ABSTRACT. Plant breeding deals with high-yielding genotypes. However, how best to choose parents of these genotypes remains an unsolved question. Here, we focus on a priori choice based on parental distances by means of agronomic and molecular data. Despite numerous theoretical and empirical studies, a priori choice continues to be a controversial procedure. Both success and failure are commonly reported. We looked at these ambiguous results in order to investigate their possible causes. A total of 139 articles on genetic divergence were sampled to examine aspects such as type and number of markers utilized. We suggest that the mean number of 160, 281 and 25 for RAPD and RFLP markers, and SSR loci, respectively, which we found in these papers, should be increased for accurate analysis. A second sample composed of 54 articles was used to evaluate the divergence-heterosis association. Most of them (28) detected positive divergence-heterosis association, whereas 26 revealed negative or inconclusive results. We examined several causes that influence a priori choice positively and negatively.

Key words: A priori choice of hybrid parents, Morphological markers, Molecular markers, Plant species

INTRODUCTION

Plant breeding deals with high-yielding genotypes. However, how best to choose parents of these genotypes remains an unsolved question. Research on parent selection may be approached in two ways (Baenziger and Peterson, 1992): a priori and a posteriori choice. The former consists of selection methods based on per se parent performance, such as midparental value, divergence according to coefficient of parentage, character complementation, multivariate analysis and parental distances, least squares, parental complementation, and ideal genotype. In the latter, parents are evaluated on the basis of F₁, F₂ data and advanced generations. A long period of time is necessary to choose parents in this way, especially in perennial plants. Here, we focused on a priori choice based on parental distances determined from agronomic and molecular data. Its predictive nature avoids the need for hundreds of crossings, as only predictively promising crosses are made and evaluated, saving labor, financial resources, materials, and time.

The a priori choice is based on the fact that heterosis is a relative measure of two generations - the parental and the progeny. For a given quantitative trait, the amount of heterosis following a cross between two populations is a function of the square of the difference of gene frequency between the populations and of the dominance deviations (Falconer, 1989). If the populations that are crossed do not differ in gene frequencies, there will be no heterosis. Likewise, loci without dominance cause neither inbreeding depression nor heterosis. Falconer (1989) argues that: i) the occurrence of heterosis is dependent on directional dominance and its absence is not sufficient grounds for concluding that the loci show no dominance; ii) the amount of heterosis is specific to each particular cross, and iii) for inbred lines, heterosis is the sum of the dominance deviations of those loci that have different alleles in the two lines.

When the divergence-heterosis association is found to be high, it is possible to use the divergence estimate as a solid criterion for parental selection and, subsequently, for the synthesis of heterotic hybrids, as argued by Dias and Resende (2001). The a priori choice was initially made based on morphological descriptors and further reinforced by data from molecular markers; this choice assumed that the divergence between any two parents expresses the allelic differences between them. Despite numerous theoretical and empirical studies, a priori choice continues to be a controversial procedure. Success and failure are common results in these studies (Dias et al., 2003). We examined these ambiguous results in order to investigate the causes.

MATERIAL AND METHODS

First, a total of 139 articles (Table 1), dealing with genetic divergence, were sampled to investigate the causes of the ambiguous results. In spite of the limitations and difficulties inherent to this type of sample, some of the aspects of this kind of study, such as type and number of markers, could be examined. Subsequently, a second sample was investigated; this time it was composed of 54 articles (Table 2), involving 21 plant species, to evaluate the divergence-heterosis association in particular.

RESULTS AND DISCUSSION

Genetic divergence

Genetic distance estimates were mostly based on morphological, biochemical (isoenzymes), and molecular markers [random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and sequence-tagged site-polymerase chain reaction (STS-PCR)] (Table 1). Some of the articles also used pedigree information (relationship coefficient) separately, or in combination with other markers. In the measures of divergence, RAPD markers were most commonly used, comprising the largest number of species and reported papers (Table 1). This observation may be due to the fact that the RAPD technique (Williams et al., 1990) facilitates automation, and has a low operational cost, besides its simplicity, speed, and good degree of polymorphism, in addition to wide genome coverage. Such features mitigate its negative properties, such as low reproducibility and dominant heritage.

RFLP markers are frequently used for estimating genetic diversity. These markers are co-dominant, reliable, and they have a high information content; however, this technique demands a considerable amount of good quality DNA, it is laborious, and it often requires the use of radioactive substances. The use of fluorescence, instead of radioactivity, with a well-equipped laboratory and good staff gives this system a further advantage. RFLP has the advantage of allowing the selection of probes that target desirable DNA sequences associated with specific yield or resistance traits. Some of the probes used in the studies that succeeded in associating genetic distance with hybrid prediction were cDNA clones. These advantages help explain why RFLP is the most commonly employed in research on genetic distance-heterosis associations (see Table 2).

When compared to other kinds of molecular markers, AFLP is promising, as it combines the specificity, resolution and sampling power of enzyme restriction, with the simplicity of PCR polymorphism. Furthermore, the large number of markers generated with this technique is obvious from Table 1. However, it gives limited genetic information per locus, and it is a dominant marker, as is RAPD, from which it differs by a need for higher quality DNA and more protocol steps (Ferreira and Grattapaglia, 1998). On the other hand, microsatellites or SSR are the most polymorphic markers. SSR polymorphism is based on differences in simple repetitive sequences that are flanked by conserved borders; they are distributed all over the genome, making them the most suitable for paternity studies. It is important to bear in mind that the information content does not differ significantly among RFLP, AFLP and SSR, as argued by Bohn et al. (1999). For a detailed review of biochemical and molecular markers, see Ferreira and Grattapaglia (1998).

In search of the optimum number of bands, Tivang et al. (1995) observed that, regardless of the restriction enzyme used, the average number of polymorphic RFLP bands provided an equivalent amount of information, although 284 to 377 bands were necessary for genetic distance estimates for 37 inbred maize lines associated with a fixed 10% coefficient of variation. Similarly, Fanizza et al. (1999) evaluated 10 accessions of Vitis vinifera and concluded that the optimum number of RAPD markers for evaluating genetic divergence was above 400. The cluster formed with 400 markers did not present distortion when compared to the cluster formed with all polymorphic markers (932 bands), and the coefficient of variation of the genetic distances was only 5%. Picoli et al. (2004) obtained similar results in a study of 84 Eucalyptus genotypes. In spite of the limitations that this value (~400 bands) may have for other gene pools or plant species, it is a reference point that should be taken into account in diversity studies. Based on these findings, the mean number (160) of markers that we found in our sample (Table 1) should have been larger.

An analogous effort was made for microsatellites, for which 44 primer pairs were required for a correlation value of 95 and 6.44% stress, comparable to a standard sample of 57 primer pairs (Moraes, 2003). This suggests that the respective mean numbers of 160, 281 and 25 for RAPD and RFLP markers, and SSR loci that we found in the articles that we examined (Table 1) should be larger to achieve accurate analysis. The low number of markers seems to be the major drawback for the use of isoenzymes (Table 1), besides the restricted coverage of the genome, weak correlation with other markers and possible environmental influence (Tsegaye et al., 1996). To some extent, morphological markers suffer these same limitations, although phenotype assessment is essential for evaluating the traits of interest.

Divergence-heterosis association

Noteworthy in the second sample of articles (Table 2), 28 of them detected positive divergence-heterosis association, whereas 26 revealed negative or inconclusive results. There are several reasons that could explain these results. Besides the deviations of dominance, genotypic divergence and complementation already reported, additional conditions for divergence-heterosis association have been inferred on the basis of simulation studies (Bernardo, 1992): i) manipulation of traits with high heritability; ii) variation of the allelic frequencies of the parents within narrow limits; iii) that at least 30 to 50% of quantitative trait loci (QTL) be linked to the markers, and iv) that less than 20 to 30% of the markers be randomly distributed or not linked to QTL.

In practice, the success of a priori choice has confirmed that moderate/high heritability of the traits is decisive (Dias and Resende, 2001; Dias et al., 2003), as much as marker linkage to QTL (Vencovsky and Rumin, 2000), based on items i and iv, above. Nevertheless, this last condition may only be partially valid in species for which linkage maps are unavailable, where wide genome coverage may result in a “blind” prediction, according to item iv. Additionally, the parents with maximum relative divergence will not necessarily originate the most heterotic crossings, in agreement with item ii. For instance, Dias and Resende (2001) and Dias et al. (2003) found higher frequencies of heterotic hybrids and a larger magnitude of heterosis for yield components in the crosses involving parents of moderate divergence. They obtained success with a priori choice of hybrid parents, using yield components, as well as DNA markers (RAPD).

There are many other conditions negatively influencing a priori choice, such as: i) increased genetic similarity in a gene pool due to strong selection pressure (Barbosa et al., 2003); ii) lack of linkage between genes controlling the traits and the markers used (Bernardo, 1992); iii) differences in the contributions of the marked DNA regions (Kwon et al., 2002a,b); iv) gene pool with a narrow genetic base (Marrof et al., 1997); v) a lack of linkage disequilibrium (Charcosset et al., 1991); vi) epistasis (Boppenmaier et al., 1992); vii) high degree of improvement of the gene pool used (Dias et al., 2003), and viii) genotype-environment interactions (Dias et al., 2003). These remarks suggest that divergence and heterosis do not always associate linearly. Non-linear relationships support this reasoning, as revealed by Sant et al. (1999), a feature that may explain the erratic results in many studies.

ACKNOWLEDGMENTS

The authors are grateful to FAPEMIG, CNPq and CAPES for financial support.

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