NSW Agriculture, Wagga Wagga Agricultural Institute, Pine Gully Road, Wagga Wagga, NSW 2650
Several traits, that otherwise involve laborious and costly procedures, have proven to be suitable candidates for marker assisted selection (MAS). The major limitation to implementation of molecular markers in the breeding program is the labour involved in DNA extraction. To enable efficient MAS, we have devised a method of DNA isolation. Grinding of leaf tissue was facilitated with chrome-plated ball bearings. This method allows a person to extract DNA from at least 200 samples in one day. The isolated DNA allows amplification of several markers currently in use in the barley breeding program of the NSW Agriculture.
Availability of rapid PCR based markers linked to different traits of interest has allowed breeders to increasingly utilise molecular markers in breeding programs. However, to ensure routine marker-assisted selection that is cost effective, it is necessary that use of such markers is linked with protocols that would allow rapid DNA isolation from the leaf tissue. Several techniques exist that allow direct amplification of plant genomic DNA from leaf, seed and root tissue (Berthomieu and Meyer, 1991; Klimyuk et al., 1993, Clancy et al., 1996; Saini, et al., 1999; Paris and Carter, 2000). These techniques, although rapid, have inherent problems of contaminants, such as polysaccharides and secondary metabolites, which indiscriminately inhibit some amplification reactions. These methods have limited use for marker-assisted selection, particularly, when large number of markers linked to various traits need to be assayed. A method for rapid DNA extraction has been modified to enable consistent PCR amplification of marker loci. The extracted DNA was used to implement PCR based Single Sequence Repeats (SSR), Cleaved Amplified Polymorphic Sequence (CAPS) and Sequence Tagged Site (STS) linked to (Al) tolerance (Raman et al., 2001), ant28 (Garvin et al., 1998), QTLs governing β-glucan, fine extract, fine/coarse difference, diastatic power and free alpha amino nitrogen (FAAN) (Karakousis et al, 2000), β-amylase (Erkkila et al, 1998), resistance to Barley Yellow Dwarf Virus (Paltridge et al, 1998; Ford et al, 1998) and Russian Wheat Aphid (Nieto-Lopez and Blake, 1994) in selected crossbred lines.
Material and Methods
Approximately 6cm leaf material was excised from either glasshouse or field grown plants, and placed inside 2ml polypropylene centrifuge tubes containing two 6.34mm diameter chrome-plated ball bearings. The samples were used either fresh after storage in 60 well racks at-80°C. DNA isolation was achieved using a modified method (Table 1). PCR reactions were performed in a final volume of 25μl that contained approximately 100ηg template DNA, 1x Red Taq (PCR buffer, 1.5 Units Red Taq DNA polymerase (Sigma Aldrich,), 0.4μM of each primer and 50μM MgCl2. CAPS marker p245 was digested with EcoRV enzyme. PCR products of high β-amylase and BYDV resistance loci were size fractionated using 3% (1% normal and 2% low melting) agarose. All products were analysed using 1.5% agarose gel except for RWA resistance marker KVI and KV2, which was analysed on 6% polyacrylamide gels.
Results and Discussion
DNA Extraction Method
To minimise time spent in breaking cell walls of individual leaf tissue samples, we have used a chrome-plated ball bearing method that has eliminated manual grinding requiring few minutes to grind 200 samples. This method provides adequate time for a person to extract 200 consistent highly purified DNA samples per day. The DNA was found to be suitable for PCR amplification using different primers and has been used for reliable assessment of several traits in different breeding populations.
Use of excess chloroform enabled us to get rid of chlorophyll and other non-polar pigments to obtain a clearer aqueous phase. Chilling of samples, soon after shaking, for 10 minutes prior to centrifugation also aided the separation of a visible whitish layer of polysaccharides at the interface of phenol-aqueous phase. The most critical and time-consuming step in the procedure, however, is transfer of the aqueous phase to a fresh tube. Normally, this step requires a considerable amount of skill to avoid trace amounts of phenol and other contaminants, such as polysaccharides entering into pipette tips along with the aqueous phase. To completely avoid any possibility of contamination, we pipette out only 350μl of aqueous phase instead of 500μl. Perhaps, automation to handle aqueous phase at this stage may allow DNA isolation from a much larger number of plants. The whole procedure requires two steps of centrifugation as against 4-5 in most similar DNA isolation protocols
To select for several malting quality attributes we have used STS based allele specific amplicons that have allowed us to select “Harrington” type homozygous and heterozygous alleles (Lee and Penner, 1997). The selected genotypes have desirable QTLs governing malting quality combined with tolerance to aluminium. However, use of allele-specific SSR markers has significantly improved MAS efficiency and has allowed us to overcome problems associated with lack of allelic variation with STS based markers. Among different allele-specific primers that show association with different malting quality traits, an STS marker based on β-amylase intron 111 sequences didn’t show polymorphism in a population of cross between good malting quality cv. Harrington and aluminium tolerant cv. Brindabella. This problem was circumvented using SSR marker HVM67.
Two STS based markers, ABG8 and KV1, KV2 on chromosomes 2 and 5 respectively of RWA resistant barleys (Lopez and Blake et al. 1994) were used to screen RWA resistant F5 lines derived from crosses between PI366449xAB200; PI366444xFranklin; PI366444xBrindabella and PI366449/AB200/AB35/Franklin (Figure 1. A).
For Al tolerance, a number of PCR based microsatellite markers closely linked with the Alp locus have been developed at NSW Agriculture (Raman et al, 2001). The locus is flanked by the microsatellite loci HVM68, Bmag 353 and Bmac 310. The microsatellite Bmag 353 was validated and implemented using an F2 population (140 plants) from a population developed from Dayton/F6 Ant28-48 cross. Large variation in allele size between Dayton and F6 Ant28-48 allowed us to assay this marker (Figure1.B).
To select lines with resistance against BYDV mediated by the Ryd2 gene, we have used two markers, Ylp and YLM, indicative of presence or absence of Ryd2 gene. A combination of both markers can be a good source to distinguish homozygous and heterozygous lines. However, Ovesna and colleagues have shown that MAS for Ryd2 resistant material in winter barley can be obtained using more tightly linked Ylp marker alone (Ovesna et al., 2000).
CAPS marker, p245, could not be used for the selection of ant28-48 gene in a doubled haploid population from a cross that involve cv. Arapiles, Caminant and Barque, or in a F2 population derived from a cross Dayton and Caminant because of lack of polymorphism. However, an EcoRV digest of the PCR amplified products has shown polymorphism between Caminant and Barque and Dayton and Caminant. This has enabled us to use this marker in the breeding program.
In summary, the protocol offers a rapid means to grind leaf material into a fine powder that ensures at least 200 samples of DNA suitable for assaying PCR based markers. Availability of various SSR, CAPS and STS markers has allowed us to use MAS more efficiently. In some of the populations, involving a range of parents, it was difficult to implement STS markers because of lack of polymorphism. SSR markers that show a high degree of genetic variability among Australian barley varieties and lines can be exploited as markers where there is allelic variation and strong linkage to the traits of interest.
The authors are thankful to NSW Agriculture and the Grains Research and Development Corporation, Australia for providing financial support for DAN352 and DAN320 projects.
1. Secure a metal sheet over the 2ml polypropylene tubes containing leaf tissue and ball bearings in the rack. Immerse the rack in liquid N2 for 2 minutes. Shake until samples turn into a fine powder. This normally takes 30 to 60secs. Leave the rack on ice to thaw before removing the cover.
2. Remove the ball bearings by swiping each tube over a heavy-duty welding magnet (FRAGRAM Tools, Australia).
3. Add 600μl of extraction buffer containing 0.05MTris, 0.3M EDTA pH 8.0, 0.9% SDS and 20μg of proteinase K. Mix for 5-10 mins at room temperature (RT).
4. Add 300μl of phenol and 500μl of chloroform. Secure the rack as in step 1. Place it on platform shaker perpendicular to the surface and shake for ½ hr to allow thorough mixing.
5. Chill the rack at –20°C for 10 minutes.
6. Spin for 5 mins at 13,000 rpm.
7. Gently transfer 350μl of supernatant into fresh 1.5ml tube. Add 1/10th vol of NaOAc pH 8.0 and 2 volumes of absolute alcohol. Mix by inverting several times.
8. Pellet the DNA by centrifuging for 8 mins. Discard the supernatant
9. Wash with 70% ethanol. Vacuum dry in desiccator for 5 mins.
10. Add 70μl of T.E (pH 8.0) containing 20μg RNAase A. Leave at RT for 1 hr before storing at -20°C.
Figure1. Amplification of isolated DNA using the method described. A. PCR amplification of ~1.6kb fragment of RWA resistance loci in doubled haploid progeny of cross between (1) PI36444 and (2) AB200. M: GeneRuler™ 1kb marker. B. PCR amplification of marker Bmag 353 linked to aluminium tolerance locus in (1) Dayton (tolerant) and (2) F6ant28-48.
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