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CELL BIOLOGY & MOLECULAR GENETICS

Relationships among Early European Maize Inbreds

IV. Genetic Diversity Revealed with AFLP Markers and Comparison with RFLP, RAPD, and Pedigree Data

^a Institute of Plant Breeding, Seed Science, and Population Genetics, Univ. of Hohenheim, D-70599 Stuttgart, Germany
^b Keygene n.v., P.O. Box 216, Wageningen, The Netherlands

luebbit{at}uni-hohenheim.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
A set of 51 elite maize (Zea mays L.) inbred lines representative of the early-maturing European flint and dent heterotic groups were assayed for amplified fragment length polymorphisms (AFLP) markers using eight primer combinations. Our main objectives were to (i) investigate the amount of variation for AFLP markers in these materials, (ii) examine the usefulness of AFLP markers for assigning inbred lines to heterotic groups, and (iii) compare the genetic similarity (GS) based on AFLP markers with Malécot's coancestry coefficient (f) based on pedigree data and with GS estimates based on restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPD) data. The eight AFLP primer combinations yielded 462 polymorphic bands. GS estimates calculated from AFLP data ranged between 0.38 and 0.77 between unrelated (f = 0) pairs of lines. All flint and dent inbreds showed a smaller mean GS to lines from the other heterotic group than to unrelated lines from the same heterotic group. For lines of mixed origin, the difference in mean GS to flint lines and to dent lines was consistent with the expected genomic proportions from each heterotic group determined on the basis of pedigrees. Principle coordinate analysis of GS estimates resulted in a separate grouping of flint and dent lines. Correlations between f and GS estimates were substantially higher for RFLPs and AFLP markers than for RAPDs. Correlations of GS estimates based on different marker systems were closest between RFLP and AFLP markers both for related (f > 0) and unrelated (f = 0) pairs of flint and dent lines. Results from this study corroborate the usefulness of AFLP markers for (i) assigning inbreds into heterotic groups and (ii) revealing pedigree relationships among lines.

Abbreviations: f, coancestry coefficient • GS, genetic similarity • GS-AFLP, genetic similarity based on AFLP markers • GS-RAPD, genetic similarity based on RAPDs • GS-RFLP, genetic similarity based on RFLPs • MI, marker index • PCR, polymerase chain reaction • PIC, polymorphic information content • r, correlation coefficient • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
INFORMATION ABOUT THE GENETIC DIVERSITY of lines and populations is fundamental in breeding and germplasm preservation. Classification of elite germplasm into heterotic groups and assignment of inbred lines to established heterotic groups are major decisions in any breeding program for hybrid maize (Hallauer et al., 1988). In the past five decades, a large number of maize inbreds have been developed from a limited number of elite lines and elite line synthetics. This engenders the danger of a loss of genetic diversity and restricts the possibility of crosses between genetically divergent parents. Knowledge of the genetic relationship among breeding materials could help to avoid the great risk for an increasing uniformity in the elite germplasm and could ensure long-term selection gains (Messmer et al., 1993).

Different methods are available to investigate the genetic diversity in breeding materials or populations. The genetic similarity of two genotypes can be estimated indirectly from pedigree information. Malécot (1948) devised the coancestry coefficient, f, as an indirect measure for the relative genetic similarity of related individuals. He defined f as the probability that two homologous genes drawn at random, one from each of the two individuals, are identical by descent. It has been widely used in autogamous crop species such as barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), oats (Avena sativa L.), soybean [Glycine max (L.) Merr.], and peanut (Arachis hypogaea L.) to examine the level of genetic diversity of a given germplasm pool, monitor trends in germplasm usage, and identify major groupings of related cultivars (Martin et al., 1991). Accurate estimation of genetic similarity by coancestry requires reliable and detailed pedigree records. However, for many maize inbreds and their progenitors, pedigree records tracing back more than two generations are rare or incomplete. In addition, some progenitors were derived from open-pollinated populations. Hence, calculation of coancestry for maize is often not feasible or dubious (Messmer et al., 1993).

Molecular markers allow a direct comparison of the similarity of genotypes at the DNA level. In maize, RFLPs have been used quite extensively for this purpose (Melchinger, 1993). The major advantage of RFLPs in maize is the large number of polymorphic loci found in breeding materials (Messmer et al., 1992). Studies with elite lines adapted to the U.S. Corn Belt and early-maturing European maize inbreds showed that RFLPs are suitable to (i) define heterotic groups, (ii) assign inbred lines to such groups, (iii) reveal genetic relationships among lines, and (iv) identify diverse germplasm sources. However, RFLP assays are labor intensive and time consuming and, therefore, increasingly substituted by other marker techniques based on the polymerase chain reaction (PCR) such as RAPDs, amplified fragment length polymorphisms (AFLPs; AFLP is a registered trademark of Keygene N.V., Wageningen, the Netherlands), and simple sequence repeats (SSRs) (Jones et al., 1997).

AFLP markers are genomic restriction fragments detected after selective PCR amplification (Vos et al., 1995). The major advantage of AFLP markers compared with RFLPs and SSRs is the generation of multiple marker bands in a single assay. In addition, AFLP markers were shown to be highly reproducible, in contrast to RAPDs, which also produce multiple banding patterns (Jones et al., 1997). The usefulness of AFLP markers for the assessment of genetic diversity was demonstrated for a number of species such as lentils—Lens culinaris Medikus (Sharma et al., 1996), rice—Oryza sativa L. (Mackill et al., 1996), soybean (Powell et al., 1996), and barley (Russell et al., 1997). In maize, AFLP markers have been employed to investigate (i) the relationship between genetic distances and hybrid performance or heterosis for yield (Ajmone-Marsan et al., 1998; Melchinger et al., 1998) and (ii) the genetic similarity of U.S. dent inbreds (Pejic et al., 1998).

In the present study, we assayed 51 maize inbreds widely used for production of commercial hybrids in Central and Northwestern Europe that had previously been analyzed with RFLPs (Messmer et al., 1992, 1993) and RAPDs (Hahn et al., 1995). Our main objectives were to (i) investigate the degree of polymorphism detected with AFLP markers in these materials, (ii) determine the level of genetic diversity for AFLP markers found within and between the flint and dent heterotic groups, (iii) evaluate the usefulness of AFLP markers for quantifying the degree of pedigree relatedness, and (iv) compare the genetic similarity of maize inbreds determined by AFLP markers with that determined by RFLPs, RAPDs, or the coancestry coefficient.

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Maize Inbred Lines
A subset of 51 out of the 57 maize inbred lines investigated in the RFLP study of Messmer et al. (1992) was analyzed for AFLP markers: 25 lines belong to the dent germplasm, 23 to the flint germplasm, and three lines were of mixed origin in that they had various proportions of both flint and dent germplasm. Pedigree information of the lines have been presented in detail by Messmer et al. (1992). The majority of flint lines had as an ancestor at least one of the European elite lines F2 and F7 from the French population Lacaune, EP1 from the Spanish population Lizargarote, or DK105 from the German land variety Gelber Badischer Landmais. Most of the dent lines trace back to North American elite inbreds, including CO125 (with unknown genetic background) from the Ottawa research station, W401 from Wisconsin, and B14 from BSSS. All lines were inbred by more than 12 selfing generations.

As a test for the reliability of the AFLP technique, each of the four lines D44, D140, D406, and D503 were included as duplicate and one line (DK105) as triplicate entries. These replicated entries have been separated after 8 to 12 selfing generations of the same inbred line and were subsequently maintained in different breeding programs for one to eight selfing generations as described by Boppenmaier et al. (1991).

AFLP Analyses
AFLP analyses were performed as described by Vos et al. (1995). Genomic DNA of the maize inbreds was digested with the restriction enzymes EcoRI or PstI and MseI. In a second step, the following adapter sequences were ligated to the restricted DNA fragments: EcoRI:5'-CTCGTAGACTGCGTACC CATCTGACGCATGGTTAA-5' PstI:5'-CTCGTAGACTGCGTACATGCA CATCTGACGCATGT-5' MseI:5'-GACGATGAGTCCTGAG TACTCAGGACTCAT-5'

The primers used for pre-amplification and amplification were similar to those described by Vos et al. (1995) with the (PstI)/MseI extensions (CTC+CTG)/CAC, (CTC+CTG)/CAG, (CTA+CTT)/CAC, (CTA+CTT)/CAG and the EcoRI/MseI extensions ACA/CAT, AAG/CAT, AAG/CCT, and AAG/CTC. In total, eight primer combinations were employed for selective amplification. The PCR products were separated by electrophoresis on a denaturing polyacrylamide gel. After drying, the gels were exposed to phospho-imager plates for 16 h. The imager plates were scanned with a phosphor-imager and polymorphic bands were coded in binary form by 1 and 0 for presence or absence in each inbred, respectively. RFLP and RAPD analyses of the same inbreds have been described in detail by Messmer et al. (1992) and Hahn et al. (1995), respectively. In the RFLP study 188 clone–enzyme combinations produced 1132 distinct RFLP bands, whereas 234 polymorphic RAPD bands were generated with 31 informative primers.

The average polymorphic information content (PIC) and the marker index (MI) were calculated for AFLP markers across assay units by applying the formulas given by Powell et al. (1996) and Smith et al. (1997):

where f_i is the frequency of the ith allele. The PIC value provides an estimate of the discriminatory power of a marker by taking into account not only the number of alleles at a locus but also the relative frequencies of these alleles. Marker indices were calculated as the product of PIC and the number of polymorphic bands per assay unit. An assay unit for AFLP markers corresponds to a primer combination. Each polymorphic DNA fragment within an AFLP assay unit is considered as a single dominant marker locus.

Statistical Analyses
Genetic similarity (GS) between two Inbreds i and j was calculated according to the formula given by Nei and Li (1979):

(1)

where N_ij is the number of bands in common between Lines i and j, and N_i and N_j are the total number of bands in Lines i and j with regard to all eight primer combinations, respectively. Mean GS of Lines i to a Set J of inbreds was obtained by averaging individual GS estimates according to following formula:

(2)

where |J| is the number of elements in Set J. Standard errors of GS estimates were obtained by the bootstrap procedure (Tivang et al., 1994).

For a given f value of two lines, the expected genetic similarity (GS_exp) was calculated according to Melchinger et al. (1991) by the following formula:

(3)

In order to estimate the mean genetic similarity () of unrelated (f = 0) pairs of lines, we averaged GS estimates across the entire set of unrelated line pairs of the respective heterotic group (Melchinger et al., 1991). For each set of related (f > 0) pairs of flint and dent lines, we computed a linear regression (GS_reg) of GS on f values. Correlations (r) among GS estimates based on AFLP markers, RAPDs (Hahn et al., 1995), and RFLPs (Messmer et al., 1992) were calculated separately for unrelated (f = 0) and related (f > 0) pairs of lines. In the latter case, correlations to the coancestry coefficient (f) were also determined. The combinations with D44 were omitted in correlation analyses because of doubts on the pedigree records of D44 (Messmer et al., 1993). In addition, GS estimates of the three marker systems for all pairs of lines were organized in matrices. Correspondence between pairs of matrices was tested with the Mantel Z statistic (Mantel, 1967). Significance of Z was determined by comparing the observed Z value with a critical Z value obtained from its permutational distribution. This distribution was derived by calculating Z values for one matrix with 1000 permuted variants of a second matrix. Associations among the maize inbred lines were revealed by principal coordinate analyses (PCoA) for AFLP-based GS estimates (Gower, 1972). These computations were performed with the computer package NTSYS-PC (Rohlf, 1992).

	Results

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Variation for AFLP Markers
The eight AFLP primer combinations resulted in 550 different amplification products with 462 polymorphic AFLP bands for the 51 European inbred lines (Table 1) . The number of polymorphic bands per AFLP primer combination ranged between 50 and 79 for EcoRI/MseI and between 43 and 54 for PstI/MseI with an overall average of 58. For each of the eight AFLP primer combinations, PIC values ranged between 0.25 an 0.31, whereas marker indices varied between 11.6 and 22.9 (Table 1). The percentage of bands observed per inbred line across all 462 polymorphic AFLP markers ranged between 27 and 34%. Individual polymorphic AFLP bands were present in between 1 and 50 out of all 51 inbred lines with a median of 11 lines. Genetic similarity (GS) estimates between replicated samples of the same inbred varied from 0.97 to 1.00 with an average of 0.98.

Genetic similarity estimates for unrelated line combinations of type flint x flint ranged from 0.47 to 0.77 and of type dent x dent from 0.45 to 0.69 with a mean of 0.57 and 0.55, respectively (Table 2) . Estimates for flint x dent combinations varied between 0.38 and 0.57 and had a significantly (P < 0.01) smaller mean of 0.49.

View larger version (59K): [in this window] [in a new window]   Fig. 1 Mean genetic similarity (GS) calculated from AFLP data of eight AFLP primer combinations for 23 European flint (A) and 25 European dent (B) inbred lines to unrelated (f = 0) lines from each heterotic group. White and solid bars refer to the mean GS in combination with lines from the same heterotic group and the opposite heterotic group, respectively. The overall mean GS for unrelated line combinations within and between heterotic groups is indicated by dashed and solid lines, respectively. CI = confidence interval

View larger version (26K): [in this window] [in a new window]   Fig. 2 Associations among 51 maize inbred lines revealed by principal coordinate analysis performed on genetic similarity (GS) estimates calculated from AFLP data of eight primer combinations. European flint and dent lines, and lines of mixed origin are designated by triangles, squares and crosses, respectively. PC1 and PC2 = first and second principal coordinates

Within the flint heterotic group, the F2-related lines (F2, D102, NS1, F240, F251, F1772, D503) were positioned in Quadrant III. Lines derived from EP1 (KW2, KW3, KW4, KW5, KW6) clustered in the upper part of Quadrant IV. Line D107 related to EP1 and F2 also fell within this spread, while lines D118, F251, and KW1 (related to EP1 and F7) were positioned adjacent to the EP1 group into Quadrant IV. Line DK105 and its backcross derivative D140 were located at the border of Quadrants III and IV.

Lines of mixed origin KW20 and CO255 were positioned within the EP1-related lines in Quadrant IV. Line F1444 with equal parts of flint and dent germplasm was placed near the center and occupied an intermediate position between flint and dent lines.

Comparison of AFLP with RFLP, RAPD, and Pedigree Data
Across all 51 inbreds, the Mantel Z statistic was significant only for the comparisons between the matrices of GS estimates of AFLP markers with RFLPs (P < 0.01) but not for the comparisons between the matrices of GS estimates of RAPDs with AFLP markers or RFLPs.

For unrelated (f = 0) pairs of lines, correlations among GS estimates based on AFLP markers, RFLPs, and RAPDs were intermediate (0.42 r 0.67) but highly significant (P < 0.01) for both flint and dent lines (Table 4) . In both groups, the largest correlation was obtained between GS estimates based on AFLP and RFLP data.

Prediction of GS based on f values was calculated by Eq. [3] (GS_exp) and by linear regression (GS_reg). Coefficients of determination (R²) for the linear regression of AFLP-based GS on f values were 0.62 for related pairs of flint lines (Fig. 3) and 0.69 for respective dent lines (Fig. 4). For both groups, GS_reg was below GS_exp. For f = 1.0, GS_reg was close to 1.0 for both flint (0.99) and dent (0.95) lines. No significant difference for the slope as well as the intercept was found between GS_exp and GS_reg in both groups of materials.

View larger version (21K): [in this window] [in a new window]   Fig. 3 Genetic similarity (GS) based on eight AFLP primer combinations as a function of coancestry (f) for 88 related (f > 0.0) pairs of flint (A) and 54 related pairs of dent (B) lines. GSEXP refers to the expected genetic similarity based on Eq. [3] and GSREG refers to the linear regression of GS on f. +: line combinations with D44

	Discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

Each of the eight AFLP primer combinations employed in this study discriminated most of the unrelated line combinations. The average marker index in our materials was 16.4 for AFLP markers (Table 1), but below 4.0 for both other marker types (Lübberstedt et al., 1999). The large marker index for AFLP markers was mainly attributable to the high average number of 69 AFLP bands per lane, while the PIC values per AFLP band were about equal or lower than those for RFLPs and RAPDs (Lübberstedt et al., 1999). Similar results were reported for U.S. maize germplasm (Pejic et al., 1998), barley (Russell et al., 1997), soybean (Powell et al., 1996), and wheat (Bohn et al., 1999). The higher marker index for EcoRI-MseI AFLP primer combinations (19.0) compared to PstI-MseI primer combinations (13.7) (Table 1) was mainly due to the larger number of polymorphic bands generated by EcoRI-MseI AFLP primer combinations (Table 1). The higher marker index of the AFLP markers in comparison with RFLPs and RAPDs together with the high reproducibility corroborates that AFLP markers are a valuable tool for identification of maize inbred lines, plant variety protection and registration as well as patenting of germplasm.

The GS values among unrelated flint x flint, dent x dent, or flint x dent lines based on AFLP markers exceeded 0.48 (Table 2) and, thus, were substantially higher than the respective RFLP-based (Messmer et al., 1992) but lower than the RAPD-based GS values (Hahn et al., 1995). A fundamental difference between these three marker systems is the mode of data scoring. Individual AFLP and RAPD bands are scored on a biallelic basis (marker band present or absent), whereas with RFLPs, usually multiple bands (alleles) per locus can be distinguished. A decreasing number of allelic marker bands per locus and an increasing frequency of "band present" scores per scoring unit (marker locus for AFLP markers or RAPDs, and marker allele for RFLPs) increases the level of GS values calculated by the formula of Nei and Li (1979). Another reason for elevated levels of GS values of AFLP markers and RAPDs might be the amplification of potentially more conserved organellar DNA sequences. However, extensive mapping experiments of two maize populations indicated a good genome coverage of AFLP markers and all of them represented nuclear DNA (Vuylsteke et al., 1999). Comparable results with respect to GS levels of different marker systems have been reported for U.S. maize germplasm (Pejic et al., 1998), while GS levels were similar between marker types for autogamous crops (Powell et al., 1996; Russell et al., 1997; Bohn et al., 1999).

Genetic Similarity of Related and Unrelated Lines
The mean of genetic similarity estimates between unrelated lines based on AFLP data was slightly higher within the flint (0.57) compared with the dent (0.55) heterotic pool (Table 2). This is in agreement with findings based on RFLP data (Messmer et al., 1992), whereas RAPDs resulted in a higher mean for the dent pool (Hahn et al., 1995). European flint germplasm originated from a few early introductions from the New to the Old World and this bottleneck presumably resulted in a limited genetic variability (Wallace and Brown, 1956). Likewise, strong selection for adaption to cooler growing conditions (Brandolini, 1969) might have narrowed down the genetic diversity of a broad range of early dent germplasm transferred from North America to Europe during the past four decades. However, the mean of GS estimates based on AFLP data for a wide range of U.S. dent inbred lines (0.53) obtained by Pejic et al. (1998) was similar to that of the European dent inbreds evaluated in this study. These findings do not support the hypothesis of a narrow genetic base within the European dent and flint pools. A comparatively broad genetic base of European inbreds might be due to a (i) wide range of flint and dent sources employed in the establishment of breeding populations, (ii) for dent materials adaption to cooler growing conditions without strict selection at the genotypic level because of the complexity of this character, and (iii) genetic variation generated de novo by accumulating recombination events or mutations.

For combinations of unrelated (f = 0) inbreds, close correlations of GS estimates based on different marker systems measuring genomic sequences "alike in state" are expected, if (i) several marker loci from both marker systems are closely linked and (ii) the average number of "band present" scores per scoring unit is similar. Substantially higher correlations were obtained between GS estimates based on AFLP markers and RFLPs both in the flint as well as in the dent pool compared to correlations with GS values based on RAPDs (Table 4). These findings are in agreement with another study in maize (Melchinger et al., 1998) but different from results obtained for wheat (Bohn et al., 1999), where correlations between GS estimates among unrelated pairs of lines determined on the basis of AFLP markers, RFLPs, and SSRs were not significant and below 0.27. Given the equal genomic distribution of the selected RFLP markers (Messmer et al., 1992), the AFLP marker loci employed in this study also seem to be evenly distributed across the genome.

For related pairs of lines, a linear relationship between f and marker based GS estimates can be expected on theoretical grounds (see Eq. [3]). The strength of the correlation r(f, GS) depends on (i) the association between the true values of f and GS and (ii) the repeatabilities of the estimates for f and GS (see Bohn et al., 1999). The latter are high, if the selected parent combinations display a large variance in f estimates and if the corresponding standard errors are small. Random drift and selection will introduce a bias and an increased standard error of f estimates. The standard error of GS estimates is reduced by (i) an equal distribution of markers across the genome, (ii) a large number of polymorphic bands, and (iii) a high scoring quality of a marker system.

Highly significant (P < 0.01) positive correlations above 0.42 were found between f and each of the four marker systems both in the flint and the dent heterotic pool (Table 4). In both germplasm pools, tighter correlations with f were obtained for RFLPs and AFLP markers (r 0.69) compared with RAPDs (r 0.49). In addition, for related pairs of lines the positive correlations between GS estimates based on RFLPs and AFLP markers were much closer than for any other combination of marker systems (Table 4). Similar findings were obtained with only four AFLP primer combinations representing about the same number of polymorphic bands as for the other two PCR-based marker types (Lübberstedt et al., 1999). In a comparable study of a wide range of dent germplasm (Pejic et al., 1998), again the closest correlations between f and GS estimates were obtained with RFLPs and AFLP markers. Therefore, RFLPs and AFLP markers seem to be more suitable than RAPDs in reflecting pedigree relationships between lines and produce a similar ranking of GS estimates. In conclusion, RFLPs and AFLP markers are convenient marker systems for (i) identification of closely related lines with unknown pedigree records, (ii) testing the validity of pedigrees in plant variety protection, (iii) determining the variation in genetic similarity among materials that show no variation for f, and (iv) planning crosses between genetically divergent parents to maximize the genetic variation in segregating generations.

Assignment to Heterotic Groups
One of the major objectives in hybrid breeding is the determination of heterotic pools, simplifying the choice of parent lines for the production of high-yielding hybrids. According to Messmer et al. (1992), it is no longer possible to classify lines as flint or dent by their geographic origin or endosperm type alone, because (i) more lines with mixed origin become available, (ii) breeders attempt to eliminate the weaknesses of the flint germplasm by introgressing dent germplasm, and (iii) traditional flint x dent hybrids are increasingly being replaced by pure dent x dent hybrids.

AFLP-based principal coordinate analysis (PCoA) clearly separated flint and dent inbred lines (Fig. 2) and placed lines of mixed origin (Table 3) according to their expected proportions of germplasm from each group (Fig. 2). Within both heterotic pools, subgroups of inbred lines were formed by PCoA according to common predominant ancestors such as F2, F7, EP1, and DK105 in the flint pool and CO125, W401, B14, and L103 in the dent pool (Fig. 2). These findings are in good agreement with RFLP-based PCoA of the same lines (Messmer et al., 1992), and confirmed by the significant Mantel Z statistic for the comparisons between the matrices of GS estimates of AFLP markers with RFLPs. This is also true with respect to Inbred D44, which was clearly separated from all other CO125 derived lines (Fig. 2; Messmer et al., 1992). Hence, RFLPs as well as AFLP markers are suitable for the determination of heterotic groups, whereas in our investigations formation of subgroups within the flint and dent heterotic pools was less obvious by RAPD-based PCoA (Hahn et al., 1995).

On the basis of mean GS estimates, all inbreds were unambiguously classified into the corresponding heterotic groups by means of AFLP markers (Fig. 1) and RFLPs (Messmer et al., 1992), whereas some lines displayed a higher mean GS with lines of the opposite pool as determined by RAPD markers (Hahn et al., 1995). In conclusion, RFLPs and AFLP markers proved to be a useful tool in grouping unknown flint and dent lines into the respective heterotic pools.

For comparison of the suitability of different marker systems for the assignment to heterotic groups, we calculated the coefficient for assignment to heterotic groups (CAH) by:

where _F, _D, and _FD are the mean GS estimates for unrelated pairs of flint, dent, and flint x dent line combinations. The highest CAH value was obtained for RFLPs (0.19), whereas intermediate (0.13) and low values (0.06) were found for AFLP markers and RAPDs, respectively. Similar CAH values can be derived from the study of Pejic et al. (1998) for all marker systems except for AFLP markers (0.23). This difference might be due to the plant materials employed or to different laboratory assay conditions for AFLP markers. In conclusion, RFLPs and AFLP markers were superior to RAPDs for the assignment of inbreds to heterotic groups.

In summary, AFLP markers and RFLPs are equally well suited and superior to RAPDs for different aspects of germplasm analysis. Operationally, AFLP markers have several advantages over RFLPs (Vos et al., 1995). Above all, the production of comparable amounts of AFLP compared with RFLP data is much less laborious and can be achieved at lower cost and in shorter time. Second, an unlimited number of AFLP markers can be generated without prior need of probe production, as is needed for RFLPs. Third, because AFLP markers are based on PCR, (i) less template DNA is required and (ii) automation is easier than for the multi-step procedure of generating RFLPs. Advantages of RFLPs are their (i) codominant inheritance and (ii) consistency over different crosses. However, AFLP markers have been shown by means of suitable software (Vuylsteke et al., 1999) to be scorable in a codominant manner. Moreover, more than 90% of the AFLP bands of identical size detected in two unrelated populations mapped to the same chromosome regions (Vuylsteke et al., 1999) indicating a good consistency of AFLP markers. In conclusion, out of the three marker systems evaluated in this study, AFLP markers seem to be most appropriate for various aspects of germplasm analysis.

	ACKNOWLEDGMENTS

 
We are indebted to Dr. M.M. Messmer for her excellent initial analyses (Messmer et al., 1992, 1993) on the same maize germplasm determined on the basis of RFLPs. RFLP analyses have been performed by Dr. J. Boppenmaier. We are also indebted to Prof. Dr. H.F. Utz for programing the bootstrap procedure.

Received for publication August 20, 1999.

	REFERENCES

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