Biology subjects

Grimplet, J.

Publications and source records attributed to Grimplet, J..

3 recordsLinked to original sources

bioRxiv2024

Genome editing in almond: A CRISPR-based approach through hairy root transformation

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has revolutionized genome manipulation for crop enhancement, providing a powerful toolkit. However, the tissue culture and plant regeneration steps that are critical to the CRISPR/Cas editing framework are often challenging, especially in some woody plant species that exhibit substantial resistance to these procedures. To address this, we have developed an injection-based protocol for inducing hairy roots in almond (Prunus dulcis, syn. Prunus amygdalus), a species known for its recalcitrance to conventional transformation methods. Notably, the hairy root induction method also proved effective in almond x peach hybrids. To evaluate its utility for gene functional analysis, we combined the hairy root transformation system with CRISPR/Cas9 gene editing technology, targeting two transcription factor genes (ERF74 and GAI). Our efforts resulted in transformants with target knock-out, suggesting the potential of this genetic transformation technology as a valuable tool for future routine gene function studies in almond.

plant biology↗

bioRxiv2022

An improved reference of the grapevine genome supports reasserting the origin of the PN40024 highly-homozygous genotype

The genome sequence assembly of the diploid and highly homozygous V. vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements of the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, despite the PN40024 genome is nearly homozygous, it still contains various heterozygous regions. Taking the opportunity of the improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences and considering that several Vitis sp. genomes have recently been assembled with these approaches, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased. The number of scaffolds decreased from 2,059 to 640 and the number of N bases was reduced by 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the amount of unplaced scaffolds were reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation to date of 35,230 genes. Finally, we demonstrate that PN40024 resulted from selfings of cv. Helfensteiner (cross of cv. Pinot noir and Schiava grossa) instead of a single Pinot noir. These advances will help maintaining the PN40024 genome as a gold-standard reference also contributing in the eventual elaboration of the grapevine pangenome.

genomics↗

bioRxiv2021

Polymorphisms and gene expression in the almond IGT family are not correlated to variability in growth habit in major commercial almond cultivars

Almond breeding programs aimed at selecting cultivars adapted to intensive orchards have recently focused on the optimization of tree architecture. This multifactorial trait is defined by numerous components controlled by processes such as hormonal responses, gravitropism and light perception. Gravitropism sensing is crucial to control the branch angle and therefore, the tree habit. A gene family, denominated IGT family after a share conserved domain, has been described as involved in the regulation of branch angle in several species, including rice and Arabidopsis, and even in fruit trees like peach. Here we identified six members of this family in almond: LAZY1, LAZY2, TAC1, DRO1, DRO2, IGT-like. After analyzing their protein sequences in forty-one almond cultivars and wild species, little variability was found, pointing a high degree of conservation in this family. Gene expression was analyzed in fourteen cultivars of agronomical interest comprising diverse tree habit phenotypes. Only LAZY1, LAZY2 and TAC1 were expressed in almond shoot tips during the growing season. No relation was established between the expression profile of these genes and the tree habit. However, some insight has been gained in how LAZY1 and LAZY2 are regulated, identifying the IPA1 almond homologues and other transcription factors involved in hormonal responses as regulators of their expression. Besides, we have found various polymorphisms that could not be discarded as involved in a potential polygenic origin of regulation of architectural phenotypes. Therefore, we have established that unlike many species, IGT family genes do not play a critical role in the control of tree habit in currently commercialized almond cultivars, with other gene families contributing to the variability of these traits.

plant biology↗