to Co. Five new simple sequence repeatsmarkers were developed (C1753-3520, C18470-25831, C6536-31519, C7223-38004 and C7629-22009). The first four markers were obtained from apple genomic sequences on chromosome-10, whereas the last (C7629-22009) was from an unanchored apple contig that contains an apple expressed sequence tag CV082943, which was identified through synteny analysis between the peach and apple genomes. Genetic mapping of these fivemarkers in four F1 populations of 528 genotypes and 290 diverse columnar selections/cultivars (818 genotypes in total) delimited the Co locus in a genetic interval with 0.37% recombination between markers C1753-3520 and C7629-22009. Marker C18470-25831 co-segregates with Co in the 818 genotypes studied. The Co region is estimated to be 193 kb and contains 26 predicted gene in the 'Golden Delicious'genome. Among the 26 genes, three are putative LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) containing transcription factor genes known of essential roles in plant lateral organ development, and are therefore considered as strong candidates of Co, designated MdLBD1, MdLBD2, and MdLBD3. Although more comprehensive studies are required to confirm the function of MdLBD1-3, the present work represents an important step forward to better understand the genetic andmolecular control of tree architecture in apple. © Springer-Verlag 2012. Source

Antanaviciute L.,East Malling Research EMR | Fernandez-Fernandez F.,East Malling Research EMR | Jansen J.,Wageningen University | Banchi E.,Istituto Agrario San Michele allAdige | And 6 more authors.
BMC Genomics

Background: A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNP-based linkage map of an apple rootstock progeny.Results: Of the 7,867 Malus SNP markers on the array, 1,823 (23.2%) were heterozygous in one of the two parents of the progeny, 1,007 (12.8%) were heterozygous in both parental genotypes, whilst just 2.8% of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the 'Golden Delicious' genome sequence. A total of 311 markers (13.7% of all mapped markers) mapped to positions that conflicted with their predicted positions on the 'Golden Delicious' pseudo-chromosomes, indicating the presence of paralogous genomic regions or mis-assignments of genome sequence contigs during the assembly and anchoring of the genome sequence.Conclusions: We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the 'Golden Delicious' reference sequence will assist in the continued improvement of the genome sequence assembly for that variety. © 2012 Antanaviciute et al.; licensee BioMed Central Ltd. Source

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