Chromosomal Organization and Sequence Diversity of Genes Encoding Lachrymatory Factor Synthase in Allium cepa L.

Lachrymatory factor synthase (LFS) catalyzes the formation of lachrymatory factor, one of the most distinctive traits of bulb onion (Allium cepa L.). Therefore, we used LFS as a model for a functional gene in a huge genome, and we examined the chromosomal organization of LFS in A. cepa by multiple approaches. The first-level analysis completed the chromosomal assignment of LFS gene to chromosome 5 of A. cepa via the use of a complete set of A. fistulosum–shallot (A. cepa L. Aggregatum group) monosomic addition lines. Subsequent use of an F2 mapping population from the interspecific cross A. cepa × A. roylei confirmed the assignment of an LFS locus to this chromosome. Sequence comparison of two BAC clones bearing LFS genes, LFS amplicons from diverse germplasm, and expressed sequences from a doubled haploid line revealed variation consistent with duplicated LFS genes. Furthermore, the BAC-FISH study using the two BAC clones as a probe showed that LFS genes are localized in the proximal region of the long arm of the chromosome. These results suggested that LFS in A. cepa is transcribed from at least two loci and that they are localized on chromosome 5

Expression of two promoter-GUS fusion genes in Brassica oleracea var. italica: A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Lincoln University

Not available for loan without author's permissionTwo chimeric gene constructs, consisting of the β-glucuronidase (gus) gene under the control pf either the asparagus (Asparagus officinalis) asparagine synthetase (AS) promoter (AS/G), or the Antirrhinum majus myb305 promoter (MYB/G), were introduced into broccoli (Brassica oleracea var. italica), so that expression patterns of these promoters could be studied by GUS histochemical assays throughout the broccoli life-cycle and during post­harvest. Multiple regeneration and transformation experiments were conducted in an attempt to develop a high efficiency Agrobacterium tumefaciens-mediated transformation system. Regeneration experiments were conducted for three explant sources (cotyledons, light­ germinated hypocotyls and dark-germinated hypocotyls); three broccoli cultivars (Shogun (Sh), Marathon (Mt) and Green Belt (Gt)); and for three concentrations of N⁶ benzylaminopurine (BA) (1.0, 2.0 and 5.0 mg/L). The treatment interactions which significantly affected variation (p :'.5: 0.05) changed over the time course of the experiment. The culture conditions that produced the best results after 28 days of tissue culture were applied to subsequent transformation experiments. At this point the treatment interactions showing significant variation were Cultivar X Explant and [BA] X Cultivar. Sh and Mt dark­germinated hypocotyls produced a significantly higher percentage of explants regenerating shoots than other explant types (81.7% and 62.8% respectively, light-germinated 26.9% and 15.3%, cotyledons 56.5% and 13.9%). Dark-germinated hypocotyls on 2 mg/L BA regenerated significantly more shoots (63.9%) than dark grown hypocotyls on 1 mg/L BA (50% ). After 44 days of tissue culture, [BA] no longer had a significant effect on regeneration. However, explant and cultivar treatments still exerted a significant effect on the percentage of explants regenerating shoots. Transformation experiments were conducted to test the effect of a number of factors on transformation. These used A. tumefaciens strain LBA4404, two explant sources (light germinated hypocotyls, dark-germinated hypocotyls), the three broccoli cultivars used previously, two acetosyringone levels (0.0 µMIL and 5.0 µMIL), two co-cultivation dish sealants (Gladwrap and Micropore tape (3M)) and co-cultivation conditions of light vs dark. The transformation efficiency in these experiments was too low for statisical analysis, but statisical analysis was conducted on data for explant mortality and shoot regeneration. The percentage of explant mortality was shown to be affected by cultivar (Experiments 9 and 12, Appendix 7) (Sh 37 .5%, 40.6%, Mt 60.1 %, 60.6%, Gt 60.1 %, 68.1 % ) and sealant (Micropore 37.9%, Gladwrap 67.2%). Thirty-five AS/G transformants from five plant cultivars and thirty-eight MYB/G transformants from two plant cultivars were obtained. Plants from sixteen lines of each were transferred to a containment greenhouse. Transformation was confirmed using growth on selective levels of kanamycin as well as by molecular analyses. Molecular analyses involved PCR and Southern hybridisation to confirm the presence of the GUS reporter gene and the npt II selectable marker gene. GUS expression was observed in both AS/G and MYB/G derived transformants using the X-Glu (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) histochemical assay. The AS promoter was sufficient to drive high levels of transgene expression. A post-harvest increase in GUS expression was found in the leaves of transformants (1-4 months in the greenhouse), 24 hours after excision. These same plants were shown to be relatively insensitive to light regulation. Expression was shown not to be dependent on post-harvest (time-24 h), and evidence for sugar dependent down-regulation and nitrogen dependent up-regulation of asparagus AS promoter activity was obtained. The myb305 promoter was also sufficient to drive gus transgene expression in broccoli. GUS expression was observed in the sepals of mature (open) flowers, in stems at leaf axils, at the base or heal of leaf petioles when tom from the stem, in the vascular tissue of leaf petioles, at the base of cauline leaves when torn from the stem and in the leaf of one MYB/G line

Genetic diversity analysis and single-nucleotide polymorphism marker development in cultivated bulb onion based on expressed sequence tag-simple sequence repeat markers

Bulb onion (Allium cepa L.) is a globally significant crop, but the structure of genetic variation within and among populations is poorly understood. We broadly surveyed genetic variation in a cultivated onion germplasm using simple sequence repeat (SSR) markers and sequenced regions flanking expressed sequence tag (EST)-SSRs to develop single-nucleotide polymorphism (SNP) markers. Samples from 89 inbred and open-pollinated (OP) bulb onion populations of wide geographical adaptation and four related Allium L. accessions were genotyped with 56 EST-SSR and four genomic SSR markers. Multivariate analysis of genetic distances among populations resolved long-day, short-day, and Indian populations. EST-SSR markers frequently revealed two major alleles at high frequency in OP populations. The median proportion of single-locus polymorphic loci was 0.70 in OP and landrace populations compared with 0.43 in inbred lines. Resequencing of 24 marker amplicons revealed additional SNPs in 17 (68%) and five SNP assays were developed from these, suggesting that resequencing of EST markers can readily provide SNP markers for purity testing of inbreds and other applications in Allium genetics

The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis

Bulb colour is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow) and chalcones (bright yellow). Flavonoid regulation is poorly characterised in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressd and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (A. sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species