A high-throughput plant DNA extraction method for marker analysis

The original publication can be found at www.springerlink.comThe use of molecular markers to improve crops depends on the availability of rapid and efficient DNA extraction methods. Here we describe a simple and inexpensive method to isolate plant DNA suitable for RFLP, AFLP, and simple sequence repeat (SSR) analysis. This procedure uses stainless steel ball bearings to grind 16 samples simultaneously using a high-speed flask shaker. The method used in routine laboratory exercises yields 120–144 DNA extractions in a day by a single person at a cost of $0.60 (AUD) per sample, doubling the throughput of conventional methods.Angelo Karakousis and Peter Langridg

A consensus map of barley integrating SSR, RFLP, and AFLP markers

A consensus map of barley combining simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) markers has been developed by combining 5 Australian barley linkage maps, Galleon × Haruna Nijo, Chebec × Harrington, Clipper × Sahara, Alexis × Sloop, and Amaji Nijo × WI2585, using the software package JOINMAP 2.0. The new consensus map consists of 700 markers, with 136 being SSRs, and has a total genetic distance of 933 cM. The consensus map order appears to be in good agreement with the Australian barley linkage maps, with the exception of a small inversion located close to the centromere of chromosome 5H. Similarly, the SSR map orders are in good agreement with SSR markers integrated into the doubled haploid linkage map of Lina × Hordeum spontaneum, Canada Park. The new consensus map provides a framework to cross examine and align partial and complete barley linkage maps using markers common to many barley maps. This map will allow researchers to rapidly and accurately select SSR markers for chromosome regions of interest for barley genetic and plant breeding studies.A. Karakousis, J. P. Gustafson, K. J. Chalmers, A. R. Barr and P. Langridg

An investigation of a rapid DNA extraction method for routine MAS in the S.A. Barley Improvement Program

A crucial, but limiting step in any MAS program is the reliable and efficient isolation of DNA . While there are numerous DNA extraction protocols and commercial DNA isolation kits available, their cost in barley breeding programs is not economically feasible. Breeders need high-through-put DNA isolation which can be performed on thousands of individuals. An alkaline method was tested for its suitability and it was found that the DNA samples are suitable for only PCR based procedures. Another limitation is that the extracted DNA needs to be processed immediately, and is not suitable for long-term storage

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F2 population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions. © 1995 Kluwer Academic Publishers

Genetic diversity in Australian wheat varieties and breeding material based on RFLP data

Restriction fragment length polymorphisms (RFLPs) have been used to characterise the genetic diversity of wheat (Triticum aestivum) germplasm. One hundred and twenty-four accessions comprising all major Australian wheat varieties and lines important for breeding purposes were assayed for RFLPs with clones of known genetic location and selected to give uniform genome coverage. The objectives of this study were to determine RFLP-based genetic similarity between accessions and to derive associations between agronomically significant traits and RFLP phenotypes. Ninety-eight probes screened against genomic DNA digested with five restriction endonucleases detected a total of 1968 polymorphic fragments. Genetic similarity (GS) calculated from the RFLP data ranged from 0.004 to 0.409 between accessions, with a mean of 0.18. Cluster analysis based on GS estimates produced four groupings that were generally consistent with available pedigree information. Comparisons of the RFLP phenotypes of accessions containing disease resistance genes present on introgressed alien segments enabled the identification of specific alleles characteristic of these regions. Associations were derived for a range of stem-rust, leaf-rust and yellow-rust resistance genes. These results suggest that RFLP analysis can be used for the characterisation and grouping of elite breeding material of wheat and RFLP profiling can identify chromosome segments associated with agronomic traits

Potential of SSR markers for plant breeding and variety identification in Australian barley germplasm

SSR markers closely linked to 18 loci that control 16 important barley traits were assessed for their applicability in Australian barley breeding programs. A panel of 40 genotypes routinely used by the South Australian Barley Improvement Program (SABIP) was used to examine the usefulness of these SSR markers for marker assisted selection (MAS). The success of monitoring a trait locus from donor to recipient lines ranged from 10 to 98%, depending on the marker. SSRs with a high polymorphic information content (PIC) value were found to be the most useful for application in MAS. The assessment also indicated that SSRs derived from genomic sequences were more successful for MAS than those designed from expressed sequence tags. A total of 130 SSR markers were screened among 2 panels of Australian barley genotypes to determine which markers would be the most useful for discriminating Australian germplasm. PIC values generated by this screening were also compared with those generated using a panel of European barley genotypes. Using ordinary correlations (parametric), rank correlations (non-parametric), and partial correlations (multi-variate), a strong association was found between the 2 Australian panels, but no or weak correlation was observed between the 2 Australian panels and the European dataset. It can therefore be concluded that PIC values generated by SSR markers screened with European genotypes cannot be used to predict the usefulness of an SSR marker for discriminating Australian genotypes. From PIC values generated in this study, 36 SSR markers have been selected for the discrimination of Australian genotypes. These markers all show high and/or consistent PIC values among Australian and European barley genotypes

Identification and mapping of a gene conferring resistance to the spot form of net blotch (Pyrenophora teres f maculata) in barley

Spot form of net blotch (SFNB) (Pyrenophora teres f maculata) is an economically damaging foliar disease of barley in many of the world’s cereal growing areas. The development of SFNB-resistant cultivars may be accelerated through the use of molecular markers. A screen for SFNB resistance in 96 lines identified four new sources of resistance, including a feed variety, ‘Galleon’, for which a fully mapped doubled haploid population was available. Segregation data indicated SFNB resistance was conferred by a single gene in the ‘Galleon’בHaruna Nijo’ cross, positioned on the long arm of chromosome 7H. This gene is designated Rpt4 and is flanked by the RFLP loci Xpsr117(D) and Xcdo673 at distances of 6.9 cM and 25.9 cM, respectively. The marker Xpsr117(D) was validated using another population segregating for Rpt4, correctly predicting SFNB resistance with more than 90% accuracy.K. J. Williams, A. Lichon, P. Gianquitto, J. M. Kretschmer, A. Karakousis, S. Manning, P. Langridge, H. Wallwor

Mapping and QTL analysis of the barley population Clipper x Sahara

A genetic linkage map consisting of 211 molecular markers has been generated using a doubled-haploid population derived from a cross between the Australian barley variety Clipper and the Algerian landrace Sahara 3771. The map was used in subsequent trait mapping studies to locate the genes conferring boron tolerance and cereal cyst nematode resistance from Sahara 3371 and to map several plant type and developmental genes. Closely linked markers to the trait loci have been identified and are now being widely implemented in Australian breeding programs