A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

DNA markers linked to yield, yield components, and morphological traits in autotetraploid lucerne (Medicago sativa L.)

We have mapped and identifed DNA markers linked to morphology, yield, and yield components of lucerne, using a backcross population derived from winter-active parents. The high-yielding and recurrent parent, D, produced individual markers that accounted for up to 18% of total yield over 6 harvests, at Gatton, south-eastern Queensland. The same marker, AC/TT8, was consistently identified at each individual harvest, and in individual harvests accounted for up to 26% of the phenotypic variation for yield. This marker was located in linkage group 2 of the D map, and several other markers positively associated with yield were consistently identified in this linkage group. Similarly, markers negatively associated with yield were consistently identified in the W116 map, W116 being the low-yielding parent. Highly significant positive correlations were observed between total yield and yield for harvests 1-6, and between total yield and stem length, tiller number, leaf yield/plant, leaf yield/5 stems, stem yield/plant, and stem yield/5 stems. Highly significant QTL were located for all these characters as well as for leaf shape and pubescence.

Gingerol content of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe)

Ginger (Zingiber officinale, Roscoe), a monocotyledonous, sterile cultigen, is widely used as a spice, flavoring agent, and herbal medicine. The pungency of fresh ginger is due to a series of homologous phenolic ketones of which [6]-gingerol is the major one. The gingerols are thermally unstable and can be converted to their corresponding shogaols, which are present in dried ginger, Fresh rhizomes of 17 clones of Australian ginger, including commercial cultivars and experimental tetraploid clones, were assayed by HPLC for gingerols and shogaols. [6]-Gingerol was identified as the major pungent phenolic compound in all samples, while [8]- and [10]-gingerol occurred in lower concentrations. One cultivar known as Jamaican contained the highest concentrations of all three gingerols and was the most pungent of the clones analyzed. Gingerols were stable in ethanolic solution over a 5-month period when stored at 4 degrees C. Shogaols were not identified in the extracts prepared from fresh rhizomes at ambient temperature, confirming that these compounds are not native constituents of fresh ginger, In contrast to previous findings, this study did not find significant differences in gingerol concentrations between the tetraploid clones and their parent diploid cultivar.

Essential oil composition of diploid and tetraploid clones of ginger (Zingiber officinale roscoe) grown in Australia

Ginger oil, obtained by steam distillation of the rhizome of Zingiber officinale Roscoe, is used in the beverage and fragrance industries. Ginger oil displays considerable compositional diversity, but is typically characterized by a high content of sesquiterpene hydrocarbons, including zingiberene, arcurcumene, beta-bisabolene, and beta-sesquiphellandrene. Australian ginger oil has a reputation for possessing a particular lemony aroma, due to its high content of the isomers neral and geranial, often collectively referred to as citral. Fresh rhizomes of 17 clones of Australian ginger, including commercial cultivars and experimental tetraploid clones, were steam distilled 7 weeks post-harvest, and the resulting oils were analyzed by GC-MS. The essential oils of 16 of the 17 clones, including the tetraploid clones and their parent cultivar, were found to be of substantially similar composition. These oils were characterized by very high citral levels (51-71%) and relatively low levels of the sesquiterpene hydrocarbons typical of ginger oil. The citral levels of most of these oils exceeded those previously reported for ginger oils. The neral-to-geranial ratio was shown to be remarkably constant (0.61 +/- 0.01) across all 17 clones. One clone, the cultivar Jamaican, yielded oil with a substantially different composition, lower citral content and higher levels of sesquiterpene hydrocarbons. Because this cultivar also contains significantly higher concentrations of pungent gingerols, it possesses unique aroma and flavor characteristics, which should be of commercial interest.

Utility of cotyledon and detached leaf assays for assessing root reactions of lucerne to Phytophthora root rot caused by Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne production but it can be managed through the use of resistant cultivars. Current resistance screening methods, using mature plants or post-emergence seedling assays, are costly and time consuming. The use of zoospore inoculum on detached leaves and intact cotyledons as an assay for plant resistance was assessed using genetically defined segregating populations. The detached leaf assay was a reproducible test, but this test could not be used for accurately predicting root ratings. The cotyledon tests using zoospores gave results at the population level that were indicative of the root responses of 19 cultivars and lines tested. The cotyledon reaction of individual plants also showed a strong association with root response. The cotyledon test, while not completely predictive of mature root responses, allowed the selection of Phytophthora resistant plants at a higher frequency than could be achieved by random selection.

Effect after introducing Bacillus thuringiensis gene on nitrogen metabolism in cotton

Bacillus thuringiensis (Bt) transgenic cotton has shown changes of vegetative and reproductive growth characteristics. The objective of this study was to investigate the physiological change of nitrogen metabolism that related closely to the growth in Bt cotton cultivars. The study Was undertaken on two 131 transgenic cotton cultivars and their parents, one conventional (Xingyang822) and recurrent parent (Sumian No. 9), the other a hybrid (Kumian No. 1) and female parent (Yumian No. 1), during the 2001 and 2002 growing seasons at the Yangzhou University Farm, Yangzhou, China. In the 2001 study, The results indicated that the Bt cotton cultivars were higher than their parents in leaf total nitrogen, free amino acid and soluble protein content, greater in NR and GPT activity, and lower in protease activity, during peak square and boll developing period. The biggest increase of total nitrogen was at peak boll period, which increased by 36.01 and 18.96% for Kumian No. I and Xingyang822, respectively. There were similar results for free amino acid and soluble protein content. The results showed further in 2002 study that NR activity increased dramatically at peak square and early boll open period, the biggest increase at early boll open period, with Kumian No. I and Xingyan,822 being 87.5 and 61.4% higher than their parent, respectively, the biggest increase of GPT activity was at peak boll period, with Kumian No. I and Xingyang822 being 39.1 and 29.1% higher than their parent, respectively. However, protease activity of Bt cultivars reduced significantly before flowering and early boll open period, the biggest decrease was before flowering period, with Kumian No. I being more than 30%, Xingyang822 being 26.5% at peak square period. Moreover, the boll total nitrogen content reduced sharply. The results suggest that the Bt cotton cultivars have higher intensity of leaf nitrogen metabolism than their parent, especially during square and boll development period. It is disadvantage for square development and earlier boll maturity under high nitrogen condition. The cultural practice should aim at reducing leaf nitrogen metabolic strength and keep the balance of vegetative and reproductive growth. (C) 2003 Elsevier B.V. All rights reserved.

Identification of a Golgi-localised GRIP domain protein from Arabidopsis thaliana

A family of Golgi-localised molecules was recently described in animals and fungi possessing extensive coiled regions and a short (similar to40 residues) conserved C-terminal domain, called the GRIP domain, which is responsible for their location to this organelle. Using the model plant Arabidopsis thaliana, we identified a gene (AtGRIP) encoding a putative GRIP protein. We demonstrated that the C-terminal domain from AtGRIP functions as a Golgi-targeting sequence in plant cells. Localisation studies in living cells expressing the AtGRIP fused to a DsRed2 fluorescent probe, showed extensive co-location with the Golgi marker alpha-mannosidase I in transformed tobacco protoplasts. GRIP-like sequences were also found in genomic databases of rice, maize, wheat and alfalfa, suggesting that this domain may be a useful Golgi marker for immunolocalisation studies. Despite low sequence identity amongst GRIP domains, the plant GRIP sequence was able to target to the Golgi of mammalian cells. Taken together, these data indicate that GRIP domain proteins might be implicated in a targeting mechanism that is conserved amongst eukaryotes.

Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures

Baculoviruses are a group of viruses that infect invertebrates and that have been used worldwide as a biopesticide against several insect pests of the Order Lepidoptera. In Brazil, the baculovirus Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) has been used experimentally to control S. frugiperda (Lepidoptera: Noctuidae), an important insect pest of corn (maize) fields and other crops. Baculoviruses can be produced either in insect larvae or in cell culture bioreactors. A major limitation to the in vitro production of baculoviruses is the rapid generation of mutants when the virus undergoes passages in cell culture. In order to evaluate the potential of in vitro methods of producing SfMNPV on a large-scale, we have multiplied a Brazilian isolate of this virus in cell culture. Extensive formation of few polyhedra mutants was observed after only two passages in Sf9 cells.

Genetic variation for carbon isotope discrimination in sunflower: Association with transpiration efficiency and evidence for cytoplasmic inheritance

Plants accumulate isotopes of carbon at different rates because of discrimination against C-13 relative to C-12. In plants that fix carbon by the C-3 pathway, the amount of discrimination correlates negatively with transpiration efficiency (TE) where TE is the amount of dry matter accumulated per unit water transpired. Therefore, carbon isotope discrimination (Delta) has become a useful tool for selecting genotypes with improved TE and performance in dry environments. Surveys of 161 sunflower (Helianthus spp.) genotypes of diverse origin revealed a large and unprecedented range of genetic variation for Delta (19.5-23.8parts per thousand). A strong negative genetic correlation (r(g)) between TE and Delta (r(g) = -0.87, P < 0.001) was observed in glasshouse studies. Gas exchange measurements of field grown plants indicated that Delta was strongly correlated with stomatal conductance to water vapor (g), (r(g) 0.64, P < 0.01), and the ratio of net assimilation rate (A) to g, (r(g) = 0.86, P < 0.001), an instantaneous measure of TE. Genotype CMSHA89MAX1 had the lowest TE (and highest Delta) of all genotypes tested in these studies and low yields in hybrid combination. Backcrossing studies showed that the TE of this genotype was due to an adverse effect of the MAX1 cytoplasm, which was inherited from the diploid perennial H. maximiliani Schrader. Overall, these studies suggested that there is an excellent opportunity for breeders to develop sunflower germplasm with improved TE. This can be achieved, in part, by avoiding cytoplasms such as the MAX1 cytoplasm.

Characterisation of Rhizoctonia solani isolates causing root canker of lucerne in Australia

The fungus causing Rhizoctonia root canker of lucerne in western Queensland has been characterised as a new subgroup within anastomosis group (AG 6) of Rhizoctonia solani. Isolates from two sites showed identical rDNA ITS sequence homology but could be differentiated based on DNA fingerprints. The lucerne isolates did not cause disease on wheat, indicating they are genetically different from the AG 6 subgroup that causes crater disease on wheat in South Africa. Root canker symptoms were produced on all commercial Australian cultivars of lucerne tested.

Incidence of Stagonospora meliloti and Acrocalymma medicaginis in Lucerne crowns and roots in eastern Australia, and their comparative aggressiveness to Lucerne and inheritance of reaction to S. meliloti in lucerne

The research presented indicates that lucerne crown and root rot caused by Stagonospora meliloti is prevalent in southern New South Wales, whereas Acrocalymma medicaginis is the more commonly observed pathogen in Queensland. Although both pathogens cause reddening of internal root and crown tissue of lucerne, they can be distinguished by symptomatology. S. meliloti causes a diffuse red blotching of the internal tissue accompanied by the presence of an external lesion, whereas A. medicaginis causes red streaking at the extremity of wedge-shaped, dry-rotted tissue. Inoculation of propagules of a susceptible lucerne clone indicated that S. meliloti was the more aggressive pathogen. Although A. medicaginis does not cause leaf disease, there was a strong relationship between the leaf and root reaction of clones to S. meliloti. Inheritance of resistance to S. meliloti in lucerne appeared to be conditioned by a single dominant gene, based on segregations observed in S-1 and F-1 populations, but not in a backcross population from the same family where an excess of susceptible individuals (74% v. expected of 50%) was obtained in a cross of a resistant F-1 individual to the susceptible parent. Resistance appears to be highly heritable, however, and amenable to population improvement by breeding. A conclusion of the research is that breeding for resistance to S. meliloti for lucernes to be grown in southern Australia would appear to be a worthwhile objective. Presently, no highly resistant cultivars exist anywhere in the world.

Does the cutting of lucerne (Medicago sativa) encourage the movement of arthropod pests and predators into the adjacent crop?

Lucerne (Medicago sativa) has been suggested as an ideal refuge habitat as part of an integrated pest management (IPM) program because it harbours high numbers of beneficial arthropods. Whether or not cutting of lucerne encourages the movement of these beneficials into adjacent target crops is unknown. Vacuum samples were used to determine the effects of cutting lucerne on arthropod abundance (pests and predators) within lucerne and adjacent soybean (Glycine max) crops. Vacuum-sample collections of arthropods were conducted before and after lucerne cutting on seven occasions in four fields over two seasons. In the lucerne, 10 m by 1 m strips parallel to the crop interface were sampled at 5, 10, 15, 20 and 30 m from the interface. In the soybean, 10 m of row were sampled at the same distances from the crop interface. The abundance of predators in lucerne was reduced immediately after cutting at all distances from the interface. Predator abundance in soybean did not show any change. The cutting of lucerne significantly reduced pest numbers within the lucerne but had little effect on pest abundance in the adjacent soybean. The temporal pattern in pest and predator abundance was very different for each field sampled. Generally, arthropods decreased in abundance after cutting and gradually increased as the lucerne grew back. In soybeans, arthropod numbers fluctuated regardless of the cutting of the lucerne. Cutting of lucerne alone does not guarantee movement of predators into the adjacent target crop. The presence of lucerne fields within a cropping area may have some impact on regional predator populations, and so still be useful for IPM programs, but this has yet to be tested critically.

Identifying and discriminating among lucerne cultivars using plant morphological characters

The use of morphological data obtained from field (plot test) and glasshouse trials to identify and discriminate among four Iranian and two New Zealand lucerne (Medicago sativa L.) cultivars was investigated, following guidelines established by the International Union for the Protection of New Varieties of Plants (UPOV) for cultivar registration and the Organisation for Economic Co-operation and Development (OECD) for seed certification. Data were collected for terminal leaflet length, width and ratio, angle of stem growth, date of first flowering, stem height at first flowering, flower colour, cutting recovery height, and disease scores. None of these characters were sufficient to identify or discriminate among the six cultivars. The results indicate a need to find cost-effective and efficient laboratory techniques to enhance the assessment of distinctness of lucerne cultivars (UPOV) and for determining cultivar purity for lucerne seed certification (OECD).

Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L(-/-) germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L(-/-) meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.

The effect of high temperature on the insecticidal properties of Bt Cotton

Bt transgenic cotton has not shown the same level of resistance to bollworm in China, as in other major Bt cotton growing areas of the world. The objective of this study was to investigate the effects of high temperature on the CryIA insecticidal protein content and nitrogen metabolism, in the leaf of Bt transgenic cotton. The study was undertaken on two transgenic cotton cultivars, one conventional (Xinyang 822) and the other a hybrid (Kumian No. 1), during the 2001 and 2002 growing seasons at the Yangzhou University Farm, Yangzhou, China. In the 2001 study, potted cotton plants were exposed to 37 C for 24 h under glasshouse conditions at three growth stages peak square, peak flowering and peak boll developing periods. Based on the 2001 results, in 2002 the same two cultivars were exposed to the same temperature for 48 h at two growth stages-peak flowering and boll developing periods. The results of the study indicated that the insecticidal protein content of the leaf was not significantly affected by the stress during the square and flowering periods. However, exposure to high temperature for 24h during the boll period reduced the CryIA protein content by approximately 51% in the cultivar Kumian No 1, and 30% in Xinyang 822 in the 2001 study, and by approximately 73 and 63% for 48 h with the same cultivars, respectively, in the 2002 study. Glutamic-pyruvic transaminase (GPT) activity, total free amino acid and soluble protein content, and the activity of protease in the leaf, showed relatively little change in response to high temperature in the flowering period. However, exposure to high temperature in the boll period resulted in the following changes - a reduction of GPT activity, a sharp increase in free amino acid content, a significant decrease in soluble protein content, and significant increases in the activity of protease. The results suggest that high temperature may result in the degradation of soluble protein in the leaf, with a resulting decline in the level of the toxin CryIA. It is believed that this may be the cause of the reduced efficacy of Bt cotton in growing conditions in China, where temperatures during the boll period often reach 36-40&DEG; C. © 2004 Elsevier B.V All rights reserved.