DOI: 10.5061/dryad.zgmsbccr3
title: A lectin receptor-like kinase controls self-pollen recognition in Phlox

description: Self-incompatibility (SI) describes a widespread collection of genetic
 mechanisms in flowering plants used to specifically recognize and reject
 self-pollen. These mechanisms are fundamental to plant sexual reproduction
 and offer valuable insight into the molecular basis of cell-cell
 communication and self-recognition more broadly. Here, we leverage an
 independent evolution of SI in the lineage containing Phlox
 (Polemoniaceae) to characterize a novel gene causing self-pollen
 recognition which we name Phlox drummondii Pistil Identity Receptor Kinase
 (PdPIRK). Recognition of self-pollen associates with a single genomic
 region containing the Phlox S-locus. We generate predictions regarding how
 S-loci must function and evolve to identify a single candidate gene within
 this S-associated region. This gene, PdPIRK, is highly and specifically
 expressed in the pistil and has exceptionally high polymorphism maintained
 by negative frequency dependent selection, two hallmarks of self-pollen
 recognition genes. Functional validation with gene silencing confirms that
 PdPIRK is necessary for self-incompatibility, and we further demonstrate
 allele specific activity, confirming its role in self-pollen recognition
 per se. PdPIRK encodes a G-type lectin receptor-like kinase, which is a
 member of the same gene family as SRK, the gene controlling self-pollen
 recognition in the distantly related Brassicaceae. Our findings suggest
 the presence of genetic constraints or paths of least resistance governing
 how S-loci evolve and add to our understanding of the diverse molecular
 mechanisms through which organisms achieve self-recognition.
description: # A lectin receptor-like kinase controls self-pollen recognition in Phlox
 Dataset DOI: [10.5061/dryad.zgmsbccr3](10.5061/dryad.zgmsbccr3) ##
 Description of the data and file structure Identifying the *Phlox*
 *S*-locus using patterns of patterns of genetic association, gene
 expression, and genetic polymorphism with functional genetic validation. 
 ### Files and variables #### File:
 shortread\_drummondiionly\_alignments.zip **Description:** amino acid
 sequence alignments for the  top 10% most highly expressed pistil genes
 within the *S*-associated region in* Phlox drummondii * #### File:
 longread\_multispecies\_aligments.zip **Description:** amino acid sequence
 alignments for three genes within the *S*-associated region using alleles
 of four *Phlox *species  #### File: lecRLKtree\_alignment.fasta
 **Description:** amino acid sequence alignment of lecRLKs  #### File:
 diallelcross\_fruitdata.csv **Description:** fruit set data for diallel
 crossing experiment associating cross-compatibility with* PdPIRK* genotype
 ##### Variables * pistil: ID for the plant used as the pistil parent *
 pollen: ID for the plant used as the pollen parent * fruit set: the total
 number of fruits that set in each cross type * number crosses: the total
 number of crosses performed for each cross type #### File:
 diallelcross\_pollentubedata.csv **Description:** pollen tube data
 for diallel crossing experiment associating cross-compatibility with*
 Phlox drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) genotype. One
 individual per predicted genotype was used to validate fruit set data. 
 ##### Variables * cross_ID: description of the cross type  * maternal: ID
 for the plant used as the pistil parent * paternal: ID for the plant used
 as the pollen parent * predicted_cross_type: prediction for whether the
 cross should be compatible or incompatible based on fruit set data in
 diallelcross_fruitdata.csv * date_crossed: date cross was performed *
 date_stained: date tubes were stained and imaged * tubes_stigma: the total
 number of pollen tubes present on the stigma #### File:
 DFR\_floralscans.zip **Description:** floral scans for all flowers crossed
 during the virus-induced gene silencing experiment using a silencing
 vector targeting dihydroflavonol 4-reductase (*DFR*) only.  #### File:
 S1\_DFR\_floralscans.zip **Description:** floral scans for all flowers
 crossed during the virus-induced gene silencing experiment using a
 silencing vector targeting dihydroflavonol 4-reductase (*DFR*) and *Phlox
 drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) allele S1.  ####
 File: S4\_DFR\_floralscans.zip **Description:** floral scans for all
 flowers crossed during the virus-induced gene silencing experiment using a
 silencing vector targeting dihydroflavonol 4-reductase (*DFR*) and *Phlox
 drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) allele S4.  ####
 File: RNAseq\_normalized\_expression.csv **Description:** normalized
 expression for five tissue types: stigma/style, anther, pollen, leaf, and
 stem. Counts are given in TMM. Gene IDs are based on the* Phlox
 drummondii* genome annotation available at
 (doi.org/10.5281/zenodo.15498773). #### File: tissuespecific\_qpcr.csv
 **Description:** qPCR confirming RNAseq  ##### Variables * Sample: ID for
 each sample which includes plant ID (number) and tissue type  * PdPIRK
 Genotype: genotype at* PdPIRK* as confirmed by allele specific PCR
 amplification * Tissue: Tissue of origin for each sample (leaf, corolla,
 or stigma/style) * Individual: Plant ID (number) * S4 1 Ct (dRn):
 technical replicate 1 for PdPIRK allele S4 amplification given in deltaRn,
 the baseline-corrected, normalized fluorescence signal.  * S4 2 Ct (dRn): 
 technical replicate 2 for PdPIRK allele S4 amplification given in deltaRn,
 the baseline-corrected, normalized fluorescence signal.  * S4 3 Ct (dRn): 
 technical replicate 3 for PdPIRK allele S4 amplification given in deltaRn,
 the baseline-corrected, normalized fluorescence signal.  * Ef1a 1 Ct
 (dRn):  technical replicate 1 for the housekeeping gene, Ef1alpha,
 amplification given in deltaRn, the baseline-corrected, normalized
 fluorescence signal.  * Ef1a 2 Ct (dRn): technical replicate 2 for the
 housekeeping gene, Ef1alpha, amplification given in deltaRn, the
 baseline-corrected, normalized fluorescence signal.  * Ef1a 3 Ct
 (dRn): technical replicate 3 for the housekeeping gene, Ef1alpha,
 amplification given in deltaRn, the baseline-corrected, normalized
 fluorescence signal.  #### File: vigs\_pollentubes.csv
 **Description:** pollen tube data for flowers crossed during the
 virus-induced gene silencing experiments using a silencing vector
 targeting 1) dihydroflavonol 4-reductase (*DFR*) only, 2)*Phlox
 drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) allele S4, or
 3) *Phlox drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) allele S1
 ##### Variables * Pistil_ID: plant ID for the pistil parent *
 Flower_Number: ID for each flower crossed (corresponds to flower number in
 floral scan images) * Flower_Color: color for each flower crossed, either
 pigmented or white * Treatment: silencing status for each flower crossed,
 either silenced or unsilenced * TRV2-vector: the silencing vector used to
 infect each plant  * Pistil_Genotype: pistil genotype at* PdPIRK* as
 confirmed by allele specific PCR amplification * Pollen_Genotype: pollen
 genotype at* PdPIRK* as confirmed by allele specific PCR amplification *
 Pollen_ID: plant ID for the pollen parent * Number_Pollen_Tubes: the total
 number of pollen tubes present on the stigma  #### File:
 vigs\_fruitset.csv **Description:** fruit set data for flowers crossed
 during the virus-induced gene silencing experiments using a silencing
 vector targeting 1) dihydroflavonol 4-reductase (*DFR*) only, 2)*Phlox
 drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) allele S4, or
 3) *Phlox drummondii* Pistil Identity Receptor Kinase (*PdPIRK*) allele S1
 ##### Variables * Pistil_ID: plant ID for the pistil parent *
 Flower_Number: ID for each flower crossed (corresponds to flower number in
 floral scan images) * Flower_Color: color for each flower crossed, either
 pigmented or white * Treatment: silencing status for each flower crossed,
 either silenced or unsilenced * TRV2-vector: the silencing vector used to
 infect each plant  * Pistil_Genotype: pistil genotype at* PdPIRK* as
 confirmed by allele specific PCR amplification * Pollen_Genotype: pollen
 genotype at* PdPIRK* as confirmed by allele specific PCR amplification *
 Pollen_ID: plant ID for the pollen parent * Fruit: binary indicator of
 fruit set (1 = fruit formed, 0 = no fruit formed) #### File:
 vigs\_qpcr.csv **Description:** qPCR data for pistil tissue from silenced
 and unsilenced flowers for all individuals used in virus induced gene
 silencing experiments ##### Variables * Sample: unique identifier for each
 sample that includes the plant ID (number) and its silencing status (u =
 unsilenced, i = silenced) * PdPIRK Genotype: genotype at* PdPIRK* as
 confirmed by allele specific PCR amplification * Individual: plant ID *
 Treatment: silencing status for each sample  * Primer pair: the allele
 specific primers used to amplify PdPIRK for each sample * PdPIRK 1 Ct
 (dRn): technical replicate 1 for PdPIRK amplification given in deltaRn,
 the baseline-corrected, normalized fluorescence signal.  * PdPIRK 2 Ct
 (dRn): technical replicate 2 for PdPIRK amplification given in deltaRn,
 the baseline-corrected, normalized fluorescence signal.  * PdPIRK 3 Ct
 (dRn): technical replicate 3 for PdPIRK amplification given in deltaRn,
 the baseline-corrected, normalized fluorescence signal.  * Ef1a 1 Ct
 (dRn): technical replicate 1 for the housekeeping gene, Ef1alpha,
 amplification given in deltaRn, the baseline-corrected, normalized
 fluorescence signal.  * Ef1a 2 Ct (dRn): technical replicate 1 for the
 housekeeping gene, Ef1alpha, amplification given in deltaRn, the
 baseline-corrected, normalized fluorescence signal.  * Ef1a 3 Ct
 (dRn): technical replicate 1 for the housekeeping gene, Ef1alpha,
 amplification given in deltaRn, the baseline-corrected, normalized
 fluorescence signal.  #### File: A5\_rnabloom.transcripts.fa
 **Description:** pistil transcriptome for individual A5 from the diallel
 cross experiment (see diallelcross_fruitdata.csv) assembled using RNAbloom
 from PacBio IsoSeq HiFi reads  #### File: A6\_rnabloom.transcripts.fa
 **Description:** pistil transcriptome for individual A6 from the diallel
 cross experiment (see diallelcross_fruitdata.csv) assembled using RNAbloom
 from PacBio IsoSeq HiFi reads  #### File: A7\_rnabloom.transcripts.fa
 **Description:** pistil transcriptome for individual A7 from the diallel
 cross experiment (see diallelcross_fruitdata.csv) assembled using RNAbloom
 from PacBio IsoSeq HiFi reads  #### File: A9\_rnabloom.transcripts.fa
 **Description:** pistil transcriptome for individual A9 from the diallel
 cross experiment (see diallelcross_fruitdata.csv) assembled using RNAbloom
 from PacBio IsoSeq HiFi reads