A comparison of molecular markers for genetic analysis of macadamia

Various marker systems exist for genetic analysis of horticultural species. Isozymes were first applied to the woody perennial nut crop, macadamia, in the early 1990s. The advent of DNA markers saw the development, for macadamia, of STMS (sequence-tagged microsatellite site), RAPD (randomly amplified polymorphic DNA), and RAF (randomly amplified DNA fingerprinting). The RAF technique typically generates dominant markers, but within the dominant marker profiles, certain primers also amplify multi-allelic co-dominant markers that are suspected to be microsatellites. In this paper, we confirm this for one such marker, and describe how RAF primers can be chosen that amplify one or more putative microsatellites. This approach of genotyping anonymous microsatellite markers via RAF is designated RAMiFi (randomly amplified microsatellite fingerprinting). Several marker systems were compared for the type, amount, and cost-efficiency of the information generated, using data from published studies on macadamia. The markers were also compared for the way they clustered a common set of accessions. The RAMiFi approach was identified as the most efficient and economical. The availability of such a versatile tool offers many advantages for the genetic characterisation of horticultural species.

Using molecular markers to assess the effect of introgression on quantitative attributes of common bean in the Andean gene pool

Progress in bean breeding programs requires the exploitation of genetic variation that is present among races or through introgression across gene pools of Phaseolus vulgaris L. Of the two major common bean gene pools, the Andean gene pool seems to have a narrow genetic base, with about 10% of the accessions in the CIAT core collection presenting evidence of introgression. The objective of this study was to quantify the degree of spontaneous introgression in a sample of common bean landraces from the Andean gene pool. The effects of introgression on morphological, economic and nutritional attributes were also investigated. Homogeneity analysis was performed on molecular marker data from 426 Andean-type accessions from the primary centres of origin of the CIAT common bean core collection and two check varieties. Quantitative attribute diversity for 15 traits was studied based on the groups found from the cluster analysis of marker prevalence indices computed for each accession. The two-group summary consisted of one group of 58 accessions (14%) with low prevalence indices and another group of 370 accessions (86%) with high prevalence indices. The smaller group occupied the outlying area of points displayed from homogeneity analysis, yet their geographic origin was widely distributed over the Andean region. This group was regarded as introgressed, since its accessions displayed traits that are associated with the Middle American gene pool: high resistance to Andean disease isolates but low resistance to Middle American disease isolates, low seed weight and high scores for all nutrient elements. Genotypes generated by spontaneous introgression can be helpful for breeders to overcome the difficulties in transferring traits between gene pools.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

Molecular introduction to head and neck cancer (HNSCC) carcinogenesis

Of all human cancers, HNSCC is the most distressing affecting pain, disfigurement, speech and the basic survival functions of breathing and swallowing. Mortality rates have not significantly changed in the last 40 years despite advances in radiotherapy and surgical treatment. Molecular markers are currently being identified that can determine prognosis preoperatively by routine tumour biopsy Leading to improved management of HNSCC patients. The approach could help decide which early stage patient should have adjuvant neck dissection and radiotherapy, and whether Later stage patients with operable lesions would benefit from resection and reconstructive surgery or adopt a conservative approach to patients with poor prognosis regardless of treatment. In the future, understanding these basic genetic changes in HNSCC would be important for the management of HNSCC. (C) 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.

Markers associated with stalk number and suckering in sugarcane colocate with tillering and rhizomatousness QTLs in sorghum

Two important factors influencing sugar yield, the primary focus of sugarcane plant breeding programs, are stalk number and suckering. Molecular markers linked to both of these traits are sought to assist in the identification of high sugar yield, high stalk number, low-suckering sugarcane clones. In this preliminary mapping study, 108 progeny from a biparental cross involving two elite Australian sugarcane clones were evaluated at two sites for two years for both stalk number and suckering. A total of 258 DNA markers, including both restriction fragment length polymorphisms (RFLPs) and radio-labelled amplified fragments (RAFs), were scored and evaluated using single-factor analysis. Sixteen (7 RFLPs and 9 RAFs) and 14 (6 RFLPs and 8 RAFs) markers were identified that were significantly associated (P < 0.01) with stalk number and suckering, respectively, across both years and sites. The seven and six RFLP markers associated with stalk number and suckering, respectively, were generated by eight different RFLP probes, of which seven had been mapped in sorghum and (or) sugarcane. Of significant interest was the observation that all seven RFLP probes could be shown to be located within or near QTLs associated with tillering and rhizomatousness in sorghum. This observation highlights the usefulness of comparative mapping between sorghum and sugarcane and suggests that the identification of useful markers for stalk number and suckering in sugarcane would be facilitated by focussing on sorghum QTLs associated with related traits.

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.

Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-1, inhibin beta A, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

Contrasting quantitative traits and neutral genetic markers for genetic resource assessment of Mesoamerican Cedrela odorata

We compared within-population variability and degree of population differentiation for neutral genetic markers (RAPDS) and eight quantitative traits in Central American populations of the endangered tree, Cedrela odorata. Whilst population genetic diversity for neutral markers (Shannon index) and quantitative traits (heritability, coefficient of additive genetic variation) were uncorrelated, both marker types revealed strong differentiation between populations from the Atlantic coast of Costa Rica and the rest of the species' distribution. The degree of interpopulation differentiation was higher for RAPD markers (F-ST 0.67 for the sampled Mesoamerican range) than for quantitative traits (Q(ST) = 0.30). Hence, the divergence in quantitative traits was lower than could have been achieved by genetic drift alone, suggesting that balancing selection for similar phenotypes in different populations of this species. Nevertheless, a comparison of pair-wise estimates of population differentiation in neutral genetic markers and quantitative traits revealed a strong positive correlation (r = 0.66) suggesting that, for C. odorata, neutral marker divergence could be used as a surrogate for adaptive gene divergence for conservation planning. The utility of this finding and suggested further work are discussed.

Genetics and race variability of the lucerne - Colletotrichum trifolii pathosystem in Australia

The Australian-bred lucerne cultivars, Trifecta and Sequel, were found to possess useful levels of resistance to both Colletotrichum trifolii races 1 and 2. Race 2 has only been previously observed in the United States and surveys did not reveal its presence in Australia. Multilocus fingerprinting using random amplified polymorphic DNA (RAPDs) analysis revealed low diversity (<10% dissimilarity) within Australian C. trifolii collections, and between the Australian race 1 isolates and a US race 2 isolate. Studies on the inheritance of resistance to C. trifolii race 1 in individual clones from Trifecta and Sequel revealed the presence of 2 different genetic mechanisms. One inheritance was for resistance as a recessive trait, and the other indicated that resistance was dominant. The recessive system has never been previously reported, whereas in the US, 2 completely dominant and independent tetrasomic genes Anl and Ant have been reported to condition C. trifolii resistance. It was not possible to fit the observed segregations from our studies to a single-gene model. In contrast to US studies, clones of cv. Sequel exhibiting the recessive resistance reacted differently to spray and stem injection with C. trifolii inoculum, being resistant to the former and susceptible to the latter, providing additional evidence for the presence of a different genetic mechanism conditioning resistance to those previously reported in the US. As C. trifolii is one of the most serious diseases of lucerne worldwide, the future development of molecular markers closely linked to the dominant and recessive resistances identified in these studies, and the relationships between these resistances and Anl and Ans as determined by genetic mapping, appear to be useful areas of future study.

Weathering of hydrocarbons in mangrove sediments: testing the effects of using dispersants to treat oil spills

This field study was a combined chemical and biological investigation of the relative effects of using dispersants to treat oil spills impacting mangrove habitats. The aim of the chemistry was to determine whether dispersant affected the short- or long-term composition of a medium range crude oil (Gippsland) stranded in a tropical mangrove environment in Queensland, Australia. Sediment cores from three replicate plots of each treatment (oil only and oil plus dispersant) were analyzed for total hydrocarbons and for individual molecular markers (alkanes, aromatics, triterpanes, and steranes). Sediments were collected at 2 days, then 1, 7, 13 and 22 months post-spill. Over this time, oil in the six treated plots decreased exponentially from 36.6 +/- 16.5 to 1.2 +/- 0.8 mg/g dry wt. There was no statistical difference in initial oil concentrations, penetration of oil to depth, or in the rates of oil dissipation between oiled or dispersed oil plots. At 13 months, alkanes were >50% degraded, aromatics were similar to 30% degraded based upon ratios of labile to resistant markers. However, there was no change in the triterpane or sterane biomarker signatures of the retained oil. This is of general forensic interest for pollution events. The predominant removal processes were evaporation (less than or equal to 27%) and dissolution (greater than or equal to 56%), with a lag-phase of 1 month before the start of significant microbial degradation (less than or equal to 7%). The most resistant fraction of the oil that remained after 7 months (the higher molecular weight hydrocarbons) correlated with the initial total organic carbon content of the soil. Removal rate in the Queensland mangroves was significantly faster than that observed in the Caribbean and was related to tidal flushing. (C) 1999 Elsevier Science Ltd. All rights reserved.

Does maintaining green leaf area in sorghum improve yield under drought? I. Leaf growth and senescence

Production of sorghum [Sorghum bicolor (L.) Moench], an important cereal crop in semiarid regions of the world, is often limited by drought. When water is limiting during the grain-filling period, hybrids possessing the stay-green trait maintain more photosynthetically active leaves than hybrids not possessing this trait. To improve yield under drought, knowledge of the extent of genetic variation in green leaf area retention is required. Field studies were undertaken in north-eastern Australia on a cracking and self-mulching gray clay to determine the effects of water regime and hybrid on the components of green leaf area at maturity (GLAM). Nine hybrids varying in stay-green were grown under a fully irrigated control, postflowering water deficit, and terminal (pre- and postflowering) water deficit. Water deficit reduced GLAM by 67% in the terminal drought treatment compared with the fully irrigated control. Under terminal water deficit, hybrids possessing the B35 and KS19 sources of stay-green retained more GLAM (1260 cm(2) plant(-1)) compared with intermediate (780 cm(2) plant(-1)) and senescent (670 cm(2) plant(-1)) hybrids. RQL12 hybrids (KS19 source of stay-green) displayed delayed onset and reduced rate of senescence; A35 hybrids displayed only delayed onset. Visual rating of green leaf retention was highly correlated with measured GLAM, although this procedure is constrained by an inability to distinguish among the functional mechanisms determining the phenotype. Linking functional rather than phenotypic differences to molecular markers may improve the efficiency of selecting for traits such as stay-green.