Crop Science 43:1036-1042 (2003)
© 2003 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
The PCR-Based Marker MWG2018 Linked to the RDG2A Leaf Stripe Resistance Gene Is a Useful Tool for Assessing Barley Resistance in Breeding Programs
L. Arru,
N. Faccini,
C. Govoni,
L. Cattivelli,
N. Pecchioni,
G. Delogu,
A. M. Stanca and
G. Valè*
Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola, Via S. Protaso 302, I-29017 Fiorenzuola d'Arda (PC), Italy
* Corresponding author (gp.vale{at}iol.it)
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ABSTRACT
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Leaf stripe, caused by the fungus Pyrenophora graminea Ito et Kuribayashi [anamorph Drechslera graminea (Rabenh. ex. Schlech.) Shoemaker], is an important seed-borne disease of barley (Hordeum vulgare L.). The objective of this study was to verify the reliability of a PCR-based marker (MWG2018) associated with the resistance gene Rdg2a and to assess the leaf stripe resistant phenotype in barley breeding programs. A large number of barley cultivars and accessions were thus evaluated for their reaction to a highly virulent monoconidial isolate (Dg2) of the pathogen and genotyped for the allele of the molecular marker. Several resistant genotypes were identified and four were shown to possess the same allele as the cultivar Thibaut (the resistant parent of the original mapping population in which Rdg2a was identified) at the marker locus. One of them, cv. Rebelle, is being used as a source of leaf stripe resistance in winter barley breeding programs. The allelic composition at the MWG2018 locus was verified in several resistant lines bred from five crosses, in which Rebelle acted directly or indirectly as donor of the resistance. The results showed that the resistant phenotype of the lines was always associated with the resistance allele of the molecular marker, thereby demonstrating its reliability for selecting leaf stripe resistance. The MWG2018 PCR-based marker can therefore be proposed as a tool to assess the resistant phenotype.
Abbreviations: BCC, barley core collection • bp, base pair • CAPS, cleaved amplified polymorphic sequence • cM, centimorgan • MAS, marker-assisted selection • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • STS, sequence tagged sites
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INTRODUCTION
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LEAF STRIPE is a common disease in barley districts characterized by a cold climate during the sowing season, such as Scandinavia, Canada (spring sowing), and the Mediterranean (winter sowing). In susceptible cultivars, the disease causes brown stripes on the leaves, stunted growth, and severe yield reductions (Tekauz and Chiko, 1980; Porta-Puglia et al., 1986). The relationship between percentage of the disease in the field and percentage of yield loss varies, according to different authors, from 1:1 (Richardson et al., 1976), to 1:0.9 (Porta-Puglia et al., 1986) to 1:0.6 (Tekauz, 1983).
Both polygenic-based partial resistance (Knudsen, 1986; Skou and Haahr, 1987; Pecchioni et al., 1996) and race-specific resistance genes have been identified. A single genetic factor conferring complete resistance to barley leaf stripe was introduced into most of the resistant North-European, two-rowed barley cultivars via cv. Vada (Skou and Haahr, 1987; Skou et al., 1994). This resistance gene, named Rdg1a, has been mapped on the long arm of barley chromosome 2 (2H) (Giese et al., 1993; Thomsen et al., 1997). A new genetic factor conferring complete resistance to the highly virulent isolate Dg2 of P. graminea has recently been identified in the six-rowed cv. Thibaut. The isolate Dg2 was the most virulent among 12 tested (Gatti et al., 1992) and inoculation with it is being used to select for P. graminea resistance in barley breeding programs in Italy. Lines resistant to this isolate are also resistant to the natural field population of the pathogen, which is spread by a naturally susceptible cultivar (G. Delogu, unpublished data). This new source of resistance, designated Rdg2a, was mapped on the telomeric region of barley chromosome 1 (7H) (Tacconi et al., 2001). In the course of the Rdg2a mapping, PCR-based sequence tagged sites (STS) and cleaved amplified polymorphic sequence (CAPS) markers were developed from the sequence of linked restriction fragment length polymorphism (RFLP) markers. The STS marker MWG2018 was mapped 2.5 centimorgans (cM) proximal from Rdg2a. Given this linkage relationship, this marker potentially represents a useful tool for selecting genotypes with leaf stripe resistance in barley breeding programs.
The use of resistant cultivars is one of the most economical methods of controlling barley leaf stripe. The six-rowed winter cv. Rebelle carries resistance to the Italian field population of P. graminea and is used as a source of resistance in conventional breeding programs. The present study verified that the allelic composition of cv. Rebelle at the MWG2018 locus is the same as that of the resistant cv. Thibaut. The reliability of the STS marker MWG2018 to assess the leaf stripe resistant phenotype was then verified in several resistant barley lines bred from the crosses between the resistant parent Rebelle, or Rebelle-derived resistant lines, and different susceptible barley cultivars. To survey the spread of the Rdg2a resistance gene in the P. graminea-resistant genotypes, the allelic composition at the MWG2018 locus was also determined in a large number of resistant genotypes of differing origin.
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MATERIALS AND METHODS
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Plant Materials and Disease Screening
Leaf stripe-resistant lines at different generations of selfing (F4, F8, and F9 lines) derived from pedigree programs originated from the following five crosses were tested (Table 1).- F3494 (a resistant F5 line derived from Rebelle) x Balkan (susceptible); the three F4 resistant lines F6573, F6574 and F6575 were selected for analysis.
- F3494 (resistant) x Tamaris (susceptible); the two resistant F4 lines F6576 and F6577 were selected.
- F3505 (a resistant F5 line derived from Rebelle) x Po609.35 (susceptible); eight resistant F4 lines were chosen, i.e., F6571 and from F6578 to F6585.
- Rebelle (resistant) x Jaidor (susceptible); the two F8 resistant lines F3485 and F3486 were used.
- Rebelle (resistant) x F1341 (susceptible); the three F9 sister lines (obtained from single plants chosen in the F4 selfing generation) F3510.A, F3510.B (both susceptible to leaf stripe) and F3510.C (resistant to leaf stripe) were selected.
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Table 1. Crosses of leaf stripe-resistant (R) and -susceptible (S) genotypes and corresponding lines obtained after the reported number of selfing generations.
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During the selection process, the resistant–susceptible phenotype of the F2 plants was assessed after Skou and Haahr (1987) following field infection of F1 plants via a susceptible infected cultivar (Perga) acting as a spreader of the disease. Selection of single F2 resistant plants was based on the absence of leaf stripe symptoms. The assessment of the resistant phenotype during the subsequent selfing generations was performed by artificial inoculation with the monoconidial isolate Dg2 by the "sandwich method" (Pecchioni et al., 1996).
Similarly, the resistant–susceptible phenotypes of 19 barley genotypes (Table 2) and of 150 barley accessions chosen from the Barley Core Collection (BCC) (Knüpffer and van Hintum, 1995) were assessed within the frame of the EU Project "Evaluation and Conservation of Barley Genetic Resources in Europe" (http://barley.ipk-gatersleben.de; verified 5 December 2002) by artificial inoculation by the sandwich method. The test was performed with the monoconidial isolates Dg2 and Dg5 (19 barley genotypes and Rebelle-derived resistant lines F3494, F3505, F3485, F3486, and F3510C) or with the isolate Dg2 only (150 barley accessions of the barley core collection). The 150 barley accessions represented a sample of the genetic variability of the species H. vulgare around the world. The isolates Dg2 and Dg5 (formerly I2 and I5) are the most virulent of a collection of 12 monoconidial isolates tested on European barley cultivars (Gatti et al., 1992; Valè et al., 1994). A randomized complete block design with three replications, each comprising 50 plants, was used to evaluate the genotypes' levels of resistance in greenhouse.
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Table 2. Barley genotypes tested for reaction (percentages of infected plants) to P. graminea isolates Dg2 and Dg5. For each genotype the country of origin, the row type (six rowed, 6 or two rowed, 2), the kernel coverage (covered, C, or naked, N) and the allele at the MWG2018 locus (A: "Thibaut-type", B: "Mirco-type") are reported.
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STS Analysis
Genomic DNA for STS analysis was isolated from barley leaves following a standard CTAB (hexadecyltrimethylammonium bromide) protocol (Murray and Thompson 1980). STS analysis was performed with primers designed from the sequence of the RFLP marker MWG2018. This marker is known to be linked to the leaf stripe resistance gene Rdg2a at a map distance of 2.5 cM (Tacconi et al., 2001). Primer sequences and PCR conditions were as reported in Tacconi et al. (2001). Briefly, STS analysis was performed in a volume of 20 µL (1x reaction buffer, 2.0 mM MgCl2, 0.2 mM of each dNTP, 0.25 µM of each primer, 0.4 units of Taq polymerase) with 90 ng of barley genomic DNA as template. The following program was used for thermal cycling: one cycle for 2 min at 94°C; 35 cycles for 40 s at 94°C, 50 s at 65°C, 1 min and 20 s at 72°C with a final extension of 6 min at 72°C. Amplification products were size-fractionated on 5% (w/v) acrylamide gels, run in 1x TBE (100 mM Tris-HCl, 100 mM boric acid, 2 mM EDTA, pH 8.3) and stained with ethidium bromide.
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RESULTS AND DISCUSSION
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Reaction to Leaf Stripe
The screening for resistance to the P. graminea monoconidial isolates Dg2 and Dg5 identified barley genotypes showing different degrees of resistance. The response of genotypes ranged from immunity (0% infected plants) to complete susceptibility (100% infected plants) (Table 2). The cv. Rebelle, Proctor, Vada, Alf, and Rondo showed resistance to both the highly virulent isolates tested. Other genotypes were highly resistant to the isolate Dg2 but susceptible to the isolate Dg5, the degree of infected plants ranging from 50% (BCC line 803) to 80% or more (Thibaut, Haruna Nijo, Galleon).
The level of resistance to the two isolates was also evaluated in the resistant lines derived from the cv. Rebelle (Table 1). In these genotypes the resistance against the isolate Dg2 was similar to that of Rebelle. When inoculated with the isolate Dg5, the lines F3494, F3505, F3486, and F3510.C showed a level of infected plants ranging from 54 to 57%; only line F3485 maintained a level of resistance against the isolate Dg5 with 10% of infected plants, which corresponded to Rebelle's level of resistance. These results suggest that resistance to the two isolates is governed by two different genes.
Survey of Rdg2a Diffusion by Analysis of the Allelic Composition at the MWG2018 Locus in Resistant Barley Genotypes
The resistant–susceptible phenotype of 19 barley cultivars following artificial inoculation with the monoconidial isolates Dg2 and Dg5 of the fungus was determined (Table 2; Pecchioni et al., 1999). The resistant–susceptible phenotype of 150 barley accessions, originated from very different barley cultivation districts and belonging to the Barley Core Collection, was also verified by artificial inoculation with the isolate Dg2. The rates of diseased plants of the 29 (out of 150 tested) highly resistant genotypes are reported in Table 2. The results of the remaining BCC genotypes will be available on the web site of the EU Project "Evaluation and Conservation of Barley Genetic Resources in Europe" (http://barley.ipk-gatersleben.de).
The allelic composition at the MWG2018 locus was verified in both resistant and susceptible genotypes (Fig. 1A and B, Table 2). Of the 19 cultivars, only Thibaut, Rebelle, Haruna Nijo, and Galleon possess the resistance allele of the marker (respectively lanes 1, 2, 13, and 14 in Fig. 1A). Notably, cv. Haruna Nijo and Galleon showed a reaction against the isolates Dg2 and Dg5 similar to that observed in the cv. Thibaut, i.e., a high level of resistance against the first isolate and an extreme susceptibility to the latter. Interestingly, the two cultivars possess the same allele as cv. Thibaut at the MWG2018 locus; the Thibaut allele (Fig. 1A, lane 1) is in fact represented by two bands of about 500 and 560 base pairs (bp), while the "susceptible allele," originally found in the susceptible cv. Mirco (Tacconi et al., 2001), is represented by two bands of about 380 and 450 bp (Fig. 1B). In addition to the two bands of 500 and 560 bp, cv. Galleon showed other amplification products: one was about 450 bp and two others were slightly smaller than those of the susceptible allele. All the susceptible cultivars showed the alternate allele at the MWG2018 locus (Fig. 1B).
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Fig. 1. Amplification products obtained by means of the STS primers MWG2018 and size-fractionated on ethidium bromide stained 5% (w/v) acrylamide gels. In (A) amplification products of the resistant genotypes: 1 Thibaut, 2 Rebelle, 3 Onice, 4 Proctor, 5 Vada, 6 Alf, 7 Rondo, 8 Diadem, 9 S3097, 10 Platine, 11 Murino, 12 Alpha, 13 Haruna Nijo, 14 Galleon. In (B) amplification products of the susceptible genotypes: 1 Mirco, 2 Jaidor, 3 CI6944, 4 Nudinka, 5 Passport. In (C) amplification products from five resistant BCC accessions. Size markers (50-bp DNA ladder, Gibco BRL) are shown at the left in the figures.
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The same analysis was performed on the 29 BCC accessions showing the highest level of leaf stripe resistance of the 150 tested. All the resistant BCC accessions showed the band pattern of the susceptible allele (data not shown). Only BCC 803 (cv. Acuario) showed the MWG2018 resistant allele (first lane in Fig. 1C). The BCC line 803 tested for reaction to the isolate Dg5 showed susceptibility to it (Table 2) and, hence, the same response as cv. Thibaut.
That the barley genotypes Thibaut, Haruna Nijo, Galleon and Acuario showed the same pattern of resistance against the two isolates of P. graminea and possess the same allele at the MWG2018 locus raises the possibility that the resistance to leaf stripe in these genotypes is governed by the same resistance gene, although allelism tests are required for corroboration. These four genotypes are resistant to isolate Dg2 and susceptible to isolate Dg5. In addition, four (F3494, F3505, F3486, and F3510.C) of the five lines derived from cv. Rebelle have lost the Dg5 resistance carried by the parent. These two observations strongly suggest that resistance against the two isolates is conferred by different genes that can be separated by recombination. Nonetheless, the Rebelle-derived line F3485 retained the resistance to isolate Dg5 even after seven generations of selfing and selection for resistance against isolate Dg2 (starting from the F3 generation). This latter finding may mean that, although separable by recombination, the two genes are localized in the same chromosomal region. By using PCR-based molecular markers which span about 8.5 cM around the Rdg2a locus, we identified no recombinants between the alleles of Rebelle and those of the Rebelle-derived Dg2-resistant lines F3494, F3505, F3485, F3486, and F3510.C (data not shown), suggesting that resistance specificity for isolate Dg5 is not closely associated with Rdg2a.
Validation of MWG2018 as Rdg2a-Linked PCR-Based Marker
The six-rowed barley cv. Rebelle shows a high degree of resistance against the leaf stripe agent P. graminea (Table 2; Pecchioni et al., 1999) and is used as a source of resistance in Italy's breeding programs. The allelic composition of the Rebelle at the locus of the Rdg2a-linked marker MWG2018 was verified (Fig. 1A, lane 2) and the results confirmed that it has the same banding pattern as Thibaut, the cultivar originally used for Rdg2a mapping (Tacconi et al., 2001). This finding indicates that the two cultivars may have the same resistance gene and that, consequently, the STS marker MWG2018 might be used to assess leaf stripe resistance in Rebelle-derived breeding lines.
Marker-assisted selection (MAS) effectiveness with the Rdg2a-linked marker MWG2018 was investigated in the progenies of five crosses (Table 1), where the donor parents of leaf stripe resistance were either Rebelle-derived lines, namely F3494 and F3505 (Fig. 2A, B) or the cv. Rebelle itself (Fig. 2C). All the resistant parents showed the resistance allele at the MWG2018 locus. In Fig. 2A, the two susceptible parents (cv. Balkan and Tamaris) showed the alternate allele at the MWG2018 locus, whereas all five resistant F4 lines bred from these two crosses (F6573, F6574, F6575 and F6576, F6577) showed the resistant allele of the marker. In Fig. 2B, the resistant parent F3505 is heterozygous at the MWG2018 locus. This is reasonable because of the fact that F3505 is an F5 line and not all of its loci are fixed at homozygosity; the susceptible parent Po609.35 showed the susceptible marker allele. Four of the resistant F4 lines (F6571, F6579, F6580, F6584) derived from this cross were heterozygous for the marker; nevertheless, all nine selected resistant F4 lines had the resistance allele at the MWG2018 locus.
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Fig. 2. STS analysis of the marker verification lines derived from five crosses. A) Cross 1 (F3494 x Balkan): lines F6573, F6574 and F6575; Cross 2 (F3494 x Tamaris): lines F6576, F6577. B) Cross 3 (F3505 x Po609.35): lines 6571 and from 6578 to 6585. C) Cross 4 (Rebelle x Jaidor): lines F3485 and 3486; Cross 5 (Rebelle x F1341): lines F3510.A, F3510.B and F3510.C. R: resistant genotype; S: susceptible genotype. Genomic DNA was amplified with the STS primers MWG2018 and size-fractionated on ethidium bromide stained 5% acrylamide gels. Size markers (50-bp DNA ladder, Gibco BRL) are shown at the left in the figures.
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In Fig. 2C, the susceptible parent Jaidor showed the alternate allele of the marker and the two selected F8 resistant lines (F3485 and F3486) derived from the cross with the resistant Rebelle possessed the allele of the resistant parent. In the last cross, Rebelle (resistant) x F1341 (susceptible), three sister lines were analyzed (F3510.A, F3510.B, and F3510.C) and two (F3510.A and F3510.B) were susceptible to leaf stripe. They were selected because of their high yield performance, while the third line, F3510.C, was found resistant to the disease. The analysis of the allelic composition at the MWG2018 locus with the STS primers revealed that the two susceptible lines have the same band pattern as the susceptible parent F1341, while the resistant sister line F3510.C shows the allelic band pattern of the resistant parent.
MAS represents a valuable tool for leaf stripe resistance breeding because, until now, selection procedures of F2 resistant single plants have relied on field inoculations performed on F1 plants with a heavily infected susceptible genotype acting as source of the disease. The efficiency of the method thus depends on spreading the conidia on all the spikes and seeds. In more advanced selfing generations, selection procedures are instead based on the artificial inoculation of many seedlings with monoconidial isolates, followed by greenhouse screening of the resistant phenotypes. Both procedures have substantial defects ascribable to the high rate of escape from the disease of F2 field-infected plants (Skou and Haahr, 1987) and to the time-consuming assay for resistance screening performed with the sandwich method.
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CONCLUSIONS
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Before a marker linked to a given trait can be used in breeding programs, it is important to determine whether the marker can detect different alleles in resistant as compared with susceptible lines and if it can predict the resistance phenotype in additional crosses other than the one used for the original mapping (Williams et al., 1999). In the present study, the reliability of the Rdg2a-linked PCR-based marker MWG2018 to detect leaf stripe resistance was followed in five crosses where cv. Rebelle, or resistant lines derived from it, acted as donors of resistance. All the parents, resistant and susceptible, proved to possess alternate alleles at the marker locus (Fig. 2A, B, and C). For the resistant lines derived from the crosses, the marker MWG2018 correctly predicted the resistant phenotype. Furthermore, in the cross Rebelle x F1341, the marker discriminated the resistant F9 line F3510.C from the two susceptible sister lines. This PCR-based marker, which lies within 2.5 cM of the resistance gene, thus proved to be suited to MAS for the resistance of both the cv. Rebelle and of the resistant lines derived from it.
The present study also reports the identification of new sources of resistance to the most virulent isolate of P. graminea, isolate Dg2 (Table 2). A large proportion of these highly resistant genotypes have naked caryopsis, demonstrating that kernel coverage does not affect the level of resistance of barley genotypes to the disease.
The barley genotypes Thibaut, Rebelle, Haruna Nijo, Galleon, and the BCC accession 803 (cv. Acuario) represent very different barley genetic backgrounds: Thibaut and Rebelle are French six-rowed winter cultivars, the BCC accession 803 was derived from Chile, Haruna Nijo from Japan, and Galleon from Australia. These findings suggest that Rdg2a is thus widespread in different regions around the world and is carried by both six-rowed (Thibaut and Rebelle) and two-rowed (BCC803, Haruna Nijo, Galleon) genotypes. The potential availability of different barley genotypes carrying the same resistance gene may thus help to counter the narrowing of genetic diversity that would result when only a few resistant parents for which either markers and/or polymorphic markers are available (Barr et al., 2000). The resistant barley cultivars identified can therefore be used as donors of Dg2 leaf stripe resistance in breeding programs of both six- and two-rowed barleys and the PCR-based marker MWG2018 can be used for selection of the resistant phenotype.
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ACKNOWLEDGMENTS
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The authors wish to thank K.J. Williams for having provided the seed of cultivars Haruna Nijo and Galleon. This work was supported by MiPA, Project: "Protezione delle piante mediante l'uso di marcatori molecolari (PROMAR)" and it was carried out in close collaboration with EU GENRES CT-98-104.
Received for publication July 13, 2001.
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ABSTRACT
INTRODUCTION
