Barley yellow dwarf luteoviruses (BYDV) are globally recognised as one of the most widespread and damaging diseases of cereal crops and pasture grasses (D'Arcy and Burnett 1995). A number of types of aphids transmit the BYDV. The disease causes discolouration of leaves, growth stunting due to reduced internode elongation, inhibition of leaf initiation and elongation, reduced tillering, suppressed heading, and sterility. These symptoms lead to significant yield losses which can be reduced by cultural practices such as controlling aphid populations and destruction of inoculum, and use of BYDV resistant cultivars. Breeding for disease resistance is a sustainable and environmentally friendly method of control, reducing significantly the application of pesticides and it is cost effective. A number of barley lines have been reported to possess the gene Ryd2 conferring resistance against BYDV and these have been extensively used in barley breeding programs (Schaller et al 1963; Burnett et al 1995). More than 17 cultivars with Ryd2 have been released all over the world (Burnett et al 1995). To develop resistant cultivars, efficient and reliable methods of selection of BYDV resistance are required. Conventional glasshouse screening using infected aphids is labourious and is highly influenced by environmental conditions. Recently linkage between the Ryd2 gene and molecular markers such as RFLP, AFLP based PCR, CAPS and ASPCR has been reported (Collins et al 1996; Paltridge et al 1998; Ford et al 1998). We are routinely implementing these markers in our breeding program (Raman and Read 1996, 1997). However all these protocols involve DNA extraction which is costly mainly because of the labour involved. Here we report the implementation of marker assisted selection (MAS) of Ryd2 in our breeding materials in conjunction with use of leaf tissue or sap as the template for the PCR.

Seedlings of Franklin (Ryd2 +), with the gene for resistance to BYDV, and Ryd2- genotypes AB 6, Rubin, Skiff, Kaputar, AB 200, PI 366444 and F₂/F₃ derivatives of crosses of Franklin × AB 200 and Franklin × PI 366 444 were raised in plastic trays. About 10 cm of leaves excised from 10 to 14 day - old seedlings were collected in 2 ml round bottom eppendorf tubes. Tubes containing tissues were frozen in liquid nitrogen and pulverised with disposable mortar (Eppendorf) into fine powder. DNA was extracted by following the method described by Guidet et al (1991) and was dissolved in sterile water/TE (100 mM pH 8.0). Backcross Franklin derived derivatives grown under field conditions were also used for marker assisted selection.

To enhance selection efficiency, crude DNA, leaf sap, and leaf tissue were used as templates. Crude DNA or precipitated sap was prepared by homogenising the frozen tissue with extraction buffer described by Guidet et al (1991) followed by precipitation with ethanol (70%). The pellets were air-dried and then dispersed into 30 microliters water/TE, and 2 microliters of this crude DNA was used as template. For leaf sap, leaf discs were punched into an Eppendorf tube and homogenised in 20 microliters of 0.5 N NaOH (10 microliters of NaOH/mg of tissue). The homogenate was diluted 1:25 with 100 mM Tris (pH 8.0) and 2.0 microliters of sap was used as template (dilution depends upon the maturity of the leaf and the level of homogenisation). Leaf discs (1-2 mm) were prepared and treated with 50 microliters of 0.25 M NaOH by following the protocol described by Klimyuk et al. 1993. Tissues were then vacuum infiltrated for 10 seconds and boiled for 15-30 seconds. Leaf tissues were then neutralised with 50 microliters of 0.25 M HCl and 25 microliters of 0.5 M Tris-HCl (pH 8.8). Macerated tissues were again boiled for 2 minutes and a small piece of tissue, 1/8 to 1/4th of the disc, was used as the template for PCR amplification.

The assay for selection of the Ryd2 resistant allele using PCR was performed by using 50 ng of DNA as described by Paltridge et al (1998) in 20 microliters reaction in Omni thermocycler and no mineral oil was used. Oligos (5' CAG GAG CTG GTG AAA TAG TGC CT 3' and 5' TTA AAG GGC TCC GTG AAG C 3') were synthesised on ABI 392 at University of Adelaide. After amplification, PCR products were separated on agarose/polyacrylamide gel (6%) avoiding any particulate leaf tissue. Plants having the marker for Ryd2 allele were selected and were further used in our breeding program at Wagga.

For RFLP analysis, six different restriction enzymes; EcoRV, EcoRI, Dra I, Bam HI, Hind III, Bgl II were used to evaluate the parents (Ryd2+ and Ryd2-); Franklin, AB6, Rubin, Skiff and Kaputar for polymorphism. About 8-10 micrograms of DNA was digested with these restriction enzymes. Digested DNA was electrophoresed on 0.9% agarose gel and alkaline blotted on Hybond N+ nylon membrane (Amersham) for 4 hrs. Probe BCD 828 closely linked (0.9 cM) with Ryd2 gene was labelled with 32P by random priming (Feinberg and Vogelstein 1983). Hybridisation and washings were performed as described by Bernatzky and Tanksley (1986). Polymorphic loci were used for single plant analysis to select the linked Ryd2 gene.

Both the RFLP and PCR based markers revealed polymorphism between Franklin, the resistant parent containing Ryd2 gene and susceptibles lacking the Ryd2 gene (Fig 1, 3). Among different restriction enzymes, EcoRV, Bam HI and Bgl II revealed polymorphism between Franklin and the susceptibles, AB 6, Rubin, Skiff and Kaputar, when probed with BCD 828.

For marker assisted selection, only EcoRV digests were probed. The Franklin backcrossed derivatives segregated for BCD 828 (Fig 2). These selected individuals were also screened with a PCR based marker for comparison. The results were highly consistent for both the RFLP and PCR based markers. To increase the efficiency of selection of Ryd2 genes, PCR based assay was used in the subsequent experiments. RFLP analysis is costly, laborious and involves radioisotopes and hence is not suitable for large scale screening of individuals. PCR based assays are efficient and easier.

PCR based assay also revealed polymorphism among Ryd2+ and Ryd2- genotypes. Franklin amplified a fragment of 89 nucleotides as compared to 100 nucleotides in susceptibles (Fig 3). The Ryd2 marker segregated in F₂ populations and heterozygotes- exhibiting both the alleles (+/-) could also be discriminated. Hence this marker could be used as a codominant marker to select heterozygotes in backcross progenies.

Similar results were found when crude DNA, sap and leaf tissue were used as templates instead of isolated 'purified' DNA. Crude DNA did amplify the Ryd2 allele (+/-) but results were not consistent which may be due to presence of PCR inhibitors. Leaf sap and the method using leaf tissue itself consistently amplified the desired PCR products (Fig 4). Sap extraction still involves grinding of the samples.

These methods do not involve use of any organic solvents such as phenol, chloroform, iso amy alcohol or proteinase treatment or serial centrifugations which are required in DNA extraction. Both the leaf tissue and sap templates can be re-used further for other PCR based markers of interest in

same cross and can be stored for months. In our hands, leaf tissue stored even after 8 months did amplified desired PCR products but required boiling for 2 minutes. Successful and consistent amplification using leaf sap and tissue was probably due to the treatment with NaOH leading to

extraction of nuclear DNA and probably elimination or reduction of potential inhibitors of PCR (Wang et al 1993). The use of direct leaf tissue in PCR amplifications has a number of advantages in marker assisted selection such as high efficiency and low cost of template preparation. Reproducibility is the same as with the good quality of DNA. We have extended this protocol to select other traits like resistance to Russian wheat aphid and leaf scald. We have also performed direct staining of PCR products amplified from leaf tissue with ethidium bromide and visualised the differential amplification of Ryd2 + and Ryd2- alleles, but this assay was found to be highly sensitive to contaminants like polysaccharides, proteins and RNA which bind to ethidium bromide non-specifically and give fluorescence under the UV transilluminator. It is concluded that direct leaf tissue/leaf sap can be used successfully as template for marker assisted selection and can reduce the cost of selection for traits.

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Acknowledgements

The authors are thankful to Ros Prangnell for Southerns and B. Carroll, University of Queensland for their help in optimisation of leaf tissue as template