Isolation of Pseudo Response Regulator genes and evaluation as candidate genes for photoperiod response loci
Liew, L.C.1, Hecht, V.1,
Weeden, N.2 and Weller, J.L.1
The genetic control of flowering in pea has been studied for more than five decades, and several loci affecting photoperiod response are known (1). Mutations at the Sn, Dne, Ppd or Hr loci result in early-flowering under short-day conditions (2, 3, 4, 5) whereas loss-of-function mutations in the PhyA or Late1 genes cause late flowering under long-day conditions (6, 7). In arabidopsis, many genes that affect photoperiodic flowering have a primary role in regulation of the circadian clock, and we recently showed that Late1 is the pea ortholog of the clock-related arabidopsis gene GIGANTEA (GI) (6). This study also showed that Late1 interacts genetically with Sn, and that sn mutant impairs the diurnal expression rhythm of Late1 (6). This provides the first direct evidence that Sn might be involved in the clock mechanism in some way.
One potential route to the identification of the Sn gene could be to assess homologs of arabidopsis circadian clock-related genes as candidates. We previously isolated several clock-related genes in pea (8), but found that none of them mapped close to known photoperiod response loci. However, the list of potential candidate genes for these loci has been extended with recent identification of additional clock-related genes in arabidopsis. Isolation of the corresponding pea genes has been greatly assisted by recent progress in sequencing of the medicago genome and advances in comparative genome analysis between medicago and pea (9, 10, 11).
Several recent studies have examined the contribution of the PSEUDO RESPONSE REGULATOR (PRR) gene family to the circadian clock mechanism (12, 13, 14, 15). This family includes the core clock gene TIMING OF CAB EXPRESSION 1 (TOC1) and four other members; PRR9, PRR7, PRR5, and PRR3. All five genes show diurnal and circadian regulation, with distinct peaks of expression that occur sequentially every 2 hours after dawn (12). This finding has suggested that like TOC1, other members of the “PRR quintet” might also form part of the central oscillator. We recently observed that the effect of the sn mutant on the expression of LATE1 is similar to the effect of the prr5 prr7 prr9 triple mutant on GI in arabidopsis (16), raising the possibility that in some respects Sn might act similarly to PRR genes. We therefore set out to isolate PRR genes in pea, in order to examine their potential role within the pea circadian clock, and also their potential identity as candidate genes for photoperiod response loci.
Materials and Methods
Sequences of PRR homologs from Medicago truncatula and other species were obtained using tBLASTn searches of the Genbank database (http://www.ncbi.nlm.gov) and the medicago EST database at http://compbio.dfci.harvard.edu. To isolate members of the PRR gene family in pea, degenerate primers were designed within conserved domains using the CODEHOP strategy (http://blocks.fhcrc.org/codehop.html) (17). The full length PsPRR37 and partial PsPRR59 cDNA were obtained by 5’ and 3’ RACE-PCR using the BD-SMART RACE cDNA amplification kit (CLONTECH). Protein alignments of various PRRs were performed with ClustalX (18) and adjusted using GENEDOC (Nicholas et al. 1997; http://www.psc.edu/biomed/genedoc). Relationships among PRR amino acid sequences were determined using phylogenetic analyses in PAUP* 4.0b10 (http://paup.csit.fsu.edu).
The origin of the WT line NGB5839 (cv. Torsdag le-3) and the sn-2 and sn-4 mutants have been described previously (19, 6). The sn-3 mutant is an additional recessive mutant isolated in the same screen as sn-4 (6). All plants were grown in the Hobart phytotron, using previously-described growth media, light sources and phytotron conditions (6).
Information and approximate map positions of pea genes in the bottom half of LGVII was obtained from several published maps (20, 21, 22). To identify medicago homologs of pea genes in this region, tBLASTx searches were performed against the medicago genomic database at the J. Craig Venter Institute (http://www.jcvi.org/cgi-bin/medicago/index.cgi). The map positions of relevant genes were obtained by using Medicago Genome Browser (http://gbrowse.jcvi.org/cgi-bin/gbrowse/medicago_imgag/).
Results and Discussion
Identification of PRR homologs in Medicago truncatula
PRR proteins are characterized by two conserved domains; the Pseudo-regulator (PR) domain and the CONSTANS, CONSTANS-LIKE, and TOC1 (CCT) domain (12). Several reports have identified PRR genes in a number of species including rice (23), Lemna gibba, L. paucicostata (24) and Populus trichocarpa (25). Phylogenetic analysis shows that these genes fall into three major groups, which can be designated as PRR1, PRR59 and PRR37, on the basis of the arabidopsis sequences they include (Figure 1). Two accessions from Lemna and one from rice (OsPRR59) show affinity to the PRR59 clade at the sequence level, but fall outside this group in the phylogenetic analysis, most probably because they do not contain complete sequences for both conserved domains.
BLAST searches of the Medicago truncatula genomic (NCBI) and EST databases identified seven distinct PRR sequences, including three genomic and four ESTs. Additional EST contigs corresponding to three of the genomic sequences were also identified. Four of these sequences were predicted to encode full-length PRR proteins, including two in the PRR59 clade and two in the PRR37 clade. The remaining three EST sequences were only partial. Two distinct ESTs from the 5’ region grouped with the partial pea TOC1 sequence described previously, while the other from the 3’ region did not show a clear relationship to any other PRR.
The three medicago BAC contigs containing the genomic PRR sequences have all been assigned map positions (www.medicago.org), on chromosome 3 (CR940305), 7 (AC150443) and 4 (AC149306). These positions predict positions for the corresponding pea sequences in the middle of LGIII, near the top of LGV, and in the bottom half of LGVII, respectively. We noted in particular that AC149306 was located in a region of chromosome 4 corresponding to the region of pea LGVII known to contain the Sn locus. Sn was previously reported to show close linkage with the amylase locus Amy1 (26) and was mapped between isozyme loci Aldo and Gal2 (27) on the lower section of pea linkage group VII. The similar positions of MtPRR59 (CR940305) and the Dne locus also suggested a possible candidate gene relationship.
Isolation of PRR homologs from pea
Using degenerate primers targeting the PR and CCT domains two distinct fragments from the PR domain were amplified by PCR. These sequences were extended to both 5’ and 3’ ends using RACE PCR to obtain one partial (70%) and one full-length coding sequence. Phylogenetic analysis showed that these sequences belonged the PRR59 and PRR37 clades, respectively, and they were designated as PsPRR59 and PsPRR37. Figure 1 shows that these sequences are apparent orthologs of the medicago PRR genes on CR940305 and AC149306.
Mapping of PRR genes and evaluation of PsPRR37 as a candidate for Sn
Sequencing of PRR59 and PRR37 from mapping parents JI1794 and “Slow” identified polymorphisms that were used to map both genes in the RIL population derived from these parents (22). The results confirmed positions for PRR59 in LGIII near Dne and for PRR37 in LGVII, consistent with the positions predicted by the location of the orthologous medicago genes. The relationship between PRR59 and Dne was not explored further, as Dne has been identified as the pea ortholog of the arabidopsis ELF4 gene (28). However, for PRR37, no recombination was detected with the Amy locus on LGVII, and as Amy was previously noted to be tightly linked to Sn (26), this indicated that PRR37 is in the region of Sn.
In order to carry out fine mapping of Sn, we also generated a new mapping population derived from a cross between the sn-4 mutant in the NGB5839 background and cv, Térèse. Unfortunately, we found that the coding region of PRR37 was identical in NGB5839 and Térèse, precluding the straight-forward mapping of PRR37 relative to Sn in this cross. Instead, the entire coding sequence of PRR was determined from the three known induced sn mutants and from their isogenic wild-type lines Borek (sn-2) and NGB5839 (sn-3 and sn-4). In all three cases the PRR37 coding sequence was identical in mutant and wild-type, indicating that the sn phenotype does not result from a mutation that affects PRR37 protein structure. It will obviously be of interest in future to isolate flanking sequence of PRR37 and identify an appropriate polymorphism that will allow the cosegregation of PRR37 and Sn to be directly tested.
A comparative map of the Sn region
As an aid to future mapping studies in the region of Sn, we generated a comparative map using markers anchored in published pea linkage maps and the medicago physical map (Figure 2). This identified a broad region likely to contain Sn, bounded approximately by the Aldo and Sod9 genes. In medicago, the physical map of this region is estimated to span approximately 5Mb, although it still contains several gaps where adjacent BAC contigs are not yet joined. We are now using this information as a basis for the design of additional markers for the mapping of Sn and for the identification of other potential candidate genes. The first step will be the mapping of Sn relative to other markers shown.
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