Acquadro, A.

Barchi, L.

Lanteri, S.

Martina, M.

Portis, E.

English

Institutional Research Information System University of Turin

Proceedings of the LXV SIGA Annual CongressPiacenza, 6/9 September, 2022ISBN: 978-88-944843-3-5Poster Communication Abstract – 6.24 APPLICATION OF K-SEQ GENOTYPING PROTOCOL IN TWO LARGE GENOMESORNAMENTAL SPECIESMARTINA M.*, BARCHI L.*, ACQUADRO A.*, LANTERI S.*, PORTIS E.* *) DISAFA, Plant Genetics and Breeding, University of Torino, 10095Grugliasco (TO), ItalyGenotyping by sequencing, genotyping, ornamentals, big genomes, reduced-representation sequencing techniquesReduced-representation sequencing (RRS) techniques generate genome-widehigh-throughput sequencing data, making it possible to obtain a largenumber of genetic polymorphisms. Thus, they find application in molecularmarker development, population genetic studies, linkage map development andQTL mapping. The most frequently applied are restriction-site associatedDNA (RADseq) and GBS (genotyping by sequencing), which are in principleapplicable to any genetic background, suitable also for non-model species.However, being patented (SBG, Keygene; EP 2292788), they require a licensefor being used, in particular for non-academic purposes.The first example of a RRS methodology not based on enzyme restriction isthe repeat Amplification Sequencing (rAmpSeq, Buckler et al. 2016,www.biorxiv.org/content/early/2016/12/24/096628), which consists in the useof primers designed on repetitive sequences to amplify middle repetitiveregions. Starting from this concept, the K-seq protocol (Ziarsolo et al.2021, Plant Methods 17, 30) was recently developed, based on Klenowpolymerase-based PCR using sets of primers designed by analyzing k-mer setsin nonrepetitive regions. This methodology can find wide application inboth genome-orphan and well-studied species, with a high level ofrepeatability. Furthermore, the license-free nature of the techniques,combined with potentially low library preparation costs, makes K-seq avaluable option as a genotyping by sequencing technique.Persian buttercup (Ranunculus asiaticus L.; 2n=2x=16; estimated genomesize: 7.6Gb) and poppy anemone (Anemone coronaria L.; 2n=2x=16; estimatedgenome size: 9.08 Gb) are ornamental, outcrossing, perennial crops marketedboth as cut flower or potted plant. To date, the application of DNA-basedtechniques in varietal development of these species is very limited, andreference genomes have not been developed. Here we investigated the K-seqprotocol application in these two species through the development of a setof primers based on the reference genome of the closely-related Aquilegiakansuensis (Brühl). K-seq protocol was applied on 10 commercial genotypesfor R. asiaticus and 6 genotypes for A. coronaria. The pipeline was theninvestigated for optimization purposes. The results for A. coronaria werecompared with the SSR-base fingerprinting published by Martina et al.,2021. Our findings make it possible to optimize genotyping protocol in sucha complex species, potentially lowering wet-lab and sequencing costs forwide genotyping projects. 

Application of K-seq genotyping protocol in two large genomes ornamental species

https://iris.unito.it/bitstream/2318/1879041/1/Application_of_K-seq_genotyping_protocol_in_two_large_genomes_ornamental_species.pdf

Application of K-seq genotyping protocol in two large genomes ornamental species

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