The evolution of intraspecific molecular diversity of genes involved in morphological variation is of major importance to understand the history and adaptation of a species. The pattern of molecular diversity of particular genes is the result of complex interactions between different evolutionary forces such as selection, genetic drift and migration among populations. Most data of intraspecific nucleotide variation have been obtained in human or Drosophila species. During the past decade, however, an increasing amount of plant nucleotide diversity has been described, mainly in Arabidopsis and maize (see Ching et al., 2002; Nesbitt & Tanksley, 2002; Yoshida et al., 2004; Henry et al., 2005; Nordborg et al., 2005; Schmid et al., 2005; Wright et al., 2005; Wright & Gaut, 2005 for a review and references therein). Although most of these studies focused on nonrandom candidate genes, only some of them showed clear-cut evidence of selection, for genes involved in resistance to pathogens or herbivores (Stahl et al., 1999; Tian et al., 2002), flower development (Olsen et al., 2002), geographical adaptation (Morrell et al., 2003) or domestication (Wang et al., 1999; Tenaillon et al., 2004). In these studies, molecular signs of selection are deduced from values of the amount of diversity, frequency distribution of polymorphisms or haplotype frequency that are unexpected under neutrality hypothesis. However, all these parameters are also strongly affected by demography, population structure or nonrandom sampling that may affect molecular diversity at the genome level. Unambiguous identification of selective events has been made possible in species where numerous genes have been surveyed and compared, such as Arabidopsis thaliana (Aguade, 2001; Kuittinen et al., 2002; Nordborg et al., 2005; Schmid et al., 2005) and maize (Whitt et al., 2002; Tenaillon et al., 2004; Wright et al., 2005).
In Zea mays, where up to 50 loci have been surveyed, nucleotide polymorphism appears to be (i) very high when compared with other species, including plants, animals and humans (Buckler & Thornsberry, 2002), and (ii) lower in cultivated maize than in its wild relative Z. mays ssp. parviglumis, as a result of both selection and demographic bottleneck during domestication (Tenaillon et al., 2004; Wright et al., 2005). Selection has been shown to affect 4–20% of these genes during domestication, including Tb1, Ts2, D8, Y1 and Bt2 (Wang et al., 1999; Whitt et al., 2002; Tenaillon et al., 2004; Wright et al., 2005). Indeed, domestication is a particularly favourable context to reveal selective events as it occurred recently, i.e. around 10 000–5000 years ago (Iltis, 1983; Doebley, 1990a; Matsuoka et al., 2002), led to rapid and drastic modification of phenotypic traits such as plant architecture or grain weight, and may easily be studied through the comparison of present wild and cultivated forms. The promoter region of Tb1, a gene that affects plant architecture and flower sex, is one of the most striking examples of selection and reduced diversity resulting from maize domestication as almost no variability remains among cultivars whereas teosintes are strongly polymorphic (Wang et al., 1999; Clark et al., 2004).
Among all morphological traits that differ between the wild and domesticated forms in Z. mays, kernel weight shows both a strong variation between wild and cultivated forms and quantitative variability within maize. Endosperm weight and composition are important factors of both wild teosinte fitness, as endosperm resources are directly involved in seedling growth, and maize yield, kernel weight being one of the main criteria for human domestication and selection. Kernel weight mainly depends on the ability of plant to accumulate starch (80% of mature endosperm weight) in its endosperm. Several studies provide evidence that Shrunken2 (Sh2) affects seed weight through starch content in maize kernel endosperm. Sh2 encodes the large regulatory subunits of the heterotetrameric ADP-glucose pyrophosphorylase (AGPase) present in endosperm, whereas the small subunits are encoded by the Brittle2 (Bt2) gene. AGPase catalyses the first step of starch synthesis in plants, i.e. the production of ADP-glucose that is polymerized into the two starch compounds amylose and amylopectin (Preiss, 1988). Evidence for the important role of this enzyme was first provided by mutants where starch deficiency is associated with the loss of AGPase activity (Tsai & Nelson, 1966) and shrunken/brittle phenotype. Quantitative variation of AGPase activity has also been reported in normally developed endosperm and correlated with starch content (Stark et al., 1992; Prioul et al., 1994; Giroux et al., 1996; Greene & Hannah, 1998). Several quantitative trait loci (QTL) mapping studies in controlled maize populations revealed that a major genetic factor for grain yield (Stuber et al., 1992), kernel starch content (Goldman et al., 1993) and amylose content (Sene et al., 2000) co-localizes with Sh2 gene on chromosome 3L, suggesting that Sh2 is a candidate gene for these trait variations among cultivated maize. Finally, in a population derived from a cross between teosinte and maize, Doebley et al. (1994) showed that a QTL for seed weight also co-localizes with Sh2. Overall, these studies highlight the important role that Sh2 diversity may play in determining phenotypic variation of fitness traits such as kernel weight and composition, both among maize cultivars and between wild and cultivated forms. An analysis of diversity pattern of several genes involved in starch biosynthesis pathway in maize inbred lines and teosintes from the parviglumis subspecies suggested that Sh2 nucleotide diversity has been shaped by selective constraints predating domestication (Whitt et al., 2002).
In order to evaluate the role of Sh2 precisely in adaptive processes, we assess the molecular diversity of this gene among both wild and domesticated forms of Z. mays. Our study includes two annual teosintes, the most closely related to cultivated forms, Z. mays parviglumis and Z. m. mexicana, as well as cultivated accessions from different levels of human selection such as landraces (i.e. traditional populations cultivated in central America) and highly selected inbred lines. The particular design of our genotype sample allowed us (i) to evaluate the extent and structure of molecular diversity in Sh2, (ii) to determine the extent to which this gene shows evidence for selective processes, as a result of natural selection in the whole species and/or artificial selection during domestication (comparing teosintes and landraces) or since domestication (comparing landraces and inbred lines), and (iii) to describe the history of Sh2 gene within Z. mays species using phylogenetic reconstruction. Based on a large data set of homologous sequences, we were first able to confirm the observation of Whitt et al. (2002) that Sh2 was submitted to selection only prior to domestication. Moreover, as our data were carefully checked for polymerase chain reaction (PCR) artefactual mutation and PCR-mediated recombination, we were able to analyse linkage disequilibrium along the gene and construct a phylogenetic tree of Sh2 alleles, allowing better insight into the understanding of the evolutionary history of Sh2 in Z. mays.