European hazelnut (Corylus avellana L.) is a diploid with 11 pairs of chromosomes (2n = 2x = 22), monoecious, dichogamous and wind-pollinated. Eastern filbert blight (EFB) caused by Anisogramma anomala is a serious disease of European hazelnut (Corylus avellana) in North America, and infection leads to cankers that girdle branches and eventual tree death. Although breeding for genetic resistance is challenging it is still viewed as the most effective long-term method of disease control and pyramiding of multiple resistance genes in a single genotype is a promising approach to increase disease resistance durability. Alignment of the sequences of mapped SSR and RAPD markers and Illumina sequences of BACs with the reference 'Jefferson' genome identified four contigs (95F, 56F, 42F, and 77F) as being in the 'Gasaway' resistance region. These selected contigs were targeted to develop new SSR markers, of which 19 segregated in the mapping population and were placed on the reference linkage map.
Twelve new sources of resistance to EFB were investigated. A 1:1 segregation ratio was observed for disease response in the 12 progenies, indicating that these sources carry resistance controlled by single loci at which the dominant alleles confers resistance, and that the resistant parents are heterozygous. Based on co-segregation with mapped SSR markers, five resistance sources were assigned to LG6 in the same region as 'Gasaway' resistance [Estrella #1, Moscow #26, OSU 1181.002 (C. heterophylla 'Ogyoo'), OSU 1185.126 (Crimea) and Moscow #23]. Two of the resistance sources (Moscow #27 and Moscow #2) were assigned to LG7 and one of them (OSU 1187.101 (Holmskij) was assigned to LG2. Single nucleotide polymorphism (SNP) markers from a double digestion protocol with two restriction enzymes (XbaI and DdeI) and genotyping-by-sequencing were used to construct a high-density linkage map for the F1 mapping population from the cross of OSU 252.146 and OSU 414.062. A total of 13,773 SNPs were identified and linkage maps were constructed for each parent using the double pseudo-testcross mapping strategy. Although SNPs from the female parent (OSU 252.146) were assigned to 11 LGs, which corresponds to the haploid chromosome number of hazelnut, SNPs from the male parent (OSU 414.062) were assigned to only 10 LGs with LG2 and LG7 merged and we attribute the merger to a reciprocal translocation involving LG2 and LG7. A pseudo-linkage between markers on LG2 and LG7 segregating from the male parent OSU 414.062 was found using bioinformatic approach and the SNP markers were placed in 13 groups in male parent (OSU 414.062). The distribution of SNP markers according to their physical positions on the 11 scaffolds in the V3 genome and their positions on the linkage maps of the parents OSU 252.146 and OSU 414.062 reveals that the markers from the ApeKI and double digestion protocol show a more uniform distribution across each scaffold compared to those from Cac8I. The translocation breakpoint on scaffold #1 was estimated to be between coordinates 14.202261 Mbp and 26.024850 Mbp and on scaffold #7 between 5.913835 Mbp and 12.015229 Mbp on the ‘Jefferson’ genome sequence (V3). From a library of bacterial artificial chromosomes (BACs), engineered to clone large DNA molecules in bacterial cells, a minimum tiling path (MTP) of BACs was identified. The ends of the BACs in the MTP were sequenced and aligned with the ‘Jefferson’ reference genome. A total of 6,807 and 7,262 BAC end sequences were aligned with the V2 and V3 genomes, respectively. The aligned BAC end sequences include 53 and 74 pairs associated with the MTP of the EFB resistance and pollen-stigma incompatibility regions, respectively, in the ‘Jefferson’ genome sequence.