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Flowering time is an important fitness trait for species with short life cycles, in that flowering at the wrong time can result in the failure of a plant to reproduce. Thus, geographically widespread plant species often show extensive variation in flowering time (Riihimaki et al., 2005; Franke et al., 2006; Matsuoka et al., 2008), exhibiting genetically based clines for flowering time along latitudinal and/or altitudinal gradients, for example Solidago spp. (Weber & Schmid, 1998), Arabidopsis thaliana (Stinchcombe et al., 2004; Montesinos-Navarro et al., 2011), and Lythrum salicaria (Montague et al., 2008). Understanding the genetic mechanisms controlling flowering time, especially identifying the main genes responsible for natural variation in flowering time between different populations, is clearly important in determining how plants adapt locally and are able to reproduce over a wide range of latitudes and altitudes.
In the model plant A. thaliana, genes belonging to four main pathways (the vernalization, autonomous, light-dependent, and GA pathways) are involved in the control of flowering time (Mouradov et al., 2002; Amasino, 2010). Several flowering time pathways (e.g. vernalization and autonomous) converge on FLOWERING LOCUS C (FLC), a MADS-box transcription regulator that represses flowering (Crevillen & Dean, 2010). FLC expression levels are correlated with flowering time (Michaels et al., 2003; Lempe et al., 2005), and a flowering time quantitative trait locus (QTL) cluster has been found in a region including the locations of the entire FLC clade of transcription factor genes (Saloméet al., 2011). FLC and its activator FRIGIDA (Johanson et al., 2000; Choi et al., 2011) have been shown to be major determinants of flowering time variation in A. thaliana raised under experimental conditions (Le Corre et al., 2002; Michaels et al., 2003; Shindo et al., 2005), although circadian clock genes may possibly play an even more important part under natural conditions. Indeed, several circadian clock-related genes, such as CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), TIMING OF CAB EXPRESSION 1 (TOC1), CYCLING DOF FACTOR 3 (CDF3) and CONSTANS-LIKE 1 (COL1), were detected in an association mapping study of flowering time in A. thaliana (Brachi et al., 2010). Gene expression variation in the light-dependent pathway has been suggested to correlate with photoperiodic flowering in nonmodel species, such as soybean cultivars (Zhang et al., 2008) and common sunflower (Blackman et al., 2011). Although less is known about the control of flowering time in other plant species, the available data suggest that the same pathways are involved, although individual genes might have different importance (e.g. Lagercrantz, 2009).
Capsella bursa-pastoris is a herbaceous, predominantly selfing, tetraploid plant, notable for its wide geographical distribution (Neuffer et al., 2011). Capsella is a small genus that contains only three species: the tetraploid C. bursa-pastoris and the two diploids Capsella rubella and Capsella grandiflora. C. bursa-pastoris and C. rubella are selfers, while C. grandiflora is an outcrosser. A recent study indicates that C. bursa-pastoris is an autopolyploid of C. grandiflora (St. Onge et al., 2012). Because Capsella is one of the most closely related genera to A. thaliana (German et al., 2009; Franzke et al., 2011), it is easy to transfer molecular genetic resources developed for A. thaliana to C. bursa-pastoris. There are both winter-annual (late-flowering) ecotypes and summer-annual (early-flowering) ecotypes in China, and investigations in Europe and America have revealed significant geographical differences in flowering time in the species (Neuffer et al., 2011). For example, ecotypes in Scandinavia and southern Spain flower early, whereas ecotypes from intermediate latitudes flower late (Neuffer & Hurka, 1986; Neuffer & Bartelheim, 1989; Neuffer & Hoffrogge, 2000). Besides, C. bursa-pastoris showed clinal differentiation in flowering time along a 2500 km latitudinal transect in Russia: the most northern and most southern provenances flowered earlier than intermediate provenances (Neuffer, 2011). Flowering time is delayed with altitude in alpine climates in the Alps in Europe (Neuffer & Hurka, 1986) and the Sierra Nevada in North America (Neuffer & Hurka, 1999), whereas populations at high elevations in subarctic regions such as Norway (Neuffer & Hurka, 1986) and at locations where summers are hot and dry, as in southern Spain, flower early (Neuffer & Hoffrogge, 2000).
The evolutionary history of C. bursa-pastoris may have contributed to the population differentiation of flowering time. C. bursa-pastoris is believed to have originated in the eastern Mediterranean region, and subsequently spread westwards to Europe where introgressive hybridization with diploid C. rubella took place, and eastwards to Asia where C. rubella does not grow (Hurka & Neuffer, 1997; Ceplitis et al., 2005; Slotte et al., 2008). C. bursa-pastoris was very recently introduced to North America by European settlers, and variation patterns of flowering time there can be traced back to the introduction of preadapted genotypes (Neuffer & Hurka, 1999). Worldwide surveys of nuclear/chloroplast genetic diversity in C. bursa-pastoris have revealed limited variation within the species and suggest that it recently went through a rapid expansion such that ecotypic differentiation of flowering time is likely to have evolved recently (Ceplitis et al., 2005; Slotte et al., 2006, 2008). The latter is likely to be true, particularly for the species in China where the species has been shown to exhibit much lower nuclear genetic diversity and a less pronounced genetic structure than in Europe (Slotte et al., 2008, 2009). Although Chinese C. bursa-pastoris is derived from European material that spread to eastern Eurasia relatively recently (21–64 ka) (Slotte et al., 2008), the species is today widely distributed across China occurring in a broad array of complex environmental conditions ranging from subtropical climatic conditions in the south to more extreme environments in the northwest and northeast. Chinese C. bursa-pastoris can thus be used as an independent replicate to studies carried out in the European part of the species range to address questions about the importance of shared ancestry and parallel evolution in the development of flowering time clines in different parts of the world.
In Capsella, Linde et al. (2001) found that three major QTLs accounted for onset of flowering, and this was the first evidence of the multigenic control on this trait. Later studies have suggested that there are both differences and similarities in the genetic control of flowering time in the European and Chinese parts of the natural range. An association study between flowering time and sequence polymorphism at FLC, FRIGIDA, CRYPTOCHROME 1 (CRY1) and LUMINIDEPENDENS (LD) revealed that single nucleotide polymorphisms (SNPs) at CRY1 and FLC were significantly associated with flowering time variation in western Eurasia, whereas in China CRY1 was monomorphic and a different SNP at FLC was significantly associated with flowering time variation (Slotte et al., 2009). Other flowering time genes, such as FRIGIDA, CRY1 and LD, were either monomorphic or exhibited almost no sequence variation in the Chinese plants studied, despite notable variation in flowering time (Slotte et al., 2009). On the other hand, Slotte et al. (2007) found good agreement of flowering time gene expression differences in comparisons between two pairs of accessions, one pair comprising an early-flowering accession from Taiwan and a late-flowering accession from northern Europe, and the other comprising both early- and late-flowering accessions from California. They noted that this could indicate that the genetic basis of expression differences is shared by common ancestry, or that similar regulatory differences have evolved in parallel. Interestingly there were many key circadian clock genes among the genes that were differentially expressed between early- and late-flowering accessions. Further analysis of gene expression differences among different flowering C. bursa-pastoris ecotypes from a broad array of environmental conditions in China would help to clarify the situation further.
In the present study, we broadened the analysis of flowering time variation in C. bursa-pastoris to samples collected from multiple environments in China. We also constructed gene expression profiles of 12 different samples representing extremes of flowering time using the Solexa/Illumina’s Digital Gene Expression (DGE) system. The DGE system allows an examination of variation in gene expression across many genes at the same time and has been successfully applied to studies of gene expression in different animal species (Harhay et al., 2010; Veitch et al., 2010; Pemberton et al., 2011), including transcriptome response to virus infection (Hegedus et al., 2009; Basu et al., 2011). A few studies have also used DGE to look at gene expression differences in plant species (e.g. maize (Eveland et al., 2010) and cucumber (Qi et al., 2012)). The aims of the study, therefore, were to examine the pattern of flowering time variation for C. bursa-pastoris in China; to examine further whether circadian clock genes are strong candidates for the evolution of adaptive flowering time variation as indicated previously (Slotte et al., 2007; K. Holm et al., unpublished); and to assess whether C. bursa-pastoris plants exhibit different patterns of genetic variation from one another for candidate genes likely to affect flowering time across the broad range of environments they face in China.