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Modern Applied Statistics With S

Wild Crop Relatives: Genomic and Breeding Resources

TDZ (N-phenyl-N’-1,2,3-thidiazol-5-ylurea) is a substituted phenylurea compound which was developed for mechanized harvesting of cotton bolls and has now emerged as a highly efficacious bioregulant of morphogenesis in the tissue culture of many plant species. Application of TDZ induces a diverse array of cultural responses ranging from induction of callus to formation of somatic embryos. TDZ exhibits the unique property of mimicking both auxin and cytokinin effects on growth and differentiation of cultured explants, although structurally it is different from either auxins or purine-based cytokinins. A number of physiological and biochemical events in cells are likely to be influenced by TDZ, but these may or may not be directly related to the induction of morphogenic responses, and hence, the mode of action of TDZ is unknown. However, the recent approaches applied to study the morphogenic events initiated by TDZ are clearly beginning to reveal the details of a variety of underlying mechanisms. Various reports indicate that TDZ may act through modulation of the endogenous plant growth regulators, either directly or as a result of induced stress. The other possibilities include the modification in cell membranes, energy levels, nutrient uptake, or nutrient assimilation. In this review, several of these possiblities are presented and discussed in light of recently published studies on characterization of TDZ-induced morphogenic effects.

Thidiazuron : A potent regulator of in vitro plant morphogenesis

Background

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species.

Results

We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.

Conclusions

This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

/pdf/in-posidonia-oceanica-cadmium-induces-changes-in-dna-3cu7uj83mr.pdf

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

Haploids are plants (sporophytes) that contain a gametic chromosome number (n). They can originate spontaneously in nature or as a result of various induction techniques. Spontaneous development of haploid plants has been known since 1922, when Blakeslee first described this phenomenon in Datura stramonium (Blakeslee et al., 1922); this was subsequently followed by similar reports in tobacco (Nicotiana tabacum), wheat (Triticum aestivum) and several other species (Forster et al., 2007). However, spontaneous occurrence is a rare event and therefore of limited practical value. The potential of haploidy for plant breeding arose in 1964 with the achievement of haploid embryo formation from in vitro culture of Datura anthers (Guha and Maheshwari, 1964, 1966), which was followed by successful in vitro haploid production in tobacco (Nitsch and Nitsch, 1969). Many attempts have been made since then, resulting in published protocols for over 250 plant species belonging to almost all families of the plant kingdom (reviewed in Maluszynski et al., 2003). In fact, under optimal conditions, doubled haploids (DH) have been routinely used in breeding for several decades, although their common use is still limited to selected species. There are several reasons for this. These might be categorized as biological, based on plant status (annual, biannual, perennial, authogamous, allogamous, vegetativelly propagated) and flower morphology or technical, which are the result of the feasibility and efficiency of DH induction protocol. Induction protocols substantially vary, in fact, not only among species but also among genotypes of the same species.

/pdf/haploids-and-doubled-haploids-in-plant-breeding-35v6rme0ry.pdf

Haploids and Doubled Haploids in Plant Breeding

The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis.

/pdf/dna-free-genome-editing-of-brassica-oleracea-and-b-rapa-516v0ew0x7.pdf

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes.

Twenty-eight basil accessions including six Ocimum species and six botanical varieties or cultivars of O. basilicum were studied using molecular markers, nuclear DNA content, and chromosome counting. This is the first study reporting the nuclear DNA content in the genus Ocimum. The results supported the existence of more infrageneric groups within the genus. The section Ocimum was further divided into two separate clades. The first clade contained the accessions belonging to different botanical varieties and cultivars of O. basilicum as well as O. minimum, indicating that the separate species rank of O. minimum was not justified. The second clade, comprising O. americanum, O. africanum, and two O. basilicum var. purpurascens accessions, could represent a set of allopolyploid species sharing some common parental genomes. O. tenuiflorum was the most divergent species according to genetic distance; it had the smallest genome size, organized in small chromosomes, and the lowest chromosome number. Chromosome data obtained in our research could indicate that the basic chromosome number for species belonging to section Ocimum is x = 12. This suggestion implies that species belonging to O. basilicum clade are tetraploids, while species belonging to O. americanum clade are hexaploids. It seems that the basic chromosome number for O. gratissimum could be x = 10 and for O. tenuiflorumx = 9. The differences in genome size and chromosome number among Ocimum species indicate that evolution of their genomes was accompanied by both sequence deletion/amplification and chromosome rearrangements and polyploidization.

Genetic Relations Among Basil Taxa (Ocimum L.) Based on Molecular Markers, Nuclear DNA Content and Chromosome Number

We used the Roche-454 platform to sequence from normalized cDNA libraries from each of two inbred lines of onion (OH1 and 5225). From approximately 1.6 million reads from each inbred, 27,065 and 33,254 cDNA contigs were assembled from OH1 and 5225, respectively. In total, 3,364 well supported single nucleotide polymorphisms (SNPs) on 1,716 cDNA contigs were identified between these two inbreds. One SNP on each of 1,256 contigs was randomly selected for genotyping. OH1 and 5225 were crossed and 182 gynogenic haploids extracted from hybrid plants were used for SNP mapping. A total of 597 SNPs segregated in the OH1 × 5225 haploid family and a genetic map of ten linkage groups (LOD ≥8) was constructed. Three hundred and thirty-nine of the newly identified SNPs were also mapped using a previously developed segregating family from BYG15-23 × AC43, and 223 common SNPs were used to join the two maps. Because these new SNPs are in expressed regions of the genome and commonly occur among onion germplasms, they will be useful for genetic mapping, gene tagging, marker-aided selection, quality control of seed lots, and fingerprinting of cultivars.

Borut Bohanec

Papers

Haploids and Doubled Haploids in Plant Breeding

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes.

Genetic Relations Among Basil Taxa (Ocimum L.) Based on Molecular Markers, Nuclear DNA Content and Chromosome Number

Transcriptome sequencing to produce SNP-based genetic maps of onion

Studies of gynogenesis in onion (Allium cepa L.): induction procedures and genetic analysis of regenerants