Articles
S-genotyping and genome editing to investigate the genetic basis of self-incompatibility in Citrus
Article number
1448_35
Pages
275 – 282
Language
English
Abstract
The genus Citrus shows a self-incompatibility (SI) gametophytic system that prevents self-fertilization.
The mechanism is based on a highly polymorphic S-ribonuclease (S-RNase) encoding a stylar glycoprotein able to inhibit the growth of the self-pollen tube along the style.
Several Citrus species, including pummelo, mandarin, and mandarin-like accessions show SI that coupled with parthenocarpy, ensures the production of seedless fruit, a trait highly appreciated for the fresh citrus market.
To date, 18 S-RNases have been reported in different citrus species.
Among them, the mutated S-RNase (Sm-RNase), characterized by a mutation causing the loss of SI has been reported in sweet orange and in several self-compatible accessions.
In the present study, a collection of 41 accessions belonging to different species and genera of the Rutaceae family has been characterized molecularly for the identification of S-alleles by using consensus primer-based PCR and Sanger sequencing.
The results allowed to define the S-genotype of the accessions analysed by the identification of 15 S-alleles.
Among them, the analysis has allowed to confirm S-alleles already found in other studies and to identify 5 new alleles not yet reported (Sz, Sx, Sq, Sy and Sr). The analysis also allowed to evaluate the degree of genetic variability and the phylogenetic relationships among accessions.
Concurrently, a CRISPR/Cas9 approach is underway in the self-compatible ‘Doppio Sanguigno’ sweet orange (an easy-to-transform and highly seeded genotype) by the edition of the Sm-RNase to revert its mutation and consequently restore the SI mechanism.
The CRISPR/Cas9 construct has been implemented by the addition of the citrus FLOWERING LOCUS T (CsFT) cassette to induce the early flowering phenotype and then speed up the subsequent fruit phenotypic characterization.
The results will help to understand the genetic basis of SI in citrus and will allow to plan marker-assisted breeding programmes or genome editing approaches to obtain new seedless genotypes.
The mechanism is based on a highly polymorphic S-ribonuclease (S-RNase) encoding a stylar glycoprotein able to inhibit the growth of the self-pollen tube along the style.
Several Citrus species, including pummelo, mandarin, and mandarin-like accessions show SI that coupled with parthenocarpy, ensures the production of seedless fruit, a trait highly appreciated for the fresh citrus market.
To date, 18 S-RNases have been reported in different citrus species.
Among them, the mutated S-RNase (Sm-RNase), characterized by a mutation causing the loss of SI has been reported in sweet orange and in several self-compatible accessions.
In the present study, a collection of 41 accessions belonging to different species and genera of the Rutaceae family has been characterized molecularly for the identification of S-alleles by using consensus primer-based PCR and Sanger sequencing.
The results allowed to define the S-genotype of the accessions analysed by the identification of 15 S-alleles.
Among them, the analysis has allowed to confirm S-alleles already found in other studies and to identify 5 new alleles not yet reported (Sz, Sx, Sq, Sy and Sr). The analysis also allowed to evaluate the degree of genetic variability and the phylogenetic relationships among accessions.
Concurrently, a CRISPR/Cas9 approach is underway in the self-compatible ‘Doppio Sanguigno’ sweet orange (an easy-to-transform and highly seeded genotype) by the edition of the Sm-RNase to revert its mutation and consequently restore the SI mechanism.
The CRISPR/Cas9 construct has been implemented by the addition of the citrus FLOWERING LOCUS T (CsFT) cassette to induce the early flowering phenotype and then speed up the subsequent fruit phenotypic characterization.
The results will help to understand the genetic basis of SI in citrus and will allow to plan marker-assisted breeding programmes or genome editing approaches to obtain new seedless genotypes.
Publication
Authors
S. Bennici, L. Poles, F. Salonia, C. Catalano, M. Di Guardo, G. Distefano, A. Gentile, S. La Malfa
Keywords
Sanger sequencing, S-RNase, new breeding techniques, genetic improvement, seedlessness
Groups involved
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