Possible Origin of Two Variants of a Carnation 1-Aminocyclopropane-1-carboxylate Synthase Gene, DcACS1a and DcACS1b, as Suggested by Intron Structure in Homologous Genes in Dianthus Species

Abstract

The intron structures of two variants of 1-aminocyclopropane-1-carboxylate synthase (ACS) genes (DcACS1a and DcACS1b) in carnation (Dianthus caryophyllus) and genes homologous to them (ACS1 homologous genes) in other 10 Dianthus species (16 strains in total) were studied by comparing the sizes of the PCR amplificates and nucleotide sequence of the introns. All 16 sequenced homologous ACS1 genes, including DcACS1 genes themselves, had five exons and four introns. The exons had similar nucleotide sequences and consequently similar deduced amino-acid sequences. The sizes of three introns (intron-1, -2, -3) were variable among the homologous genes, whereas that of the fourth intron (intron-4) was almost identical. The variation in introns was probably caused by the insertion (and deletion) of nucleotide fragments of given lengths. Interestingly, the 3'-UTR of DcACS1a was different from that of DcACS1b, and the latter was similar to other 14 ACS1 homologous genes. Moreover, the length of Thr repeat in the C-terminal region was long in DcACS1a protein but short in DcACS1b protein, and the latter resembled ACS1 homologous proteins in other Dianthus species. The present findings suggest that (1) the variation in intron structure between two variants of carnation DcACS1 is reminiscent of the variation that occurred universally in Dianthus species, (2) DcACS1a is probably a gene intrinsic to carnation, and (3) DcACS1b was acquired from another, as yet unknown, Dianthus species, in the course of breeding modern carnation cultivars.

Allelic Nature of Two Carnation Variants 1-Aminocyclopropane-1-carboxylate (ACC) Synthase Genes, <i>DcACS1a</i> and <i>DcACS1b</i>

“…The first group showed the same band pattern as that of 'LPB', which includes 'Cherry Tessino', 'Scarlett Ostera', and 'Carnet'. 'LPB' was shown to have DcACS1a and DcACS1b (Harada et al, 2011;Satoh et al, 2011). In this group, PCR products of 510 nt, 950 nt and 730 nt for region 1, region 2, and region 3, respectively, came from DcACS1a, whereas those of 590 nt, 630 nt, and 670 nt, for region 1, region 2, and region 4, respectively, came from DcACS1b (Harada et al, 2011;Satoh et al, 2011).…”

“…Then, to confirm complete nucleotide sequences, we amplified full length genomic DNAs with primers derived from both ends of the composite genomic DNAs and total DNAs obtained from corresponding Dianthus species. The forward and reverse primer pair for full length cloning of DcACS1 corresponding genes was as described in Satoh et al (2011). PCR products covered from 5'-UTR until 3'-UTR.…”

“…'LPB' was shown to have DcACS1a and DcACS1b (Harada et al, 2011;Satoh et al, 2011). In this group, PCR products of 510 nt, 950 nt and 730 nt for region 1, region 2, and region 3, respectively, came from DcACS1a, whereas those of 590 nt, 630 nt, and 670 nt, for region 1, region 2, and region 4, respectively, came from DcACS1b (Harada et al, 2011;Satoh et al, 2011). The second group, including the 'Toy' cultivar, was one in which the bands corresponding to the DcACS1b in 'LPB' were not detected, suggesting the 'Toy' cultivar had only DcACS1a.…”

“…Moreover, it was revealed that three other wild-type Dianthus (wild pink) species had ACS genes orthologous to DcACS1 genes; they had a similar genomic structure with five exons and four introns, but with different intron nucleotide sequences (Harada et al, 2011;Satoh et al, 2011). Each Dianthus species had single to multiple variants of ACS1-like genes, and among wild pink species tested, D. petraeus (KBG-2 strain) had four variants of ACS1-like genes.…”

“…PCR was performed using rTaq DNA polymerase (Takara Bio Inc., Shiga, Japan) with 25 ng genomic DNA as a template in a 10 μL reaction mixture. The forward and reverse primer pairs used for amplification of regions 1-4 including intron 1, intron 2, 3'-UTR found in DcACS1a, and 3'-UTR in DcACS1b, and the PCR products were the same size as described previously (Harada et al, 2011;Satoh et al, 2011). Conditions were 94°C for 4 min followed by 30 cycles of 30 sec at 94°C, 30 sec at 53°C, and 1 min at 72°C.…”