DOI: 10.5061/dryad.3gm4500
title: Data from: Identification of a gene encoding polygalacturonase expressed specifically in short styles in distylous common buckwheat (Fagopyrum esculentum)

description: Common buckwheat (Fagopyrum esculentum) is a heteromorphic
 self-incompatible (SI) species with two types of floral architecture:
 thrum (short style) and pin (long style). The floral morphology and
 intra-morph incompatibility are controlled by a single genetic locus, S.
 However, the molecular mechanisms underlying the heteromorphic
 self-incompatibility of common buckwheat remain unclear. To identify these
 mechanisms, we performed proteomic, quantitative reverse-transcription
 PCR, and linkage analyses. Comparison of protein profiles between the long
 and short styles revealed a protein unique to the short style. Amino-acid
 sequencing revealed that it was a truncated form of polygalacturonase
 (PG); we designated the gene encoding this protein FePG1. Phylogenetic
 analysis classified FePG1 into the same clade as PGs that function in
 pollen development and floral morphology. FePG1 expression was
 significantly higher in short styles than in long styles. It was expressed
 in flowers of a short-homostyle line but not in flowers of a
 long-homostyle line. Linkage analysis indicated that FePG1 was not linked
 to the S locus; it could be a factor downstream of this locus. Our finding
 of a gene putatively working under the regulation of the S locus provides
 useful information for elucidation of the mechanism of heteromorphic
 self-incompatibility.
description: Fig 1Floral morphotypes of
 common buckwheat (Fagopyrum esculentum) and method of sampling for 2D-PAGE
 analysis. (A) Pin flower has long styles and low anthers (left panel);
 thrum flower has short styles and high anthers (right panel). (B) Sampling
 for 2D-PAGE. The stigmas and styles separated from ovules were
 collectedFig 22D-PAGE analysis of
 proteins extracted from upper parts of pistils of pin and thrum plants.
 Total proteins from stigmas and styles were separated by 2D-PAGE and
 silver-stained. SP1 and SP2, thrum-specific spots.Fig 3Amino-acid sequence of
 SP1 (FePG1) and multiple sequence alignment analysis. The predicted
 amino-acid sequence of FePG1 was aligned with those of four PG orthologues
 from Arabidopsis thaliana (At), Brassica napus (Bn), Solanum lycopersicum
 (Sl), and Turnera subulata (Ts). Conserved domains of PGs are indicated as
 regions I to IV. N-terminal and internal amino-acid sequences obtained by
 Edman sequencing and MS/MS analysis are indicated as N1, Int1, Int2, MS1,
 and MS2. The putative cleavage site was predicted by
 ExPASy.New_Fig.3_0411.tifFig 4Relative expression levels of FePG1. (A) The mRNA abundances of FePG1 in different organs of four floral morphotypes were detected by semi-qRT-PCR (40 cycles). The housekeeping gene Histone H3 (H3) was used as a loading control. L, leaves; S, stems; R, roots; F, flowers. (B) The mRNA abundances of FePG1 in pin and thrum styles were compared by real-time qRT-PCR. Values are relative to the H3 transcript level. Data are means ± s.e. for three technical replicates (bulked RNA from at least four independent plants). *P &lt; 0.05 (Student’s t test)Fig 5DNA polymorphism of the FePG1 genomic region in ‘KSC7’ (long homostyle). (A) Gene structure of FePG1 of ‘KSC7’. ‘KSC7’ has a 692-bp insertion in the 3rd exon. Black boxes, exons; bars, introns. (B) DNA polymorphism at the insertion region detected by agarose gel electrophoresis. P, pin; T, thrum; LH, long homostyle; SH, short homostyleFig 6Cis-elements of FePG1 and Fes_sc0001894.1.g000002.aua.1 A 2-kb region upstream of the translation initiation site and intron regions were analyzed. Triangles indicate cis-elements: LUX-binding site (LBS), evening element (EE), morning element (ME), and G-box (G). The lowercase letters are sequences different from the consensus sequence of each cis-element; +, forward strand; –, reverse strandNew_Fig.6_0411.tifSupplementary Fig. S1Phylogenetic tree constructed with the sequences of glycoside hydrolase family 28 domains of 51 buckwheat PG and 56 PG proteins from various plant species. Bootstrap values (1000 replicates) are shown under the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The PG proteins used in this analysis are listed in Supplementary Tables S3 and S4Sup Table S1Plant materials used in proteomic analysis, quantitative RT-PCR, and linkage analysis.Sup Tab S1.pdfSup Table S2Sequences of primers used for cloning, quantitative RT-PCR, and linkage analysis.Sup Tab S2.pdfSup Table S3Accession numbers for amino acid sequences used for phlogenetic analysis.Sup Tab S3.pdfSup Table S4Buckwheat PGs used for phylogenetic analysis.Sup Tab S4.pdf