Published in Crop Sci 39:1707-1710 (1999)
© 1999 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Crop Science 39:1707-1710 (1999)
© 1999 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Molecular Mapping of Russian Wheat Aphid Resistance from Triticale Accession PI 386156
A.K. Fritza,
S. Caldwellb and
W.D. Worrallb
a Texas A&M Univ., Southern Crop Improvement Facility, College Station, TX 77843-2123 USA
b Texas A&M Univ. Research and Extension Center, P.O. Box 1658, Vernon, TX 76384 USA
afritz{at}pop.tamu.edu
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ABSTRACT
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The Russian wheat aphid (RWA), Diuraphis noxia Mordvilko, is an economic pest of wheat (Triticum aestivum L.) in some western growing regions. Sources of resistance occur within the wheat gene pool, but there are also high levels of resistance in wheat relatives, including rye (Secale cereale L.). An F2 population between PI 386156, a Russian wheat aphid-resistant triticale (xTriticosecale Wittmack) from Russia and NE88T222, a susceptible triticale, was used to examine the inheritance of resistance. Five distinct classes of reaction to the RWA were observed, suggesting this trait is not controlled by a single dominant gene in this population. Bulked segregant analysis was performed with DNA from plants representing the most resistant (`R') and most susceptible (`S') classes. A co-dominant marker amplified by primer OP-M09 explained more than 55% of the variation observed for resistance. Sequencing results revealed a deletion in the `S' parent relative to the `R' parent. Using a set of wheat-rye addition lines, we mapped the target fragment to chromosome 4R, which has previously been identified as the critical chromosome for RWA resistance from PI 386156 and other Russian triticales.
Abbreviations: RWA, Russian wheat aphid • RAPD, random amplified polymorphic DNA • PCR, polymerase chain reaction • bp, base pairs
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INTRODUCTION
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THE RUSSIAN WHEAT APHID, Diuraphis noxia, has become a serious economic pest of Triticum aestivum L. em. Thell., since its introduction to the USA in 1986 (Stoetzel, 1987). By 1991, it had caused in excess of $657 million of damage to the U.S. wheat crop (Baker et al., 1992). Among the symptoms of RWA infestation are chlorotic streaking of the leaves and leaf rolling. The leaf rolling character limits the efficacy of chemical control for this pest. The most economically and environmentally sound method of control is genetic host plant resistance. Six genes for RWA resistance have been identified in wheat (Dong et al., 1997; DuToit, 1989a; Marais and DuToit, 1993; Nkongolo et al., 1991a,b; Saidi and Quick, 1996); however, some of the most effective Russian wheat aphid resistance genes are found outside wheat's immediate gene pool. Several triticale accessions carrying RWA resistance have been identified. PI 386148, PI 386149, and PI 386156 are among the Russian triticales identified as resistant to the RWA by Nkongolo et al. (1992). They concluded resistance from each accession was conditioned by a single dominant gene and that each of the three accessions carried the same resistance gene. In subsequent cytological research, Nkongolo et al. (1996) identified chromosome 4R as the critical chromosome for RWA resistance derived from these accessions. Puterka et al. (1992) examined biotypic variation of the RWA by testing various isolates of the insect on resistant accessions of wheat, barley and triticale. They identified one isolate that demonstrated differences in virulence on PI 386148 and PI 386156. Among their conclusions was the presence of genetic complexity in the RWA resistance of PI 386156.
The objective of this research was to identify molecular marker(s) associated with RWA resistance from PI 386156. Such a marker would be useful for marker assisted selection and transfer of the gene to wheat. PI 386156 was used as the source of resistance for this research.
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Materials and methods
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The resistant parent PI 386156 was used as the female in a cross with NE88T222, a triticale line susceptible to the RWA. The F1 progeny were selfed to obtain F2 seed. Both parents, the F1 and 100 F2 plants were screened for resistance to the Russian wheat aphid following the protocols of Caldwell and Worrall (1996). Plants were grown in individual pots in a growth room under 400-W sodium lights and 14-h photoperiod. Temperature varied from 16°C (dark cycle) to 21°C during the 14-h photoperiod. Aphids were preconditioned on TX78V2290-36-1, an experimental wheat line with the pedigree TX69A509-2\\Blueboy II\Fox. This line is highly susceptible to the RWA. Four aphids were placed directly on each plant at the three leaf stage. A plant damage scale modified from DuToit (1989b) was used to characterize reaction to the RWA. Plants were rated as follows: 1 = no damage; 2 = small to intermediate chlorotic lesions; 3 = intermediate to large chlorotic lesions; 4 = slight streaking and curling in addition to chlorosis; 5 = well defined streaking and curling. Caldwell and Worrall (1996) showed that differences between classes were most clear 8 wk after infestation. Unfortunately, allowing the infestation to progress for 8 wk results in the death of plants in the more susceptible categories. To recover plants for DNA isolation, plant damage readings were taken at weekly intervals for 4 wk. The 4-wk data were used for statistical analyses. The results are given in Table 1
. In the interval from screening to DNA isolation, five plants from the most resistant class (1, 4, 28, 35, and 76), four plants from the intermediate class (31, 70, 86, and 95) and two plants from the most susceptible class (26 and 78) died and were unavailable for further analyses.
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Table 1 Summary of the F2 screening results for individual plants from the cross between the RWA resistant accession PI386156 and NE88T222, a susceptible line
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DNA was extracted from the 89 surviving F2 plants following the protocol outlined by Gill et al. (1991). The DNA was quantified using a Beckman spectrophotometer (Beckman Coulter, Inc., Fullerton, CA) and diluted to a working concentration of 10 ng/µL for subsequent PCR reactions.
We used bulked segregant analysis (Michelmore et al., 1986) with RAPD markers to identify DNA markers associated with RWA resistance. Resistant and susceptible DNA bulks were created. The resistant bulk consisted of equal amounts of DNA from plants 5, 16, 43, 44, 46, 48, 51, 61, and 64 (Table 1). The susceptible bulk contained DNA from each of the nine surviving susceptible plants (8, 10, 14, 22, 23, 30, 34, 67, and 91). Ten-mer primers were obtained from Operon Technologies (Sunnyvale, CA). The RAPD-PCR procedures followed those developed for Sorghum bicolor (L.) Moench by Pammi et al. (1994). All PCR reactions were performed with a Perkin-Elmer Applied Biosystems (Foster City, CA) 9600 PCR machine.
The resulting PCR products were separated on 5 or 6% (w/v) denaturing polyacrylamide gels. DNA fragments were detected by silver staining on the basis of the method of Bassam et al. (1991), with modifications. Prior to pouring the gel, the small glass plate was treated with 3.5 mL of silanizing solution (995 mL L-1 ethanol, 5mL L-1 glacial acetic acid, 1 µL L-1 
-methacryloxypropyltrimehtoxysilane) (Laywood et al., 1994). The solution was evenly dispersed over the plate with a small piece of Parafilm (American Can Co., Greenwich, CT) until the solution had evaporated. The plate was rinsed with distilled water, then with 95% (v/v) ethanol, before pouring the gel. All procedures of Bassam et al. (1991) were followed through the development step. After development, the gel was placed directly in the 10% (v/v) acetic acid fix/stop solution for 2 min with gentle agitation. The gel was then transferred to distilled water for two minutes, before being placed in a 2% (w/v) sodium hydroxide solution for approximately 3 to 5 min or until the edges of the gel began to loosen from the plate. The gel was then gently moved to a 3% (v/v) acetic acid solution for 2 min. Following this step, excess solution was allowed to drain from the gel. The gel was removed from the plate by means of Whatman 3MM paper (Whatman, Inc., Clifton, NJ) and dried, allowing retention of the gel as the permanent record (Fritz et al., 1995).
A total of 500 RAPD primers were screened against the bulks. Primers that produced bands differentiating the resistant and susceptible bulks were tested in three independent reactions to verify the presence of the polymorphism(s). Following confirmation of polymorphism between the bulks, DNA from each individual plant was tested as the template in a PCR reaction with the primer that had produced the polymorphism. Gels were visually scored by three independent observations.
A simple regression analysis was performed with the reaction to the RWA as the independent variable and marker score as the dependent variable. The data set included information for all 89 plants from which DNA was isolated. The analyses were carried out by PROC GLM of SAS (SAS Institute, 1987), as well as the AGROBASE (Agronomix Software, Inc., Winnipeg, MB, Canada) statistical program.
The target bands were excised from the polyacrylamide gel and reamplified by means of the original primer. The PCR product was cloned using the TA Cloning Kit (Invitrogen, San Diego, CA), following the manufacturer's directions.
Plasmid DNA was isolated from selected colonies with the Qiaprep Spin Miniprep Kit (Qiagen, Chatsworth, CA). The PCR fragment was excised from the plasmid, digested with EcoRI and analyzed on a 0.8% (w/v) agarose gel to confirm that the insert was of the appropriate size. Sequencing was performed by PCR techniques with 250 ng of plasmid DNA, following the protocols of Perkin-Elmer Applied Biosystems (Foster City, CA), using fluorescent-labeled dideoxynucleotides. The PCR products were analyzed on an Applied Biosystems 373 automated DNA sequencer, following the manufacturers' specifications.
The sequence data was entered into the `Primer' program (Whitehead Institute) to design primers that would amplify the target sequence. The sequences of the forward and reverse primers were 5'-GTCTTGCGGACTCTTGAAGC-3' and 5'-TCTTGCGGATATGCCCT-3', respectively. Primers were purchased from Genosys Technologies (The Woodlands, TX).
Seed of the Chinese Spring-Imperial wheat-rye addition lines for S. cereale chromosomes 1R, 2R, 3R, 4R, 5R, 6R, and 7R was obtained from Dr. Neal Tuleen (Texas A&M University, College Station, TX). DNA was isolated from each addition line by the same isolation protocol as was used for the F2 plants. Each addition line and PI 386156 were used as templates for amplification with the fragment specific primers.
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Results and discussion
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The RWA screening results for the F2 plants are shown in Table 1. All plants of PI 386156 were given a rating of 1, while all plants of NE88T222 were given a rating of 5. The F1 plants were given a rating of 2. While they were quite resistant, small to intermediate chlorotic lesions were visible on the leaves of all F1 plants.
Of the 500 RAPD primers tested, only OP-M09 produced a fragment that clearly and consistently differentiated the resistant and susceptible bulks. This primer produced what appeared to be a co-dominant marker at approximately 220 bp and was subsequently tested on all individuals of the population. Figure 1
shows the banding pattern for all resistant and susceptible plants. Regression analysis indicated that this marker was significantly associated with Russian wheat aphid score (P < 0.001) and explained 55.6% of the variation observed. The bulks were restructured in an attempt to find markers that would explain more of the observed variation. The new `R' bulk consisted of the DNA from plants 21, 46, 50, 55, 65, 66, 89, 94, and 96. These plants from the two most resistant classes were heterozygous for the OP-M09220 marker. The new `S' bulk consisted of the DNA from plants 8, 23, 72, 75, 77, 85, 90, 92, and 100. These plants from the two most susceptible classes were also heterozygous for the OP-M09220 marker. The same set of 500 primers were screened against these bulks. Using this approach, we hoped to find additional markers that would explain more of the variation. We did not find any bands that were polymorphic between the two new bulks. There are several possible reasons for our inability to identify additional markers. It is possible that the remaining variation is due to the action of several genes, each with a small effect. Because of the initial assumption that resistance was simply inherited, our experiment was not designed for the detection of quantitative trait loci. It is also possible that the primers used did not detect polymorphism in the region of additional gene(s) associated with RWA resistance.
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Fig. 1 RAPD pattern of the resistant parent PI 386156 (RP), susceptible parent NE88T222 (SP), and the most resistant and most susceptible F2 plants, using primer OP-M09. Lane numbers for resistant mad susceptible plants correspond to individual plant identification numbers given in Table 1
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The genetic basis of resistance in this population is not clear. It is likely that there is a major gene linked to OP-M09220, working in concert with other, undefined modifying factors. We did observe that the average RWA score for plants heterozygous at the OP-M09220 locus was intermediate to the scores for the two homozygous classes (Table 2)
. This would suggest incomplete dominance or additive gene action. The inability to separate plants into distinct resistant and susceptible classes prevented us from performing genetic linkage analysis.
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Table 2 Average Plant Damage Score for the F2 population of PI 386156 x NE88T222, based on their genotype at OP-M09220
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To confirm the co-dominant nature of the RAPD fragments generated by OP-M09, the bands from PI 386156 and NE88T222 were excised from the gel and sequenced. Sequence analysis revealed a deletion in the band amplified from NE88T222 relative to the band amplified from PI 386156 (Fig. 2)
. Primers were generated to amplify this segment and serve as STS markers for this locus. When these primers were tested against the set of wheat-rye addition lines, a fragment of the appropriate size was amplified from the 4R addition line (Fig. 3)
. This result supports previous research by Nkongolo et al. (1996), who had identified chromosome 4R as the critical chromosome for Russian wheat aphid resistance from this accession. Our results, however, suggest a more complex mode of inheritance for RWA resistance from PI 386156. There are several possible explanations. It is possible that NE88T222 carries minor factors for RWA resistance that are not obvious in the absence of the gene in PI 386156. It is also possible that differences in screening methodology could have influenced the results. While these remain viable explanations, we believe our results are supportive of Puterka et al. (1992), who suggested complexity in RWA resistance from PI 386156.
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Fig. 2 The DNA sequence analysis of the OP-M09220 fragment amplified from PI 386156 and NE88T222 is shown. Relative to PI 386156, there is a deletion of a `G' nucleotide in the fragment amplified from NE88T222. The arrow identifies the single nucleotide polymorphism
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Fig. 3 The STS primers derived from the sequence of OP-M09220 amplified a fragment from the resistant parent, PI 386156 (RP) and the 4R wheat-rye addition line. Lanes labeled 1 though 7 are wheat-rye addition lines for rye chromosomes 1R through 7R, respectively
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Though we were unable to explain all the variation for RWA resistance in this population, we believe this marker still has potential for use in marker assisted selection and the transfer of the resistance from triticale to wheat. If selection were based on the presence of the band in a homozygous state, 86% of the resistant plants, 58% of the moderately resistant plants, and 26% of the intermediate plants in this study would have been selected. All moderately susceptible or susceptible plants would have been discarded. It is likely that the three most resistant classes would provide adequate field protection against the RWA. We are currently using this marker to assist in the introgression of resistance from triticale to wheat.Michelmore Paran Kesseli 1991
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ACKNOWLEDGMENTS
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The authors acknowledge the Texas Wheat Producers' Board for their financial support of this project. This research was also funded, in part, by a grant from the Texas Grain and Grass Initiative.
Received for publication December 18, 1998.
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ABSTRACT
INTRODUCTION
