Dion K. Harrison

Single-site mutations in the carboxyltransferase domain of plastid acetyl-CoA carboxylase confer resistance to grass-specific herbicides

Grass weed populations resistant to aryloxyphenoxypropionate (APP) and cyclohexanedione herbicides that inhibit acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) represent a major problem for sustainable agriculture. We investigated the molecular basis of resistance to ACCase-inhibiting herbicides for nine wild oat (Avena sterilis ssp. ludoviciana Durieu) populations from the northern grain-growing region of Australia. Five amino acid substitutions in plastid ACCase were correlated with herbicide resistance: Ile-1,781-Leu, Trp-1,999-Cys, Trp-2,027-Cys, Ile-2,041-Asn, and Asp-2,078-Gly (numbered according to the Alopecurus myosuroides plastid ACCase). An allele-specific PCR test was designed to determine the prevalence of these five mutations in wild oat populations suspected of harboring ACCase-related resistance with the result that, in most but not all cases, plant resistance was correlated with one (and only one) of the five mutations. We then showed, using a yeast gene-replacement system, that these single-site mutations also confer herbicide resistance to wheat plastid ACCase: Ile-1,781-Leu and Asp-2,078-Gly confer resistance to APPs and cyclohexanediones, Trp-2,027-Cys and Ile-2,041-Asn confer resistance to APPs, and Trp-1,999-Cys confers resistance only to fenoxaprop. These mutations are very likely to confer resistance to any grass weed species under selection imposed by the extensive agricultural use of the herbicides.

Molecular taxonomic clarification of Ptilotus exaltatus and Ptilotus nobilis (Amaranthaceae)

A complete botanical key for the genus Ptilotus R. Brown ( family Amarathaceae) has not yet been published. Identifying the 100 or more Ptilotus species using morphological characters has been difficult because plants often exhibit slight morphological differences and intermediate characteristics common to several species, subspecies, varieties and forms. Ptilotus exaltatus Nees and P. nobilis ( Lindl) F. Muell share many morphological characteristics, but are classified as different species predominantly based on inflorescence colour. The current study involved a molecular phylogenetic analysis of 14 Ptilotus species using sequence data from the internal transcribed spacer ( ITS) regions ITS 1 and ITS 2 within the 18S-26S nuclear rDNA. Of the 39 accessions analysed, all except those identified as P. exaltatus and P. nobilis clustered according to their respective species based on their morphological taxonomy. In contrast, all 18 P. exaltatus and P. nobilis accessions formed a distinct monophyletic clade with 99% bootstrap values and a low level of sequence variation ( GD=0.002). Taking into account the lack of reliable morphological characters for separating P. exaltatus and P. nobilis, together with the ITS sequence data showing little genetic divergence or genetic structure, we propose that P. exaltatus and P. nobilis are conspecific.

The effects of host defence elicitors on betacyanin accumulation in Amaranthus mangostanus seedlings

The effect of elicitors associated with host defence on betacyanin accumulation in Amaranthus mangostanus seedlings was investigated. Under the conditions of the experiments, betacyanin accumulation was generally enhanced by light. Methyl jasmonate (MeJA) treatment increased betacyanin synthesis in a concentration-dependent response. Seedlings treated with ethylene as 5mM Ethephon also had elevated levels of betacyanin. In contrast, salicylic acid (SA) and H(2)O(2) treatments had no influence on betacyanin contents in light or dark. Combined MeJA with Ethephon or H(2)O(2) had an additive effect on betacyanin accumulation in dark-grown seedlings. However, a decline was recorded in light-grown seedlings. Moreover, an antagonistic effect on betacyanin synthesis was found when MeJA and SA were added simultaneously. Our results indicate that betacyanin content in A. mangostanus seedlings can be upregulated by MeJA and ethylene. Both additive and antagonistic effects in regulating betacyanin synthesis in A. mangostanus seedlings were observed between MeJA and other elicitors.

Characterisation of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species

Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae). RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD). In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II). PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene family evolution.

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

BackgroundThe purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls.MethodsGenomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (Hpa II). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates.ResultsApproximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P ≤ 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P ≤ 0.05).ConclusionsThe powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the Hpa II locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.

Analysis of genetic diversity in Cassia brewsteri with Randomly Amplified DNA Fingerprints (RAFs)

Genetic diversity in Cassia brewsteri (F. Muell.) F. Muell. ex Benth. was assessed with Randomly Amplified DNA Fingerprints (RAFs). Thirty accessions of C. brewsteri collected from throughout its natural distribution were analysed with three random decamer primers, along with three accessions of C. tomentella (Benth.) Domin and a single accession of each of C. queenslandica C. T. White and C. marksiana (F. M. Bailey) Domin. The three primers yielded a reproducible amplification profile of 265 scorable polymorphic fragments for the 35 accessions. These molecular markers were used to calculate Nei and Li similarity coefficients between each pair of individuals. A matrix of dissimilarity of each pair of individuals was examined by multidimensional scaling (MDS). The analysis supports the division of C. brewsteri into two subspecies and the suggestion that intergradation of C. brewsteri and C. tomentella can occur where the distributions of these species meet.

Low genetic diversity in the ground parrot (Pezoporus wallicus) revealed by randomly amplified DNA fingerprinting

The ground parrot (Pezoporus wallicus) is a vulnerable species that occurs in isolated pockets of heathland and sedgeland of Australia. This study used randomly amplified DNA fingerprinting (RAF) to examine genetic diversity in the eastern population of the ground parrot. The seven primers used produced an average of 68 markers per primer, and the number of unambiguous polymorphic markers per primer averaged 6.3 (9.2%). Overall genetic similarity was 0.978 ± 0.03. The low level of genetic diversity revealed by RAF is comparable to the lower end of diversity found in species that are declared endangered.

Stringent programming of DNA methylation in humans

We describe a PCR-based method called Amplified Methylation Polymorphism (AMP) for scanning genomes for DNA methylation changes. AMP detects tissue-specific DNA methylation signatures often representing junctions between methylated and unmethylated DNA close to intronexon junctions and/or associated with CpG islands. Identical AMP profiles are detected for healthy, young, monozygotic twins.

Reproductive biology and intergeneric breeding compatibility of ornamental Portulaca and Calandrinia (Portulacaceae)

Portulaca grandiflora Hook and P. umbraticola Kunth (Portulacaceae) are popular garden annuals, and have been bred for improved ornamental value. However, limited research has been published on hybridisation of Portulaca, with no reports on intergeneric hybridisation. Calandrinia balonensis Lindley and Calandrinia sp. nov. (not yet fully classified) are floriferous Australian Portulacaceae species, with potential as novel flowering pot plants, and are potential candidates for breeding with ornamental Portulaca. We studied the reproductive biology of these four species and breeding compatibility for reciprocal crosses of P. grandiflora × C. balonensis (2n = 18) and P. umbraticola × C. sp. nov. (2n = 24). All four species produced seeds for intraspecific outcrosses. P. grandiflora and C. sp. nov. are partially self-compatible whereas P. umbraticola and C. balonensis are highly self-incompatible. Autogamy was detected only for P. grandiflora. Reciprocal crosses of P. grandiflora × C. balonensis and P. umbraticola × C. sp. nov. with similar chromosome numbers did not produce seeds, primarily because of pollen–pistil incompatibility that prevents pollen-tube growth within the stigmata. Methods to overcome hybridisation barriers of these species combinations need to be established to create novel products for ornamental horticulture.

Expression of FGF2 and TGFα and testis morphology during testicular hypertrophy subsequent to hemicastration in the neonatal boar

The objective was to ascertain fibroblast growth factor‐2 (FGF2), epidermal growth factor (EGF), and transforming growth factor‐α (TGFα) mRNA expression and testis morphology during accelerated testicular growth after hemicastration in the neonatal boar. On Day 10 after birth (Day 0), boars were assigned to control (n = 28), no treatment; hemicastrated (n = 28), left testis removed. The right testis in both groups (n = 7) was removed on Days 5, 10, 15, and 20. Expression of mRNA for FGF2, EGF, and TGFα was determined by qRT‐PCR using TaqMan®. Testicular morphology was determined on Day 15. On Day 10, hemicastrated boars had a greater (P = 0.01) testis weight (6.2 ± 0.8 g; mean ± SEM) than controls (4.3 ± 0.4 g) and on Day 15 testis weight in hemicastrated boars (8.8 ± 0.8 g) was twice (P < 0.01) that of control boars (4.2 ± 0.3 g). Seminiferous tubule volume was approximately doubled in hemicastrated boars (P < 0.01) and was associated with an increase (P < 0.01) in Sertoli cell number. Interstitial compartment volume was greater (P < 0.01) in hemicastrated boars. Leydig cell numbers were similar (P = 0.14) but volume was greater (P < 0.01) for hemicastrates. There were no differences (P > 0.05) between control and hemicastrated boars in TGFα or FGF2 expression on Day 5 or Day 10, and EGF was not detected. It was concluded that upregulation of TGFα or FGF2 expression is not a pre‐requisite for enhanced testicular growth and increased Sertoli cell proliferation that occurs subsequent to hemicastration in the neonatal boar. Mol. Reprod. Dev. 75: 961–966, 2008.