PCR amplification can be used to generate DNA fragments suitable for use as genetic markers. The principle of the technique involves the use of two primers, which recognize sites some distance apart, and the amplification of the sequence flanked by them. AFLP fragments can also be isolated from the designed polyacrylamide gels and SCAR primers. The fragments can be excised directly from the gel after localization by autoradiography (MEKSEM, et al., 1995), or after silver staining (CHO, et al., 1996).
Plant material , ,
Greenhouse screening procedure
All root systems were weighed and dissected, and the nematode eggs were extracted with 1% (w/v) NaOCI. The total number of eggs produced was determined for each root system by counting an aliquot of the egg. DNA was isolated from freeze-dried leaf material by a modified CTAB extraction procedure (DELLAPORTA, et al., 1983).
The DNA was precipitated from the aqueous phase with 0.66 volume isopropanol at room temperature and centrifuged for 15 min at 12,000 x g. After overnight incubation at -20°C, the DNA was recovered by centrifugation at 12,000 x g for 15 min and washed twice with 70% (v/v) ethanol for 5 minutes. The DNA was resuspended in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and treated with 0.11 µg IJL·1DNase-free RNase, prepared by heating a stock solution of 10 mg mL-1RNase (Roche Boehringer Mannheim), Randburg, South Africa) for 10 minutes at 94°C.
DNA extraction with SOS 62
RFLP analysis ,
The plasmid DNA containing the probe fragment was isolated from overnight bacterial cultures by an amine prep procedure. Overnight cultures were centrifuged at 3500 x g and resuspended in STET (50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 8% (w/v) sucrose, 5% (v/v) Triton-X100). The bacterial cell walls were lysed by adding 10 µg lysozyme and heating at 94°C for 1 min. The samples were cooled immediately on ice and the nuclear DNA was removed by centrifugation at 12000 x g for 20 min. The probe DNA was denatured at 94°C for 5 minutes and immediately cooled on ice before dilution in DIG Easy Hyb solution at a concentration of 0.5-0.75 µL. The membranes were hybridized overnight in a volume of 3.5 ml 100 cm -2 of this solution at 50°C. The Rf values of both the Prima control plants and the F2 population showed a large variation (Figure 3.3). The Rf value calculated for Gazelle was low and varied between 0.25 and 2.31 with an average value indicating good resistance to the nematode.
AFLP is a PCR-based technique capable of detecting more than 50 loci in a single reaction, making it very valuable for detecting markers in a relatively short time. The use of this technique in combination with BSA and selective genotyping can be an effective tool to generate molecular markers for specific traits in separating populations with minimal cost and time requirements. The bulk samples were constructed by mixing equal amounts (m/m) of DNA from 5 F2 plants, each corresponding to two factors: (a) Plants from each of the two homozygous genotypes (AA and BB), as determined by RFLP- analysis with probe B212. b) The bile index values for these plants, where the resistant plants have a value of 1 and the susceptible plant a value of 9. Marker A808 was judged as the dominant marker. The patterns produced were compared with images downloaded from the soybean database (SOY8ASE, 1995).
The markers were grouped into 9 groups with 34 markers unlinked to any group (Figure 4.1). The 7AFLP markers, identified by analysis of variance as closely linked to resistance to gall formation, clustered together with marker 8212, which could be anchored on LG-F. Map development in soybeans followed along the history covered extensively in Chapter 1. The current soybean map includes 24 linkage groups, 8 of which have only 4 markers or less. Apolymorphism (600 bp) was amplified between Gazelle and Prima at an annealing temperature of 60°C with an aMg2+ ion concentration of 3mM (Figure 5.3). The enzyme concentration had no visible effect on the band pattern.
Both markers identified accounted for 41-42% of the gall index variation individually, which was of the same magnitude as the heritability estimate for other soybean populations (h TAMUlONIS et al., 199?b).
F 2 characterization : PG3-1, PG3-2
The F2 population showed values between the two parents (Figure 3.3), with the population mean within the lower limit of standard deviation of the Prima controls. Analysis of variance indicated a statistically significant difference (P=O.05) between the bile index values as well as the Rf values of the two parental populations. Comparison of means of Prima and F2 progeny with a t-test indicated a statistically significant difference between the means of the two populations at the 95% confidence level (t=4.4).
These (216) primers were used to screen the two bulk DNA samples comprising individual plants for the two phenotypic extremes. The effect was clearly seen in the number of polymorphisms detected between the two bulks (of 51 primers), which was much less than between the two parents (216). The RAPD analysis was repeated with these 51 primers with DNA from the 12 individual plants included in the 2 bulks (Table 3.1). Three putative markers were indicated (Table 3.3) with analysis of the variance of the Rf values of individual plants included in the two DNA pools. The fragments OPT-08(1) and OPC-16(1) could each account for 42.8%.
Re-evaluation ofF 1 ••
The use of two large groups grouped informant individuals to equalize genetic background so that the groups would theoretically differ primarily for the gene(s) in question. Since this analysis was done on unbiased selection of plants from the two extremes, this would be expected to be a gross overestimate of the marker contribution to the phenotypic effect. This quantitative difference was reproducible and may be due to more than one fragment of the same molecular weight migrating together.
RAPD analysis was repeated with these 3 primers with 40 additional randomly selected F2 plants, but no linkage with P<0.05 to the resistance trait could be confirmed (Table 3.3), based on reproduction factor values. Likewise, no significant linkage between markers and bile index values of the F2 population could be established. Likewise, no segregation of bands could be confirmed in randomly selected F2 plants (results not shown).
Discussion 71
Of the 377 polymorphic isms amplified between the two parents, 47 were informative in the bulk samples. Three of the markers (E-AAG/M-CTA5, E-ACC/M-CTC2 and E-AGC/M-CTG6) are linked to the resistance gene in linkage phase, and 4 markers are linked in repulsion phase. The RFLP marker 8212 was closely linked to the resistance trait and accounted for 62% of variation in gall index.
Three of the AFLP markers were found to be associated with biliary index resistance in this population in the coupling phase and four in the repulsion phase. Marker E-AAC/M-CAT1 caused the greatest variation in gall numbers (42%) and was linked to the resistance trait in the rejection phase. An additional AFLP marker was linked to the resistance trait, using a combination of 8SA and selective genotyping methods.
INTRODUCTION
MATERIALS AND METHODS 94
Isolation and cloning of AFLP fragments , , ,
APUYA, N.R, FRAZIER, B.L., KEIM, P., ROTH, E.J. and LARK, K.G. 1988. Restriction fragment length polymorphisms as genetic markers in soybean, Glycine max (L.) Merrill. Theoretical and Applied Genetics 75:889-901. LUll, B.M., TAMULONIS, JP., HUSSEY, RS. and BOERMA, HR.1995b. Inheritance of Javanese root nematode resistance in soybean. Crop Science. MAHESWARAN, M., SUBUDHI, P.K., NANDI, S., XU, J.C., PARCO, A., YANG, D.C. and HUANG, N. 1997. Polymorphism, distribution and segregation of AFLP markers in a duplicated haploid rice population. Theoretical and Applied Genetics 94:39-45.
SCHACHERMAYR, G., SIEDLER, H., GALE, M.D., WINZELER, H., WINZELER, M. and KELLER, B.1994. Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat. 1994.Constructing aplant genetic linkage map with DNA markers.In: DNA-Based Markers inPlants.Phillips, R.L. and Vasil, I.K. VU, K., PARK, S.J. and POYSA, V. 1999. Use of molecular markers to aid selection of common bean resistant to common bacterial blight (Xanthomonascampestrispv. Phaseoll). Annual Report of the Bean Improvement Corporation 42:3-4.
Restriction digestion of PCR products 98
DEVELOPMENT OF A SCAR FROM AN RFLP PROBE (B212) 99
The pB212 probe insert was sequenced from both ends and new primers were designed near the ends to amplify the bracketed fragment between them (Figure 5.2). The pstl sites used to clone the cDNA probe were accurately sequenced and the ends of the cDNA could be identified. No palindromic sequences were found within the primers, although some crossover dimers can be expected.
PCR analysis of 50 F2 plants showed that the B212600 fragment was not associated with variation in gall index values (P=0.406). Restriction analysis of PCR products with a wide range of enzymes produced informative digestion patterns with HindIII, Taq/, AlvI and Oral between Prima and Gazelle. Digestion patterns of PCR products with HindIII, TaqI and Oral did not reveal any significantly linked fragments (P>0.05).
DEVELOPMENT OF SCARS FROM AFLP FRAGMENTS 101
Analysis of the progeny did not reveal any significant association of these fragments with the resistance trait (P>0.05). A total of 5 fragments were amplified at 55°C (2 mM MgCl 2 ), two of which were monomorphic and three polymorphic between the two parents. Two bands between 500 and 1 500 bp separated in th~F plants, but no significant linkage with gall index variation could be established (P>0.05).
Six fragments were amplified with the designed primers for pSOJA9 at 50°C (2 mM MgCl ), with a polymorphism between Gazelle and Prima at <500 bp. The polymorphism segregated in the F2 offspring, but was not significantly linked to biliary index variation (P>O.05). The cloned pSOJA6 and pSOJA7 fragments were labeled with DIG and used as probes to hybridize with fcoRI-digested genomic DNA from the parent and F2 progeny plants.
DiSCUSSiON
The SOJA6 marker amplified three polymorphic fragments that segregated codominantly in an F2 population following a Mendelian segregation pattern. Although a single locus is unlikely to be amplified by the sequence-specific primers, the 240-bp fragment co-segregated with variation in the bile index. The inheritance of resistance to root knot nematodes in soybeans is quantitative and has a moderate to high heritability (TAMULONIS, et al., 199?b).
The aim of this study was to develop a practically applicable marker in the soybean breeding program, thus focusing on the most important aspects influencing nematode resistance. The SCAR markers explained 88% of the lower estimate (0.48) and the RFlP marker explained 62%, accounting for 100% of the average heritability estimate (0.62). Additional Oll.s would probably be of minor importance in terms of their contribution to explaining the variation in the bile index.
CONCLUSiONS ,
ADAM-BLONDON, A.F., SEVIGNAC, M., BANNEROT, H. and DRON, M.1994. SCAR, RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean. Theoretical and Applied Genetics 88: 865-870. BECKER, J., VOS, P., KUIPER, M., SALAMINI, F. and HEUN, M. 1995. Combined mapping of AFLP and RFLP markers in barley. Molecular and General Genetics 249: 65-73. BONIERBALE, MW., PLAISTED, RL. and TANKSLEY, S.o. 1988. RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics.
HALEY, S.D., MIKLAS, P.N., STAVELY, J.R., BYRUM, J.and KELLY,J.D.1993.identification of RAPD markers linked to an unusual rust resistance gene block. KEIM,P.,DIERS,BW.,OLSON,T.C.and SHOEMAKER,RC.1990a.RFLP mapping in soybean: relationship between marker location and variation in quantitative traits. Genetics. MARTIN,G.B.,WILLlAMS, J.G.K.and TANKSLEY, S.D.1991.Rapid identification of markers linked to the Pseudomonas resistance gene in tomato using random and near-isogenic primers. Proceedings of the National Academy of Sciences, USA.
RIGGS, RD.and SCHMITT, D.P.1987.Nematodes.In: Wilcox, JR (Ed.)Soybeans: Improvement, production and uses, 2nd edition -Agronomy Monograph no.16. WILLLAMS, JG.K., KUBELLK, A.R., L1VAK, K.J., RAFALSKI, JA. and TINGEY, S.V. 1990. The DNA polymorphism probe of random primers are useful as genetic markers. Nucleic Acids Research.
