The Long Ashton Legacy: Characterising United Kingdom West Country cider apples using a genotyping by targeted sequencing approach

Abstract

Societal Impact Statement The English West Country is the home of cider making, providing the region with jobs and industry, as well as cultural reference points such as Laurie Lee's Cider with Rosie. Many important cider apple varieties were developed at Long Ashton Research Station (LARS), near Bristol, UK, including 29 varieties known collectively as ‘The Girls’. After its closure, some of the knowledge and expertise acquired at Long Ashton was lost, including the pedigree of ‘The Girls’. We sampled LARS’ derived trees and, using a novel genotyping technique, rediscovered the pedigree of ‘The Girls’, ensuring that this important cider apple collection will be available for future generations. Summary Our research had two objectives: (a) record the influence of Long Ashton Research Station on the introduction of new cider apple cultivars to the UK; (b) rediscover the parentage of the cider apple cultivars known collectively as ‘The Girls’. For rapid, cost effective and accurate genotyping, we used the recently developed, medium density, single nucleotide polymorphism‐based genotyping procedure, SEQSNP®, to characterize the cultivars. We generated a medium density (1,500 markers), whole genome genotype for 245 apple cultivars that allowed us to determine the relationship between cultivars and, in so doing, rediscover the parentage of ‘The Girls’. We show that SNP genotyping is an efficient tool for the analysis of genetic diversity in cider apples and apples in general, and that the cider apple breeding programme carried out at Long Ashton Research Station made, and continues to make, a unique contribution to UK cider production.

Development of a minimal KASP marker panel for distinguishing genotypes in apple collections

“…Following an earlier study of 372 accessions with 1,300 SNP markers [ 13 ], we considered the possibility of identifying a minimum set of markers that would be sufficient to identify all cultivars studied. To achieve this, we used a bioinformatics approach ( Fig 1 ).…”

“…Numerous samples assayed in this study (372) had been genotyped previously using the SeqSNP ® protocol [ 13 ]. Given that the 21 KASP markers were designed directly from the SeqSNP markers used in that study, we expected complete concordance between the SNP profiles produced by the two assays.…”

“…Principally, these came from the National Fruit Collection at Brogdale in Kent (2,104 trees sampled) and the Welsh Collection at the National Botanic Garden of Wales in Llanarthney (143 trees samples). Samples were collected from a further 428 trees from various orchards and gardens: these included, a small number of trees from small, local orchards in Bristol, and all the samples (372) used in the study of Harper et al [ 13 ] ( S1 File : spreadsheet ‘ Samples studied ’). Based on name alone, the samples represented 2,287 different varieties: for 2,058 of these varieties apparently a single tree was sampled (these are referred to as ‘nominal singletons’); for the other varieties, again based solely on name, we had samples taken from more than one tree (often these were from different orchards or gardens).…”

“…We used a Perl script (available at https://github.com/pr0kary0te/minimalmarkers.git ) together with test data from Harper et al . [ 13 ], to identify a set of 21, highly polymorphic SNP markers capable of differentiating all 372 cultivars used in that study. The script first selects the marker with the highest minor allele frequency then evaluates all remaining markers to see which one differentiates the highest number of cultivars that were not split by the first marker.…”

“…For each of the 21 markers identified by the perl script (see above), two allele-specific forward primers and one common reverse primer were designed using the same primer sequence used in Harper et al . [ 13 ]; (primer sequences are available in S1 File : spreadsheet ‘ KASP probes ’). Genotyping was performed using the KASP ™ system (LGC Genomics) scaled for 1,536 format.…”