Barley Genetics Newsletter Vol. 28 RAPD-markers linked to the BaMMV-resistance gene ym9

A. Schiemann1, E. Bauer2, A. Graner3, W. Friedt1 and F. Ordon1

1Institute of Crop Science and Plant Breeding I, Ludwigstr. 23, D-35390 Giessen

2Institute for Resistance Genetics, Graf-Seinsheim-Str. 23, D-85461 Grünbach

3Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben

Due to a constant spread and high yield losses frequently observed in susceptible winter barley crops, barley yellow mosaic virus disease - caused by a complex of at least three viruses, i.e., barley mild mosaic virus (BaMMV), barley yellow mosaic virus (BaYMV) and BaYMV-2 (cf. Huth 1990, Huth & Adams 1990) - is a serious threat to winter barley cultivation in Europe. Due to transmission of these viruses by the soil-borne fungus Polymyxa graminis (Toyama & Kusaba 1970) chemical measures against the disease are neither efficient nor acceptable for ecological and economical reasons. Growing resistant cultivars is the only way to ensure winter barley cultivation in the increasing area of infested fields, and breeding for resistance against the members of the barley yellow mosaic virus complex is therefore of special importance to barley breeding. In general, selection for resistance is carried out in extensive greenhouse tests using mechanical inoculation with BaMMV followed by DAS-ELISA or in time consuming field tests in later generations (F4). However, as symptom development strongly depends on the climatic conditions during spring and winter time reliable selection cannot be carried out each year. Therefore, PCR-based markers fitting the high throughput requirements in practical barley breeding programmes and facilitating efficient marker based selection procedures in early generations have to be considered as a very useful tool. In this respect different resistance genes against barley yellow mosaic virus disease have already been integrated into the RFLP-map of barley (Graner &Bauer 1993, Graner et al. 1995, Bauer et al. 1997), and RAPD-markers have been developed for some of them including ym9 (OP-C04H910 cf. Fig. 1) located on chromosome 4HL (Ordon et al. 1995, 1997, Weyen et al. 1996, Schiemann et al. 1996). However, as this RAPD-marker shows an additional fragment on resistant plants (ym9) it is not suited for marker based selection in segregating F2-populations. Therefore, further studies aimed at developing markers amplifying a fragment linked to the dominant allele encoding susceptibility, facilitating the identification of homozygous resistant plants in F2.

For this purpose F2 progeny comprising 60 lines of a cross between the BaMMV-resistant variety 'Bulgarian 347' (ym9) and the susceptible cv. 'Alraune' were used. Plants were scored for resistance in F2 using mechanical inoculation with BaMMV followed by DAS-ELISA. Ten progeny of each F2-plant were retested in F3 in order to confirm the F2-results and to differentiate susceptible plants into homozygous and heterozygous ones. Screening for RAPD-polymorphism was carried out by bulked segregant analysis (Michelmore et al. 1991) with bulks containing DNA of 11 F2-plants each. PCR reaction was performed in a volume of 25ul consisting of 25ng genomic DNA, 0.4mM dNTPs, 6mM MgCl2, 0,3uM of a random 10mer primer and 1.5U AmpliTaq DNA-polymerase Stoffel-fragment (Perkin Elmer) and its corresponding reaction buffer. PCR was conducted in a thermal cycler (TC9600, Perkin Elmer). An initial denaturation step (94deg.C/4min) was followed by 45 cycles of 94deg.C/1min, 36deg.C/1min, and 72deg.C/2min, respectively. The heating rate from 44deg.C to 72deg.C was restricted to 5deg.C/min. PCR products were separated on a 2% agarose gel (NuSieve, FMC) by electrophoresis and visualized by ethidium bromide staining and UV illumination (286 nm). Linkage analysis was carried out by using MAPMAKER (Lander et al. 1987) and crossover units were converted into map distances (cM) by applying the Kosambi function (Kosambi 1944).

Besides OP-C04H910 (cf. Bauer et al. 1997) the RAPD-markers OP-I10H1270, OP-M04H610, and OP-P08H790 showing linkage to ym9 have been identified out of 500 RAPD-primers tested in these studies (Fig. 1). Like OP-C04, primers OP-P08 and OP-I10 lead to the amplification of an additional fragment on resistant plants. Due to this reason and the quite loose linkage (about 25 cM) these markers are of limited value in practical breeding programs in contrast to OP-M04 which shows an additional fragment on susceptible plants and is well suited for screening large populations for resistance encoded by ym9 in F2, therefore. Furthermore, using both markers (OP-C04 and OP-M04) heterozygous carriers of ym9 may be identified and the offspring of these selected F2-plants may be retested in F3 in order to identify resistant ones.

However, as resistant exotic varieties are of inferior agronomic performance (Ordon & Friedt 1994) backcrossing procedures are needed in order to combine resistance with superior agronomic traits. This process may be abridged by using marker OP-C04H910, thereby identifying heterozygous carriers directly after backcrossing and avoiding the selfing step needed to detect resistant plants in conventional backcross breeding programs. Once combined with superior agronomic performance marker OP-M04H610 is an efficient tool for selecting homozygous resistant plants in F2 in future breeding programs. Therefore, these markers are well suited for application in practical barley breeding in order to broaden the genetic base of resistance to BaMMV. Furthermore, these markers offer the opportunity of a marker-assisted combination of ym9 with different resistance genes like ym4 and ym5 which are located on chromosome 3HL and tagged by different RFLP and PCR-based markers.

References

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Kosambi DD (1944) The estimation of map distances from recombination values. Ann Eugen 12: 172-175.

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Ordon F, Bauer E, Friedt W, Graner A (1995) Marker-based selection for the ym4 BaMMV-resistance gene in barley using RAPDs. Agronomie 15: 481-485.

Ordon F, Weyen J, Schiemann A, Pellio B, Bauer E, Graner A, Friedt W (1997) RAPD-based selection in breeding for resistance against soil-borne viruses in barley. In: Buerstmayer H, Ruckenbauer P (eds) Application of marker aided selection in cereal breeding programms. Book of Abstracts EUCARPIA Cereal Section, Sept 22-23 1997, Tulln Austria, pp 26-27.

Ordon F, Friedt W (1994) Agronomic traits of exotic barley germplasms resistant to soil-borne mosaic-inducing viruses. Genetic Resources and Crop Evolution 41: 43-46.

Schiemann A, Graner A, Friedt W, Ordon F (1996). Specificity enhancement of a RAPD marker linked to the BaMMV/BaYMV resistance gene ym4 by randomly added bases. Barley Genet Newsletter 26: 63-65.

Toyama A, Kusaba T (1970) Transmission of soil-borne barley yellow mosaic virus. 2. Polymyxa graminis Led. as vector. Ann Phytopath Soc Japan 36: 223-229.

Weyen J, Bauer E, Graner A, Friedt W, Ordon F (1996b) RAPD mapping of the distal portion of chromosome 3 of barley, including BAMMV/BaYMV resistance gene ym4. Plant Breeding 115: 285-287.

Figure 1. Genetic map of the distal portion of chromosome 4HL including the resistance gene ym9 based on 60 F2 plants of the cross 'Bulgarian 347' x 'Alraune', and banding pattern of the respective RAPD-markers.