Variability among inbred lines and RFLP mapping of sunflower isozymes

Eight isozyme systems were used in this study: acid phosphatase (ACP), alcohol dehydrogenase (ADH), esterase (EST), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), phosphoglucoisomerase (PGI), 6-phosphogluconate dehydrogenase (PGD), and phosphoglucomutase (PGM). The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map.


Introduction
Sunflower (Helianthus annuus L.) is a diploid (2n = 34) species, which is second only to soybean in its importance as an annual oil seed crop.Despite its economic value, the number of simply inherited genes identified in sunflower is relatively small (Miller, 1992), and there is no classical genetic map for this species.Several sunflower RFLP linkage maps were published recently (Berry et al., 1995;Gentzbittel et al., 1995Gentzbittel et al., , 1999;;Jan et al. 1998), but unfortunately the relationship between these different maps is unknown, because the vast majority of the markers are not publicly available.The mapping of major gene loci, such as the Rf1 locus (Gentzbittel et al., 1995;Berry et al., 1997;Jan et al., 1998), the downy mildew resistance gene cluster (Vear et al., 1997), Orobanche resistance (Lu et al., 2000) and the Hyp-1 locus (León et al., 1996), as well as protein markers, such as seed storage proteins (Serre et al., 2001) and isozymes, will provide landmarks allowing the alignment of some linkage groups.
Isozymes have been used to assess genetic variation in both domesticated and wild sunflower populations (Dry and Burdon, 1986;Rieseberg et al., 1990;Cronn et al., 1997), as well as to establish phylogenetic relationships and speciation mechanisms within the genus Helianthus (Rieseberg et al., 1991;1998).They have also been used to study intra specific variation in both wild (Carrera and Poverene, 1995;Carrera et al., 1996) and cultivated sunflowers (Carrera and Poverene, 1991;Quillet et al., 1992), as well as to identify interspecific hybrids (Carrera et al., 1996).In cultivated sunflower, Quillet et al. (1992) demonstrated that eight polymorphic isozyme loci could discriminate between distantly related inbred lines, whereas Tersac et al. (1994) were able to group cultivated populations according to their geographic origin by using isozyme data.Unfortunately, the relatively small number of isozyme systems limits their use in genetic mapping studies.However, isozymes are still routinely used by seed companies for seed purity testing of both inbred and hybrid seed lots, because, in comparison with RFLP markers, the assays are relatively cheap and quick to run.Therefore, knowledge about the linkage arrangement of isozyme loci would allow a more even sampling of the sunflower genome.Berry et al., (1997) released a RFLP sunflower map for public research, comprising 81 loci covering all 17 linkage groups.To date, two classical genetic markers, the CMS restoration Rf1 gene (Kinman, 1970) and the HAG-5 gene controlling 2S albumin (Allen et al., 1987), were included in this public map.The objectives of this study were: i) to investigate the level of genetic variation of isozymes in 25 elite sunflower inbred lines, and ii) to map polymorphic isozyme loci onto the public RFLP linkage map of cultivated sunflower.

Plant material
Twenty-five inbred lines were studied by electrophoretic assay (Table I), including the parents of four F 2:3 populations, which had previously been used for RFLP mapping studies.Ten seeds from each inbred line were analysed, in order to test for uniformity within the lines.The genotype of each F 2 plant was re-created by bulking 8-9 seeds from their F 3 progenies obtained by selfing.

Data analysis and map construction
The genetic similarity between two inbred lines was estimated according to Nei (1987), using the BIOSYS-1 program (Swofford and Selander, 1981) The genetic distance matrix was used to construct a phenogram by the UPGMA clustering method.The reliability of the UPGMA tree was examined using bootstrap resampling analysis, through a new version of the same BIOSYS-2 program, to construct a consensus tree through Phylip3.5C(Felsenstein, 1995).Various standard measures of genetic variation were also calculated, including the proportion of polymorphic loci (P), the mean number of alleles across all loci (A), the mean number of alleles per polymorphic locus (Ap), and the polymorphic index (PI; defined as 1 -Σp i 2 , where "pi" is the frequency of the i th allele).
The isozyme loci were mapped onto the public RFLP sunflower map.To construct this map, 81 genomic and cDNA probes were selected, and detected loci covering approximately 1200 cM of the sunflower genome, arranged into 17 linkage groups, as was demonstrated by segregation data from nine different F 2 populations (Berry et al., 1997).Isozyme linkage maps were constructed using data from four out of nine F 2 populations.The F 2 plants were self-pollinated to produce F 2:3 families, which were used as mapping populations (Table III).The segregation of the alleles at each locus was checked against the expected ratios for a codominant marker in a F 2 population, using a chisquare test with a significance level of 5%.The linkage analyses were (performed with) made using Mapmaker version 3.0 (Lander et al., 1987).A constant LOD score of 3.0 was used.The Haldane function was used to obtain the cM values.The isozyme loci were placed in the map by using the "try" command.The "error detection" command was used to check for mistakes in scoring and data entry, and for double crossover events.Linkage group and RFLP loci are named according to Berry et al. (1997).

Polymorphism and genetic distance
The eight enzyme systems assayed revealed 19 loci, with a total of 28 alleles (A = 1.47).The average number of alleles per polymorphic locus (Ap) was 2.125.All polymorphic loci were bi-allelic, except for Est-1, which was tri-allelic.Eleven loci were monomorphic in the tested lines (Adh-1, Adh-2, Gdh-1, Pgm3,, and eight loci were found to be polymorphic (Acp-1, Est-1, .The proportion of polymorphic loci (P) was therefore 0.42.There were 17 different genotypes, 12 (48%) of which were unique within this set of material (Table II).The PI ranged from 0.08 for Pgi-2 to 0.57 for Est-1, the only system revealing three alleles, with a mean value of 0.36.No intra-line heterogeneity was observed, except for ZENR15 (W21), which had two Acp-1 genotypes.The isozyme data were used according to Nei (1987), to calculate the genetic distance between all possible pairs of lines, and the result was used to construct the phenogram shown in Figure 1.The consensus tree, obtained by 100 bootstrap resampling of loci (not shown), resembled the original topology showed in Figure 1.Major groups of restorer and maintainer lines were similar in both the UPGMA and the consensus tree, although groups were resolved with low bootstrap values.
Variability and mapping of sunflower isozymes 67

Zymograms
Pgd-3 zymograms obtained from artificial bulks showed that a given genotype could be detected even in samples with a 1:8 ratio.The sensitivity of the method was lower for the other enzymes, and so three bulks of three seeds for each F 2:3 family were used.The patterns of the Mdh-1, Mdh-4 and Pgm-1 loci could not be resolved in the F 2:3 populations.These enzymes are under the genetic control of several loci encoding for cytoplasmic and organelle variants.Moreover, enzyme monomers coded by different genes can combine to produce intergenic heterodimers, making the identification of homo-and heterozygous individuals uncertain.The following five loci were selected for linkage analysis: Acp-1, Est-1, Gdh-2, Pgd-3, and Pgi-2.Examples of zymograms obtained in the F 2:3 mapping populations are shown in Figures 2 and 3.The number, spacing and relative intensity of bands of heterozygous individuals were in agreement with the expected quaternary structure of active enzymes and the ploidy level.Acp-1, Pgd-3 and Pgi-2 presented either a single band or a three-banded pattern consistent with a dimeric structure.Gdh-2 patterns had one or multiple bands according to a tetrameric structure, but low resolution was observed in heterozygotes at this locus, due to the small distance between the bands.Est-1 patterns were particularly complex, with single alleles coding for multiple bands, but allelic variants could be unambiguously identified.

Mapping
The five polymorphic loci (Acp-1, Est-1, Gdh-2, Pgd-3, and Pgi-2) showed co-dominant expression, and their segregation in the F 2 populations fitted the expected 1:2:1 ratio (Table III).These isozyme loci were mapped to linkage groups 2, 3, 9, 14 of the public RFLP map (Figure 4).The Acp-1 and Pgd-3 loci were found linked to the marker ZVG0005 on group 2, and the distance between these loci was estimated to be 11.3 cM.The Est-1 locus mapped to group 3, in the interval flanked by ZVG0009 and ZVG0010, and this position was confirmed in two separate mapping populations (Table III).Gdh-2 was located 2 cM above ZVG0062 in group 14, and Pgi-2 mapped to group 9, between ZVG0040 and ZVG0041.

Genetic variation at sunflower isozyme loci
The following 15 isozyme systems have been reported to reveal useful polymorphism in cultivated sunflower germplasm: aconitase (ACO), acid phosphatase (ACP), alcohol dehydrogenase (ADH), esterase (EST), glutamate dehydrogenase (GDH), glutamate oxaloacetate transaminase (GOT), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), malic enzyme (ME), peroxidase (PRX), phosphogluconate dehydrogenase (PGD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM) and shikimate dehydrogenase (SKDH) (Lay et al., 1988;Quillet et al., 1992;Tersac et al., 1994;Cronn et al., 1997;Mestries et al., 1998).Eight of these systems were screened in this study, revealing nineteen loci, eight of which were polymorphic.The observed values of P (0.42), A (1.47) and Ap (2.125) were very similar to those reported by Cronn et al. (1997), who studied isozyme polymorphism in 700 plants from both oilseed and confectionary sunflower accessions.Except for Mdh-3, the loci that were found to be monomorphic in the 25 elite inbred lines had previously also been found to be monomorphic in cultivated sunflower germplasm (Carrera andPoverene, 1991, 1995;Quillet et al., 1992).Chi-square probability compared to the expected 1 :2: 1 segregation ratio.In comparison with the results obtained by Quillet et al. (1992), this set of isozyme markers was far less informative.Quillet et al., (1992) were able to distinguish 45 out of 52 lines (86.5%) by using eight systems, whereas, in this survey, only 12 out of 25 lines (48%) could be uniquely distinguished.This apparent reduction in discriminatory power was probably due to a narrower sampling of the sunflower germplasm represented by this small set of inbred lines (Table I).

Comparison between isozyme and RFLP markers in sunflower
The mean number of isozyme alleles per polymorphic locus (Ap = 2.125) was lower than the values reported for RFLP markers (Berry et al., 1994;Zhang et al., 1995).A number of closely related lines was used in this study (e.g., all the B lines in the cluster ZENB8 to HA89 are sister lines derived from a cross between these two females).This narrow sampling of germplasm (would) could reduce the level of polymorphism at the isozyme loci studied.One allelic form was found to predominate at each isozyme locus (Table II), as reported by Cronn et al. (1997) in a much larger germplasm survey.This is reflected by the low PI values for each isozyme marker (Table II), which made it necessary to use a number of different F 2:3 populations, in order to map as many loci as possible (Table III).
Cluster analysis (both UPGMA and bootstrap) of the isozyme fingerprinting data (Figure 1) revealed that the eight polymorphic loci were capable of separating the majority of CMS maintainer (B) lines from the restorer (R) lines.What makes the statistical separation possible are the differences in allele frequency between the B and R germplasm pools.For example, the Est-1a allele was only found in the B lines.However, the discrimination between males and females was not absolute.For (example) instance, the males ZENR8 and ZENR10 clustered more closely with the females, (and) whereas SD and 0043 clustered with the males.This undoubtedly reflects the narrowness of the germplasm used, the low level of isozyme polymorphism, and the poor genome coverage offered by the eight polymorphic isozyme systems.Studies with large numbers of molecular markers (Berry et al., 1994, Zhang et al., 1995;Hongtrakul et al., 1997) give a better resolution and tend to reflect the pedigree more closely.

Genetic mapping
There have been several reports on the inheritance of isozyme loci in populations of both wild and cultivated sunflower (Kahler and Lay, 1985;Torres, 1983;Rieseberg et al., 1993 and1995;Quillet et al., 1995), and several pairs of loci have been found to be linked (Table IV ).Mestries et al. (1998) were the first authors who reported the mapping of a number of isozyme loci onto a proprietary RFLP linkage map.These authors mapped the following 6 isozyme loci to five different linkage groups in a single F 2 population: Got-1, Idh-I, Me, Pgd-2, Pgi-3 and Sdh.Pgi-3 and Idh-1 were found to be linked (Table IV).However, the present report is the first one on the mapping of sunflower isozyme loci onto a public linkage map, comprising 17 linkage groups.The five polymorphic isozyme loci, Acp-1, Est-1, Gdh-2, Pgd-3, and Pgi-2, mapped to four different linkage groups, with Acp-1 and Pgd-3 linked to group 2. Genetic linkage between an acid phosphatase locus (Acp-1) and a phosphogluconate dehydrogenase locus (Pgd-1) was originally reported by Lay et al. (1988), with a recombination fraction of 0.05.This value of 0.05 equates to approximately 5 cM, using either Kosambi or Haldane mapping functions (Lynch and Walsh, 1998), and is similar to the figure of 11.3 cM determined in this study.However, Lay et al. (1988) used a different nomenclature system to code their isozyme loci, which is an important consideration when comparing loci in different publications.Thus, it is possible, for example, that the PGD and PGI loci mapped by Mestries et al. (1998) are the same as those reported here.
Several isozyme systems revealed duplicated loci (Figure 2) in the sunflower genome, but unfortunately none of these duplications could be mapped in the F 2:3 populations.RFLP markers also detect duplicated loci (Berry et al., 1996), and these observations are consistent with the hypothesis that sunflower is an ancient polyploid (Jackson and Murray, 1983).The adoption of a standard nomenclature system for isozyme loci in sunflower, and the continued mapping of additional loci onto existing RFLP linkage maps, will provide an additional set of freely available public markers.Mapped isozyme loci, along with classical genetic markers and public microsatellites, will certainly help to integrate the various published sunflower maps.

Figure 1 -
Figure 1 -Dendrogram of Neil distances constructed using UPGMA method for the 8 polymorphic isozyme systems.

Figure 4 -
Figure 4 -The map position of five isoenzyme loci on four sunflower RPFL linkage groups.Linkage group and RFLP locus nomenclature is according to Berry et al. (1997) and map distances are given in centi Morgans.Isozyme loci are indicated in bold.

Table I -
Origin of the sunflower inbred lines used in this study.

Table II -
Allozyme genotypes observed in 25 sunflower lines and the polymorphic index (PI) value for each of the isozyme marker loci.

Table III -
The segregation ratio of the five polymorphic isozyme marker loci in the F 2:3 mapping populations