Use of RFLP markers for identi cation of individuals homozygous for resistance to Meloidogyne arenaria in peanut

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1 * Nematology, 2000, Vol. 2(5), Use of RFLP markers for identi cation of individuals homozygous for resistance to Meloidogyne arenaria in peanut Gregory T. CHURCH 1, Charles E. SIMPSON 2, Mark D. BUROW 3, Andrew H. PATERSON 3 and James L. STARR 1,* 1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX ,USA 2 Texas Agricultural Experiment Station, Stephenville,TX 76401, USA 3 Plant Genome Mapping Laboratory, University of Georgia, Athens, GA 30602, USA Accepted for publication: 2 July 2000 Summary To increase the ef ciency of breeding peanuts resistant to Meloidogyne arenaria, we determined the utility of two RFLP loci linked to a single gene for resistance in identifying individuals putatively homozygous for resistance. DNA was extracted from leaf samples collected from each of 548 individuals from three segregating BC 7 F 2:4 breeding populations (TP , TP296-4 and TP ). The DNA was then digested with Eco RI, and Southern blotted to Hybond-N+ membranes. The membranes were probed with the RFLP probe R2430E, the autoradiographs scored for resistance genotype, then stripped and re-probed with the probe R2545E. Samples from which no data were obtained due to problems in extraction, digestion, or hybridization ranged from a low of 14.4% for TP probed with R2430E to a high of 38.9% for TP probed with R2545E. Locus R2430 identi ed 27.6, 65.1 and 29.5% of populations TP , TP and TP , respectively, as being homozygous for resistance. The second locus, R2545E, identi ed 24.5, 50 and 23.5%, respectively,of these populationsas homozygous for resistance. In glasshouse tests of nematode reproduction on progeny of individualsidenti ed as homozygous for resistancebased on RFLP patterns,all 15 individualsof each of the 11 single plant progeny lines tested were resistant. Conversely, all progeny from an individual identi ed as susceptible to M. arenaria based on RFLP patterns supported high levels of nematode reproduction. Résumé Utilisation de marqueurs RFLP pour l identi cation d individus homozygotes pour la résistance envers Meloidogyne arenaria chez l arachide Pour accroître l ef cacité dans les croisements d arachides résistantes à Meloidogyne arenaria, nous avons démontré l utilité de deux loci RFLP liés à un seul gène de résistance en identi ant des individus potentiellement homozygotes pour cette résistance. L ADN a été extrait d échantillons de feuilles prélevés sur chacun des 548 individus provenant de trois populations appartenant à des croisements séparant BC 7 F 2:4 (TP , TP et TP ). L AND été ensuite digéré par ECO RI et transferré par Southern blotting sur des membranes Hybond-N+. Ces membranes ont été sondées grâce à une sonde RFLP (CE243OE), les autoradiographiesrepérées pour le génotype de résistance, puis dépouillées et sondées à nouveau à l aide de la sonde R243OE. Les échantillons ne fournissant aucune donnée cela dû à des problèmes d extraction, de digestion ou d hybridisation représentent de 14,4% pour la population TP sondée par R2430E à 38,9% pour la population TP sondée par R2545E. Le locus R2430 a identi é 27,6, 65,1 et 29,5% des populations TP , TP et TP3O1-1-8, respectivement, comme homozygotes pour la résistance. Le second locus, R2545E, a identi é 24,5, 50 et 23,5%, respectivement, de ces trois populations comme homozygotes pour la résistance. Lors d expériences en serre concernant la reproduction du nématode sur la descendance d individus identi és, sur la base de pro ls RFLP, comme hymozygotes pour la résistance, chacun des 15 individus de chacune des 11 lignées monoparentales testées se sont montrés résistants. A l inverse, tous les descendants d un individu identi é, sur la base des pro ls RFLP, comme sensible à M. arenaria, permettent un taux élevé de reproduction du nématode. Keywords Arachis hypogaea, host resistance, marker-assisted selection, root-knot nematode. The rst peanut cultivar (COAN) with a high level of resistance to the root-knot nematode Meloidogyne arenaria (Neal) Chitwood was released by the Texas Agricultural Experiment Station, Stephenville, TX, USA in 1999 (Simpson & Starr, 1999). The resistance in cv. COAN was derived from Arachis cardenasii Krapov & Gregory (Burow et al., 1996) via the interspeci c hybrid TxAG-6 (Nelson et al., 1989; Simpson, 1991) and introgressed into cultivated peanut by a backcrossing programme using cv. Florunner as the recurrent parent (Starr Corresponding author, j-starr@tamu.edu c Koninklijke Brill NV, Leiden,

2 G.T. Church et al. et al., 1995). During the development of the breeding populations from which cv. COAN was selected, all selections for nematode resistance were based on direct evaluation of nematode reproduction on individual plants in glasshouse tests (Starr et al., 1995). Glasshouse data were subsequently con rmed in elds naturally infested with M. arenaria(starr et al., 1999). After determining that the breeding population(tp ), from which cv. COAN was subsequently selected, possessed suf cient yield potential, in addition to nematode resistance, it was necessary to make a nal selection to ensure that nematode resistance was a xed trait. This was achieved by screening ten progeny from each of 137 individuals that had been selected for yield potential and growth habit in eld plots. In those glasshouse tests, which required nearly 8 months to complete, progeny of seven of the 137 individual plant progeny lines were found to be still segregating for the resistance trait. Seed of those lines that were still segregating for resistance were then eliminated from the population that was used to produce breeder seed of COAN. The resistance in cv. COAN is inherited as a single dominant gene and RFLP markers linked to the gene have been identi ed (Choi et al., 1999). Because these markers were tightly linked to the resistance locus and relatively easy to score, it was suggested that they could be used in a breeding programme to increase the ef ciency with which individualshomozygousfor resistance could be identi ed. There are numerous reports that identify molecular markers linked to genes for resistance to nematodes (cited in Vrain, 1999), but few data are available that compare the ef ciency of marker-assisted selection procedures to other selection techniques. Herein, we report on the relative ef- ciency of marker-assisted selection, using the previously identi ed RFLP markers, for the identi cation of individuals putatively homozygous for resistance. In this effort we used breeding populations from the seventh backcross generation, the most advanced generation available, whereas cv. COAN was selected from the fth backcross generation of the same breeding populations. Because the yield potential of COAN is slightly less than that of its recurrent parent cv. Florunner(Simpson & Starr, 1999; Starr et al., 1999), a second objective was to determine if two additional backcross generations increased the yield potential of these breeding populationsthat are also resistant to M. arenaria. Materials and methods Seeds of three BC 7 F 2:4 breeding populations (TP , TP , and TP ) were planted with 91 cm spacing within and between rows at the Texas A&M University Agricultural Research and Extension Center in Stephenville, TX, USA in June Two unexpanded tetrafoliolate leaves were collected from each plant on 21 July. Leaves that had any visible necrosis or insect damage were not collected, except in a few instances where it was not possible to avoid leaves with symptoms of damage. The leaves were placed individually in 1.5 ml microcentrifuge tubes and stored on ice during transport to the laboratory in College Station, TX, USA where the tissue was stored at 80 C until DNA extraction was completed. DNA was extracted from each sample by the protocol of Choi et al. (1999). DNA concentration for each sample was estimated by electrophoresis of a 5 ml sample on 0.8% agarose. Band intensity was compared with that of concentration standards following staining with ethidium bromide. Following extraction of the rst sample, the second tissue sample was extracted from those individuals for which less than 2 mg was available. For the few plants for which neither the rst nor the second sample yielded suf cient DNA, a third tissue sample was collected for extraction. After completion of all extractions, the DNA was digested with Eco R1 according to the manufacturer s instruction (New England Biolabs, Beverly, MA, USA) and the fragments separated on 0.8% agarose gels. The DNA was then Southern blotted on to Hybond-N+ (Amersham Pharmacia Biotech, Piscataway, NJ, USA) membranes and probed with the R2430E, which is 4.2 cm distant from the resistance locus (Choi et al., 1999). Membranes were stripped after development of autoradiographs and then re-probed with R2545E, which is 5.2 cm distant from the resistance locus (Choi et al., 1999). Individuals were scored homozygous (RR) for resistance if only the band associated with resistance was present, heterozygous(rr) for resistance if the band associated with resistance and the band associated with susceptibility were present, and susceptible (rr) if the band associated with resistance was absent. To con rm the results of genotype determination based on RFLP, 15 seeds (BC 7 F 4:5 ) from each of 12 individuals were tested in the glasshouse for resistance based on nematode reproduction according to established protocols (Starr et al., 1995). These progeny lines included 11 for which the parent was putatively RR and one line from a putative rr parent. The cvs Florunner and COAN were included in this test as susceptible and resistant controls, respectively. 576 Nematology

3 Marker-assisted selection in peanut The development of nematode population densities on the three breeding populationsin eld plots was compared to that of cvs Florunner and COAN prior to and after selection of the putative RR individuals. Field plots were two 3.8 m long rows with 91 cm between the rows, and were established in two elds in 1998 (prior to selection) and in two elds in 1999 (after selection). Seed for the 1998 tests were of the BC 7 F 2:4 generation, whereas seed for the 1999 tests were of the BC 7 F 4:6 generation. There were four replications of each cultivar and breeding population in each test. Each test site was determined to be infested with M. arenaria based on galls present on peanut roots the year preceding the test. Composite soil samples (15 cores, each 2.5 cm diam. 25 cm deep) were collected from each plot approximately 2 weeks prior to crop maturity. Second-stage juvenile nematodes were extracted from soil by elutriation (Byrd et al., 1976) and centrifugation(jenkins, 1964). Eggs were extracted from roots recovered during elutriation by the NaOCl method (Hussey & Barker, 1973). Pod yield was measured also in Nematode count data and yield data were subjected to analysis of variance using the SAS (SAS Institute, Cary, NC, USA) general linear model procedure to determine main treatment effects with mean separations by Duncan s multiple range test. Results A single unexpanded tetrafoliolate leaf from eldgrown plants yielded suf cient DNA for Southern analysis (> 2.0 m g DNA/sample) for 82.5% of the samples. When a second sample from individual plants that did not yield suf cient DNA in the rst extraction was processed, the percentage of total individuals for which it was estimated that suf cient DNA was available for Southern analysis increased to greater than 90%. Collection and extraction of a third sample increased the ef ciency to greater than 95% of all samples. After completion of digestion, electrophoretic separation, blotting to membranes, and hybridizationof the speci c probes, the RFLP genotypes could be scored for 65 to 86% of the individuals(table 1). No data were obtained from the other individuals due to a combination of incomplete digestion, poor hybridization,or high backgroundon the developed autoradiographs. The numbers of individuals identi ed as RR with probe R2430E ranged from a low of 27.6% for breeding population TP to a high of 65.1% for populationtp (table 1). Probe R2545E Table 1. Genotype analysis of three BC 7 F 2:4 peanut breeding population using RFLP loci R2430E and R2545E that are tightly linked to the Meloidogyne arenaria resistance locus. RFLP Genotype 1 Breeding Population locus TP TP TP (n = 163) (n = 172) (n = 213) R2430E RR Rr rr No data R2545 RR Rr rr No data Values are percentage of individuals of each population that were identi ed as RR = putatively homozygous for resistance; Rr = putatively heterozygous for resistance; and rr = putatively homozygous for susceptibility based on RFLP genotype. identi ed a lower percentage of individualsas RR for each population than did R2430E. For populations TP and TP , the observed ratios of putative RR, Rr, and rr individuals (Table 1) did not differ (x 2 < 5.99, P = 0.05) from the expected ratio of 3 : 2 : 3 in a self-pollinated F 4 generation derived from a Rr parent in the F 2 generation. For TP , that all individuals were scored as RR by probe R2430E and greater than 81% were RR with R2545E indicates that the F 2 individual from which this population was derived was homozygous for resistance. In glasshouse tests to con rm resistance genotypes based on RFLP analysis, M. arenaria produced a mean of 1099 eggs/g of root fresh weight on the susceptible cv. Florunner, whereas the mean on the resistant cv. COAN was six eggs/g root fresh weight. Mean egg production on progeny of the 11 individuals identi ed as putatively RR ranged from 0 to 11 eggs/g roots and was not different from egg production on COAN (P < 0.05). All progeny from individuals with a resistant RFLP genotype had a resistant phenotype based on nematode reproduction. The progeny of the single individual with the putative rr genotype had a susceptible phenotype and produced a mean of 756 eggs/g root fresh weight, which was not different (P > 0.05) from reproduction on cv. Florunner. In eld tests in 1998 prior to selection for resistance genotype, nal populationdensities of M. arenaria on cvs Florunner and Tamrun 96 were greater than 1500 eggs and J2/500 cm 3 soil at site 1 and greater than 8000 eggs Vol. 2(5),

4 G.T. Church et al. (A) (B) Fig. 1. Comparison of nal population densities of Meloidogyne arenaria on three nematode-resistant breeding populations of peanut with those on the susceptible cvs Florunner and Tamrun 96, and the resistant cv. COAN in eld plots. A: 1998, prior to selection for individuals homozygous for resistance using RFLP markers linked to resistance; B: 1999, after selection for individuals putatively homozygous for resistance. and J2/500 cm 3 soil at site 2 (Fig. 1A). At both locations, nal nematode population densities on cv. COAN and TP were less than 10% of the population densities on the two susceptible cultivars. Final nematode population densities on TP and TP were not different (P > 0.05) from those on the susceptible controls. In 1999, after selection for individuals homozygous Fig. 2. Comparison of pod yield of TP , TP , and TP peanut breeding populations following selection of individuals homozygous for resistance to Meloidogyne arenaria using RFLP markers linked to resistance to pod yield of the cvs Florunner, Tamrun 96 and COAN. Within each test site, bars with the same letter are not different (P > 0.05) according to Duncan s multiple range test. for resistance, nal nematode population densities on the susceptible cultivars ranged from 4000 to 9000 eggs and J2/500 cm 3 soil across both test sites (Fig. 1B). At each site, the nal nematode populationdensities for cv. COAN and the three breeding populations were all less than 10% of the population densities on the susceptible cultivars. At both test sites in 1999, the pod yields of the three resistant breeding populations and cv. COAN were greater (P < 0.05) than the yields of the two susceptible cultivars. The resistant breeding populations had pod yields that ranged from 1201 to 2386 g/plot across both locations, whereas yield of the susceptible cultivars ranged from 145 to 424 g/plot (Fig. 2). Mean pod yield across all three breeding lines were and 134.5% of cv. COAN at sites 1 and 2, respectively. Discussion The use of RFLP markers linked to a single gene for resistance to M. arenaria for selection of individuals homozygous for resistance improved our ef ciency relative to that of direct assessment of resistance based on nematode reproduction in greenhouse tests. In this test the genotypes for nearly 500 individuals were determined in ca 2 months compared with 8 months required to estimate 578 Nematology

5 Marker-assisted selection in peanut the genotypes for 137 individuals based on direct measurement of nematode reproduction. A major bene t of the marker-assisted selection was that it was based on the plants producing the seed rather than waiting until maturity of that crop to assay the seed directly. This represents a time saving of 2 to 3 months. In the production of breeder seed of cv. COAN, the nal selections were not completed until plots had been planted for seed increase in a winter nursery in Puerto Rico. All single plant progeny lines being tested were planted in the nursery, but only those lines for which no evidence of segregation was found in the glasshouse tests were harvested. Thus, in addition to a saving of time, the use of marker-assisted selection for resistance genotype saved the expense of planting, maintenance and harvesting plots for which the seed was subsequently abandoned. A problem with the use of the markers that are linked to the resistance gene is the probability that the linkage will be broken in some individuals and individuals lacking the resistance gene will be scored as resistant. That marker R2430E is 4.2 cm distant from the resistance locus indicates that this error rate will be ca 4%. Glasshouse tests for resistance based on nematode reproduction may also give incorrect data. Escapes can occur, due primarily to errors in the inoculation procedure, that will result in susceptible plants being scored as resistant. Although precise data on the frequency of such escapes in the greenhouse tests are lacking, we believe it to be less than 4% but not very different from that for the RFLP markers. The rigour of the marker-assisted selection procedure will be improved by the identi cation of marker(s) anking the resistance gene. Both R2430E and R2545E are located on the same side of the resistance locus. Using markers that are more tightly linked to the resistance locus or derived from the resistance gene itself will also improve the rigour of the test. Despite the fact that the use of RFLP loci that do not ank the resistance locus as marker results in data that is not unambiguous, the direct assessment of progeny of individuals selected as homozygous for resistance based on these markers failed to provided evidence of segregation for this trait. In the 1998 eld tests, breeding populations TP and TP that supported nal nematode population densities that were not different from those on the susceptible cvs Florunner and Tamrun 96, were also the two populations that had the greatest numbers of susceptible individualsin the populations based on the RFLP genotype. In contrast, TP had no detectible susceptible individualsbased on RPLP genotype and in 1998 supported nal nematode population densities that were not different from those on resistant cv. COAN. In 1999, after selection using the RFLPs, all three breeding populations supported nal nematode populations that were not different from those on cv. COAN. If high quality seed are available for glasshouse tests, one would expect to be able to obtain data from more than 90% of the seed planted. With the use of RFLP markers there was a greater frequency of lost data due to several factors, including insuf cient DNA available, incomplete digestion, and poor hybridization.if one were able to use glasshouse grown plants, which are subjected to less stress than the eld grown plants in this study, then it is likely that high quality DNA would be obtained from a greater proportion of the plants. This increased frequency of missing data with RFLPs relative to direct measurement of resistance is not a problem because the number of individuals available for testing usually far exceeds the capacity for testing by either procedure. Additionally, as demonstrated in this study, testing a large number of individuals using RFLP markers was readily accomplished. Therefore, one can compensate for expected missing data by testing a larger number of individuals. Other problems associated with the use of RFLP markers in the selection process include the time and effort needed to identify the markers. The markers used in these experiments were the result of nearly 6 years of research effort and research costs that exceeded US $ , excluding the salaries of the senior scientists (Paterson, Simpson and Starr). However, as the genetic data base for peanut improves, it will become progressively easier to identify markers linked to traits of interest. The use of RFLPs and Southern analysis is more cumbersome than other procedures such as the use of speci c PCR primers that amplify DNA sequences linked to resistance, but RFLP markers have the bene t of co-dominance.most other procedures for marker-assisted selection will identify the resistance phenotype but not the genotype. Even with the problems associated with the use of marker-assisted selection, we believe that the use of this technologyincreased the ef ciency of our breeding efforts and resulted in the selection of peanut breeding lines that are apparently homozygous for resistance. Additionally, the yield potential of these breeding lines was greater than that of cv. COAN. This increase in yield potential after two additional backcross generations suggests that resistance to M. arenaria is not tightly linked to genes that have a negative effect on yield. Vol. 2(5),

6 G.T. Church et al. Acknowledgement This material is based in part upon work supported by the Texas Advanced Technology Program under Grant No b References BUROW, M.D., SIMPSON, C.E., PATE RSON, A.H. & STARR, J.L. (1996). Identi cation of peanut (Arachis hypogaea L.) RAPD markers diagnostic of root-knot nematode (Meloidogyne arenaria [Neal] Chitwood) resistance. Molecular Breeding 2, BYRD, D.W., JR, BARKER, K.R., FERRIS, H., NUSBAUM, C.J., GRIFFIN, W.E., SMALL, R.H. & STONE, C.S. (1976). Two semi-automatic elutriators for extracting nematodes and certain fungi from soil. Journal of Nematology 8, CHOI, K., BUROW, M.D., CHURCH, G., BUROW, G., PATE R- SON, A.H., SIMPSON, C.E. & STARR, J.L. (1999). Genetics and mechanism of resistance to Meloidogyne arenaria in peanut germplasm. Journal of Nematology 31, HUSSEY, R.S. & BARKER, K.R. (1973). A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reporter 59, JENKINS, W.R. (1964). A rapid centrifugal- otation technique for separating nematodes from soil. Plant Disease Reporter 48, 692. NELSON, S.C., SIMPSON, C.E. & STARR, J.L. (1989). Resistance to Meloidogyne arenaria in Arachis spp. germplasm. Journal of Nematology 21, SIMPSON, C.E. (1991). Pathways for introgression of pest resistance into Arachis hypogaea. Peanut Science 18, SIMPSON, C.E. & STARR, J.L. (1999). Development and release of a root-knot nematode resistant peanut variety. Proceedings American Peanut Research and Education Society 31, 68. [Abstr.] STARR, J.L., SIMPSON, C.E. & LEE, T.A., JR (1995). Resistance to Meloidogyne arenaria in advanced generation breeding lines of peanut. Peanut Science 22, STARR, J.L., SIMPSON, C.E. & LEE, T.A., JR (1999). Yield of peanut genotypes resistant to root-knot nematodes. Peanut Science 25, VRAIN, T.C. (1999). Engineering natural and synthetic resistance for nematode management. Journal of Nematology 31, Nematology