An efficient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions : A case study in the Prunus salicina LG3-MYB10 genes cluster
Fiol, Arnau (Centre de Recerca en Agrigenòmica)
Jurado-Ruiz, Federico (Centre de Recerca en Agrigenòmica)
López-Girona, Elena (The New Zealand Institute for Plant and Food Research Limited (Plant & Food Research))
Aranzana, Maria José (Institut de Recerca i Tecnologia Agroalimentàries)

Date:	2022
Abstract:	Background: Genome complexity is largely linked to diversification and crop innovation. Examples of regions with duplicated genes with relevant roles in agricultural traits are found in many crops. In both duplicated and non-duplicated genes, much of the variability in agronomic traits is caused by large as well as small and middle scale structural variants (SVs), which highlights the relevance of the identification and characterization of complex variability between genomes for plant breeding. Results: Here we improve and demonstrate the use of CRISPR-Cas9 enrichment combined with long-read sequencing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina). This region, which has a length from 90 to 271 kb according to the P. salicina genomes available, is associated with fruit color variability in Prunus species. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in five plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies (ONT) run, yielding 194 Mb of sequence. The enrichment was confirmed by aligning the long reads to the plum reference genomes, with a mean read on-target value of 4. 5% and a depth per sample of 11. 9x. From the alignment, 3261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed detection, at the haplotype level, of the variability in this region. Conclusions: CRISPR-Cas9 enrichment is a versatile and powerful tool for long-read targeted sequencing even on highly duplicated and/or polymorphic genomic regions, being especially useful when a reference genome is not available. Potential uses of this methodology as well as its limitations are further discussed.
Grants:	Agencia Estatal de Investigación BES-2016-079060 Agencia Estatal de Investigación PRE2019-087427 Agencia Estatal de Investigación RTI2018-100795-B-I00 Agencia Estatal de Investigación PID2021-128885OB-I00 Ministerio de Economía y Competitividad SEV-2015-0533 Ministerio de Ciencia e Innovación CEX2019-000902-S
Note:	Altres ajuts: CERCA Programme/Generalitat de Catalunya; ERDF A way of making Europe
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Published in:	Plant methods, Vol. 18 (August 2022) , art. 105, ISSN 1746-4811

DOI: 10.1186/s13007-022-00937-4
PMID: 36030243

16 p, 2.7 MB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Experimental sciences > CRAG (Centre for Research in Agricultural Genomics)
Articles > Research articles
Articles > Published articles