Dataset DOI: 10.5061/dryad.3bk3j9kx2

Description of the data and file structure

This repository provides the code and some of the data files to recreate the analyses associated with the manuscript under review (Field et al., in review).

In this study, we developed a novel rapid method for geographic cline estimation (FastClines script) and showcase the approach with a whole genome (re-analysed) poolSeq data set of six demes along a transect (from Tavares et al., 2018) through a hybrid zone between two varieties of Antirrhinum majus spp majus that differ in flower colour. The goal was to use geographic clines to examine how barriers are distributed along the genome, in relation to known colour genes and differentiation statistics (SlidingWindows script). Secondarily, we use this technique to highlight a new colour locus linked to Flavanol synthase and validate its effects with an RNA-seq dataset to look at differential expression, controlled F2 and F3 crosses, and associations between genotype using a targeted SNP KASP genotype panel (Amajus_KASP_publishV1) and colour phenotyping from wild individuals in the hybrid zone (Amajus_FlowerPhenoMeasures).

References

Field, D. L., Stankowski, S., Reiter, T., Polechova, J., Bradley, D., Richardson, D., Pal, A., Shipilina, D., Boell, L., Pickup, M., Xue, Y., Coen, E., & Barton, N. (2025). Genome-wide cline analysis identifies new locus contributing to a barrier to gene flow across an Antirrhinum hybrid zone (p. 2025.02.17.638607). bioRxiv. https://doi.org/10.1101/2025.02.17.638607

Tavares, H., Whibley, A., Field, D. L., Bradley, D., Couchman, M., Copsey, L., Elleouet, J., Burrus, M., Andalo, C., Li, M., Li, Q., Xue, Y., Rebocho, A. B., Barton, N. H., & Coen, E. (2018). Selection and gene flow shape genomic islands that control floral guides. Proceedings of the National Academy of Sciences, 115(43), 11006–11011. https://doi.org/10.1073/pnas.1801832115

Files and variables

File: Amajus_PoolSeq_popDetails.txt

Description: An input file needed to run FastClines analysis

Variables

• Pool: pool number
• N: number of individuals in the pool
• Dist: deme span
• ID: pool ID name
• Lat: latitude position of pool
• Long: longitude position of pool
• Description: species name of pool

File: pool_info.csv

Description: additional meta information regarding all the poolSeq datasets

Variables

• Population: population name
• Species: species in pool
• Latitude: latitude position of pool
• Longitude: longitude position of pool
• Coen_ID: JIC ID code
• #_in_pool: number of individuals in the WGS pool

File: Amajus_KASP_publishV2.csv

Description: KASP SNP genotype data and location information for larger data set for 6 colour loci at the hybrid zone

Variables

• PlantID_final: final ID for plant
• RametIDs: the set of PlantIDs (ramets) that belong to the same genet. These are all the duplicate identical ramets in same location likely the same plant accidently re-sampled and re-genotyped. The PlantID_final was simply the first RametID to occur in time.
• Latitude: the final one to use for the genet (best position)
• Longitude: the final one to use for the genet (best position)
• Altitude: the final one to use for the genet (best position)
• Easting: the final one to use for the genet (best position)
• Northing: the final one to use for the genet (best position)
• ID_Track_Decision: 
• Score_phase1: quality score for GPS data & genotype. From -9 to 10 (best). See above, section 4 Phase 1 (Choosing best GPS data per genet and assigning track score).
• Population: broad population name, e.g hybrid zones = Planoles, Cadi, Mar, LAV/Ave. Also, various allopatric populations (e.g., Ave, Par). Needs some updating as some are still missing, but you can always use lat and long.
• Location: finer detail within some populations. e.g. mowerFreePatch. Most blank or Unk (Unknown). I will update this later for upper/lower road and other details.
• GenoID: old ID not generally used
• GenoScore: old quality score not used
• s1187_290152: CREMOSA locus. Alleles coded as 0 (hom), 1 (het), 2 (alternative hom), -9 (missing), -10 (not genotyped)
• s31693292: FLAVIA_1 locus upstream of coding sequence. Alleles coded same as above.
• s316_257789: FLAVIA_2 locus downstream of coding sequence. Alleles coded same as above.
• s91_39699: SULFUREA locus. Alleles coded same as above.
• s261_720757: RUBIA locus. Alleles coded same as above.
• ros_assembly_543443: ROSEA locus. Alleles coded same as above.
• ros_assembly_715015: ELUTA locus. Alleles coded same as above.
• missing: total missing genotypes from full SNP panel (not provided)
• NGY: total not genotyped yet from full SNP panel (not provided)
• totals: total from full panel available
• propMissing: proportion of loci with missing data
• totals_available: total loci available
• multiLoc_Hap_6by7: multilocus haplotype for 2 sets of loci
• multiLoc_Hap_24by7: multilocus haplotype for 3 sets of loci
• multiLoc_Hap_33by21: multilocus haplotype for all colour loci

File: Amajus_FlowerPhenoMeasuresV2.csv

Description: Flower colour phenotype data of Antirrhinum plants from the hybrid zone

Variables

• ID: Unique plant ID

• PhenoCat: colour score category based on Red and Yellow scores (FR = Full red, FO = Full Orange, WO = Weak orange, WR = weak red, W = White, Y = yellow)

• Red: red score (see manuscript)

• Yellow: red score (see manuscript)

• randomNum: random number used to pick out plants to measure

• Date: date of plant sample

• Face: fileID for photo of face of flower

• Side: fileID for photo of side of flower

• Under: fileID for photo of the under of the flower

• Top: fileID for photo of top of flower

• Venation: scoring for venation (see manuscript)

• PhotoComments: general comments and observations

• Latitude: position of the plant in latitude

• Longitude: position of the plant in longitude

• Altitude: altitude in metre of the plant position

• CorrectedLatitude: position of plant in latitude after corrections

• CorrectedLongitude: position of plant in longitude after corrections

• CorrectedAltitude: position of plant in altitude after corrections 

• Easting: projected position Easting

• Northing: projected position Northing

• Measurement_size.pixels.: area in pixels where colour measurements taken for 1st position on flower

• Area.1R: area in mm where Red colour channel measurements taken for the 1st position on the flower

• Mean.1R: mean Red colour channel measurements taken for 1st position on flower

• StdDev.1R: standard deviation of Red colour channel, where colour measurements taken for the 1st position on the flower 

• Mode.1R: mode of red colour channel taken for 1st position on flower

• Min.1R: minimum value of red colour channel taken for 1st position on the flower

• Max.1R: maximum value of red colour measurements taken for 1st position on flower

• IntDen.1R: intensity value of red colour measurements taken for 1st position on flower

• RawIntDen.1R: raw intensity value of red colour measurements taken for 1st position on the flower

• X: colour of the following colours

• Area.1G: area in mm where green colour channel measurements taken for 1st position on flower

• Mean.1G: mean green colour channel measurements taken for 1st position on flower

• StdDev.1G: standard deviation of green colour channel where colour measurements taken for 1st position on flower 

• Mode.1G: mode of green colour channel taken for 1st position on flower

• Min.1G: minimum value of green colour channel taken for 1st position on flower

• Max.1G: maximum value of green colour measurements taken for 1st position on flower

• IntDen.1G: intensity value of green colour measurements taken for 1st position on flower

• RawIntDen.1G: raw intensity value of green colour measurements taken for 1st position on flower

• X.1: colour of the following columns

• Area.1B: area in mm where blue colour channel measurements taken for 1st position on flower

• Mean.1B: mean blue colour channel measurements taken for 1st position on flower

• StdDev.1B: standard deviation of blue colour channel where colour measurements taken for 1st position on flower 

• Mode.1B: mode of blue colour channel taken for 1st position on flower

• Min.1B: minimum value of blue colour channel taken for 1st position on flower

• Max.1B: maximum value of blue colour measurements taken for 1st position on flower

• IntDen.1B: intensity value of blue colour measurements taken for 1st position on flower

• RawIntDen.1B: raw intensity value of blue colour measurements taken for 1st position on flower

  The following series of columns are repeats of the previous but for the next five positions on the flower as indicated in the number in the second last position (i.e., before characters R, G, B) (e.g., Mean.2R). These series continue through flower positions 2, 3, 4, 5, 6, as indicated in S15 Fig. [note: the columns X.2, X.3, X.4, X.5,...X.16 - numbers do not relate to positions on the flower but are simply uniquely labelled with values indicating the colour channel of the next 8 columns. These colour channels are also indicated in the final character of header columns (e.g., R, G, B)]

• X.2

• Area.2R

• Mean.2R

• StdDev.2R

• Mode.2R

• Min.2R

• Max.2R

• IntDen.2R

• RawIntDen.2R

• X.3

• Area.2G

• Mean.2G

• StdDev.2G

• Mode.2G

• Min.2G

• Max.2G

• IntDen.2G

• RawIntDen.2G

• X.4

• Area.2B

• Mean.2B

• StdDev.2B

• Mode.2B

• Min.2B

• Max.2B

• IntDen.2B

• RawIntDen.2B

• X.5

• Area.3R

• Mean.3R

• StdDev.3R

• Mode.3R

• Min.3R

• Max.3R

• IntDen.3R

• RawIntDen.3R

• X.6

• Area.3G

• Mean.3G

• StdDev.3G

• Mode.3G

• Min.3G

• Max.3G

• IntDen.3G

• RawIntDen.3G

• X.7

• Area.3B

• Mean.3B

• StdDev.3B

• Mode.3B

• Min.3B

• Max.3B

• IntDen.3B

• RawIntDen.3B

• X.8

• Area.4R

• Mean.4R

• StdDev.4R

• Mode.4R

• Min.4R

• Max.4R

• IntDen.4R

• RawIntDen.4R

• X.9

• Area.4G

• Mean.4G

• StdDev.4G

• Mode.4G

• Min.4G

• Max.4G

• IntDen.4G

• RawIntDen.4G

• X.10

• Area.4B

• Mean.4B

• StdDev.4B

• Mode.4B

• Min.4B

• Max.4B

• IntDen.4B

• RawIntDen.4B

• X.11

• Area.5R

• Mean.5R

• StdDev.5R

• Mode.5R

• Min.5R

• Max.5R

• IntDen.5R

• RawIntDen.5R

• X.12

• Area.5G

• Mean.5G

• StdDev.5G

• Mode.5G

• Min.5G

• Max.5G

• IntDen.5G

• RawIntDen.5G

• X.13

• Area.5B

• Mean.5B

• StdDev.5B

• Mode.5B

• Min.5B

• Max.5B

• IntDen.5B

• RawIntDen.5B

• X.14

• Area.6R

• Mean.6R

• StdDev.6R

• Mode.6R

• Min.6R

• Max.6R

• IntDen.6R

• RawIntDen.6R

• X.15

• Area.6G

• Mean.6G

• StdDev.6G

• Mode.6G

• Min.6G

• Max.6G

• IntDen.6G

• RawIntDen.6G

• X.16

• Area.6B

• Mean.6B

• StdDev.6B

• Mode.6B

• Min.6B

• Max.6B

• IntDen.6B

• RawIntDen.6B

• Hue360_1

• Hue360_2

• Hue360_3

• Hue360_4

• Hue360_5

• Hue360_6

• Hue_1: Colour Hue of position 1

• Hue_2: Colour Hue of position 2

• Hue_3: Colour Hue of position 3

• Hue_4: Colour Hue of position 4

• Hue_5: Colour Hue of position 5

• Hue_6: Colour Hue of position 6

• Int_1: Colour Intensity of position 1

• Int_2: Colour Intensity of position 2

• Int_3: Colour Intensity of position 3

• Int_4: Colour Intensity of position 4

• Int_5: Colour Intensity of position 5

• Int_6: Colour Intensity of position 6

• SD_1: Standard deviation of Intensity of position 1

• SD_2: Standard deviation of Intensity of position 2

• SD_3: Standard deviation of Intensity of position 3

• SD_4: Standard deviation of Intensity of position 4

• SD_5: Standard deviation of Intensity of position 5

• SD_6: Standard deviation of Intensity of position 6

• Sat_1: Saturation of position 1

• Sat_2: Saturation of position 2

• Sat_3: Saturation of position 3

• Sat_4: Saturation of position 4

• Sat_5: Saturation of position 5

• Sat_6: Saturation of position 6

File: Amajus_shortSummarySNPsV2.csv

Description: 

Variables

• LocusName: KASP locus name
• ColourGene: colour gene linked to locus
• LG: linkage group/chromosome
• cM: centimorgan position on linkage map
• scaffold: old scaffold name from v2 of genome
• position: position on the old scaffold from v2 of the genome
• Oligo: oligo in IUPAC code. Polymorphic site in brackets
• Allele_1: first allele
• Allele_2: second allele

Code/software

Analyses and figures for the main text and supplementary materials are conducted in a series of R markdown scripts contained in the main folder of https://github.com/dfield007/genome_wide_clines

• FastClines_Amajus_main.Rmd
• FastClines_Amajus_RNAseq.Rmd
• FastClines_Amajus_GenoPheno.Rmd
• FastClines_Amajus_MLEclines.Rmd
• FastClines_Amajus_simulations.Rmd

and also a number of Python scripts called within the markdowns

• SlidingWindows.py at https://github.com/dfield007/slidingWindows
• FastClines.py at https://github.com/dfield007/fastClines

Access information

Other publicly accessible locations of the data:

https://github.com/dfield007/genome_wide_clines

• Input datasets:
  • Antirrhinum Reference genome: reference genome V3.0 is available at the NGDC Genome Warehouse under accession number GWHBJVT00000000
  • Antirrhinum Annotated genome: GWHBJVT00000000.gff (GFF annotations have been uploaded to NCBI WGS under accession number SUB15081867)
  • Whole Genome PoolSeq dataset: (SRA under accession number SUB15081578)