Search

One of the most promising New Plant Breeding Techniques is genome editing (also called gene editing) with the help of a programmable site-directed nuclease (SDN). In this review, we focus on SDN-1, which is the generation of small deletions or insertions (indels) at a precisely defined location in the genome with zinc finger nucleases (ZFN), TALENs, or CRISPR-Cas9. The programmable nuclease is used to induce a double-strand break in the DNA, while the repair is left to the plant cell itself, and mistakes are introduced, while the cell is repairing the double-strand break using the relatively error-prone NHEJ pathway. From a biological point of view, it could be considered as a form of targeted mutagenesis. We first discuss improvements and new technical variants for SDN-1, in particular employing CRISPR-Cas, and subsequently explore the effectiveness of targeted deletions that eliminate the function of a gene, as an approach to generate novel traits useful for improving agricultural sustainability, including disease resistances. We compare them with examples of deletions that resulted in novel functionality as known from crop domestication and classical mutation breeding (both using radiation and chemical mutagens). Finally, we touch upon regulatory and access and benefit sharing issues regarding the plants produced. [ABSTRACT FROM AUTHOR]

In the original publication of the article, Li et al. has been incorrectly cited in the following sentence “Interestingly, a complex trait par excellence, yield, was also shown to be amenable to an SDN-1 approach. Li et al. (2017) used CRISPR-Cas9 in rice to mutate the regulatory genes Gna1, DEP1, and GS3 and obtained plants with increased grain numbers, dense erect panicles plus semi-dwarf phenotype, and larger grains, respectively.” [ABSTRACT FROM AUTHOR]

Four classes of apple allergens (Mal d 1, −2, −3 and −4) have been reported. By using PCR cloning and sequencing approaches, we obtained genomic sequences of Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin) from the cvs Prima and Fiesta, the two parents of a European reference mapping population. Two copies of the Mal d 2 gene ( Mal d 2.01A and Mal d 2.01B) were identified, which primarily differed in the length of a single intron (378 or 380 nt) and in one amino acid in the signal peptide. Both Mal d 2.01A and Mal d 2.01B were mapped at identical position on linkage group 9. Genomic characterization of four Mal d 4 genes ( Mal d 4.01A and B, Mal d 4.02A and Mal d 4.03A) revealed their complete gDNA sequences which varied among genes in length from 862 to 2017 nt. They all contained three exons of conserved length: 123, 138, and 135 nt. Mal d 4.01 appeared to be duplicated in two copies and located on linkage group 9. Mal d 4.02A and Mal d 4.03A were single copy genes located on linkage group 2 and 8, respectively. [ABSTRACT FROM AUTHOR]

Fresh apples can cause birch pollen-related food allergy in northern and central European populations, primarily because of the presence of Mal d 1, the major apple allergen that is cross-reactive to the homologous and sensitizing allergen Bet v 1 from birch. Apple cultivars differ significantly in their allergenicity. Knowledge of the genetic basis of these differences would direct breeding for hypoallergenic cultivars. The PCR genomic cloning and sequencing were performed on two cultivars, Prima and Fiesta, which resulted in 37 different Mal d 1 gDNA sequences. Based on the mapping of sequence-specific molecular markers, these sequences appeared to represent 18 Mal d 1 genes. Sixteen genes were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG 16. One gene was mapped on LG 6, and one remained unmapped. According to sequence identity, these 18 genes could be subdivided into four subfamilies. Subfamilies I–III had an intron of different size that was subfamily and gene-specific. Subfamily IV consisted of 11 intronless genes. The deduced amino acid sequence identity varied from 65% to 81% among subfamilies, from 82% to 100% among genes within a subfamily, and from 97.5% to 100% among alleles of one gene. This study provides a better understanding of the genetics of Mal d 1 and the basis for further research on the occurrence of allelic diversity among cultivars in relation to allergenicity and their biological functions. [ABSTRACT FROM AUTHOR]

Non-specific lipid transfer proteins (nsLTPs) ofRosaceaefruits, such as peach, apricot, cherry, plum and apple, represent major allergens for Mediterranean atopic populations. As a first step in elucidating the genetics of nsLTPs, we directed the research reported here towards identifying the number and location of nsLTP (Mal d 3) genes in the apple genome and determining their allelic diversity. PCR cloning was initially performed on two cultivars, Prima and Fiesta, parents of a core apple mapping progeny in Europe, based on twoMal d 3sequences (AF221502 and AJ277164) in the GenBank. This resulted in the identification of two distinct sequences (representing two genes) encoding the mature nsLTP proteins. One is identical to accession AF221502 and has been namedMal d 3.01, and the other is new and has been namedMal d 3.02. Subsequent genome walking in the upstream direction and DNA polymorphism analysis revealed that these two genes are intronless and that they could be mapped on two homoeologous segments of linkage groups 12 and 4, respectively. Further cloning and sequencing of the coding and upstream region of bothMal d 3genes in eight cultivars was performed to identify allelic variation. Assessment of the deduced nsLTP amino acid sequences gave a total of two variants at the protein level forMal d 3.01and three forMal d 3.02. The consequences of our results for allergen nomenclature and the breeding of low allergenic apple cultivars are discussed. [ABSTRACT FROM AUTHOR]