Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

The role of physiological understanding in plant breeding; From a breeding perspective

The role of physiological understanding in improving the efficiency of breeding programs is examined largely from the perspective of conventional breeding programs. Impact of physiological research to date on breeding programs, and the nature of that research, was assessed from (i) responses to a questionnaire distributed to plant breeders and physiologists, and (ii) a survey of literature abstracts. Ways to better utilise physiological understanding for improving breeding programs are suggested, together with possible constraints to delivering beneficial outcomes. Responses from the questionnaire indicated a general view that the contribution by crop physiology to date has been modest. However, most of those surveyed expected the contribution to be larger in the next 20 years. Some constraints to progress perceived by breeders and physiologists were highlighted. The survey of literature abstracts indicated that from a plant breeding perspective, much physiological research is not progressing further than making suggestions about possible approaches to selection. There was limited evidence in the literature of objective comparison of such suggestions with existing methodology, or of development and application of these within active breeding programs. It is argued in this paper that the development of outputs from physiological research for breeding requires a good understanding of the breeding program(s) being serviced and factors affecting its performance. Simple quantitative genetic models, or at least the ideas they represent, should be considered in conducting physiological research and in envisaging and evaluating outputs. The key steps of a generalised breeding program are outlined, and the potential pathways for physiological understanding to impact on these steps are discussed. Impact on breeding programs may arise through (i) better choice of environments in which to conduct selection trials, (ii) identification of selection criteria and traits for focused introgression programs, and (iii) identifying traits for indirect selection criteria as an adjunct to criteria already used. While many breeders and physiologists apparently recognise that physiological understanding may have a major role in the first area, there appears to be relatively Little research activity targeting this issue, and a corresponding bias, arguably unjustified, toward examining traits for indirect selection. Furthermore, research on traits aimed at crop improvement is often deficient because key genetic parameters, such as genetic variation in relevant breeding populations and genetic (as opposed to phenotypic) correlations with yield or other characters of economic importance, are not properly considered in the research. Some areas requiring special attention for successfully interfacing physiology research with breeding are discussed. These include (i) the need to work with relevant genetic populations, (ii) close integration of the physiological research with an active breeding program, and (iii) the dangers of a pre-defined or narrow focus in the physiological research.

Evaluating plant breeding strategies by simulating gene action and dryland environment effects

Functional genomics is the systematic study of genome-wide effects of gene expression on organism growth and development with the ultimate aim of understanding how networks of genes influence traits. Here, we use a dynamic biophysical cropping systems model (APSIM-Sorg) to generate a state space of genotype performance based on 15 genes controlling four adaptive traits and then search this spice using a quantitative genetics model of a plant breeding program (QU-GENE) to simulate recurrent selection. Complex epistatic and gene X environment effects were generated for yield even though gene action at the trait level had been defined as simple additive effects. Given alternative breeding strategies that restricted either the cultivar maturity type or the drought environment type, the positive (+) alleles for 15 genes associated with the four adaptive traits were accumulated at different rates over cycles of selection. While early maturing genotypes were favored in the Severe-Terminal drought environment type, late genotypes were favored in the Mild-Terminal and Midseason drought environment types. In the Severe-Terminal environment, there was an interaction of the stay-green (SG) trait with other traits: Selection for + alleles of the SG genes was delayed until + alleles for genes associated with the transpiration efficiency and osmotic adjustment traits had been fixed. Given limitations in our current understanding of trait interaction and genetic control, the results are not conclusive. However, they demonstrate how the per se complexity of gene X gene X environment interactions will challenge the application of genomics and marker-assisted selection in crop improvement for dryland adaptation.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

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.