Abstract
This work evaluated the genetic relationships among 442 single-seed descent accessions, representing eight Lactuca spp., including five wild Lactuca relatives (WLRs) (Lactuca georgica, L. altaica, L. saligna, L. serriola, L. aculeata), L. tuberosa, L. undulata, and the domesticated lettuce, L. sativa, 437 of them representing a core subset of the Institute of Evolution's Wild Lettuce Gene Bank (IoE's WLGB) collection. The analysis was performed by profiling 115 single-nucleotide polymorphism markers with the fluorescent KASP genotyping assay. KASP marker fragments were scored as either allele “A” or allele “B”, and then treated as bi-allelic data, but the dataset also included a relatively high number of U-scores, noting the absence of the specific sequence, that were treated as missing data. Often U-scores were specific for a certain species. Data analysis of the five WLRs showed that allele frequencies of 103 (97.2%) out of 106 differentiating loci varied significantly among the species, where 59.7% of the KASP marker diversity was between species. A neighbor-network analysis involving samples of the five WLRs and a single L. sativa cv. clearly clustered all 430 samples in accordance with their taxonomic determination. These results obtained from multiple complementary analyses of large natural populations and individuals, question the assignment of L. georgica to the primary lettuce gene pool. Together with our previous results obtained by using TRAP markers and hybridization experiments, we conclude that L. georgica is a constituent of the secondary lettuce gene pool. Our analyses shed also light on the proper placement of L. altaica.
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Data availability
The dataset generated and analyzed during the current study is available from the author.
References
Aiken HH (1955) Tables of cumulative binomial probability distribution. Harvard University Press, Cambridge, MA
Beharav A (2021) Lactuca georgica, a new wild source of resistance to downy mildew: comparative study to other wild lettuce relatives. Eur J Plant Pathol 160:127–136
Beharav A, Ben-David R, Doležalová I, Lebeda A (2008) Eco-geographical distribution of Lactuca saligna natural populations in Israel. Israel J Plant Sci 56:195–220
Beharav A, Ben-David R, Doležalová I, Lebeda A (2010) Eco-geographical distribution of Lactuca aculeata natural populations in north-eastern Israel. Genet Resour Crop Evol 57:679–686
Beharav A, Hellier B (2020) Bolting and flowering response of Lactuca georgica, a wild lettuce relative, to low temperatures. Amer J Plant Sci 11:2139–2154
Beharav A, Hellier B, Richardson KL, Lebeda A, Kisha T (2018a) Genetic relationships and structured diversity of Lactuca georgica germplasm from Armenia and the Russian Federation among other members of Lactuca L., subsection Lactuca L., assessed by TRAP markers. Genet Resour Crop Evol 65:1963–1978
Beharav A, Khalifa S, Nevo E (2018b) New insights into the range, morphology, and natural hybridization of wild Lactuca aculeata in Israel. Israel J Plant Sci 65:175–185
Beharav A, Malarz J, Michalska K, Ben-David R, Stojakowska A (2020) Variation of sesquiterpene lactone contents in Lactuca altaica natural populations from Armenia. Biochemical Syst Ecol 90:104030
Beharav A, Stojakowska A, Ben-David R, Malarz J, Michalska K, Kisiel W (2015) Variation of sesquiterpene lactone contents in Lactuca georgica natural populations from Armenia. Genet Resour Crop Evol 62:431–441
Bryant D, Moulton V (2004) Neighbor-Net: an agglomerative method for the construction of phylogenetic networks. Mol Biol Evol 21:255–265
Chu R, Xu XM, Lu ZW, Ma YG, Cheng H, Zhu SX, Bakker FT, Schranz ME, Wei Z (2022) Plastome-based phylogeny and biogeography of Lactuca L. (Asteraceae) support revised lettuce gene pool categories. Front Plant Sci 13:978417. https://doi.org/10.3389/fpls.2022.978417
Crow JF, Kimura M (1970) An introduction to population genetics theory. Harper & Row Publishers, New York
Earl DA, VonHoldt BM (2012) Structure Harvester: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 4:359–361
Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:2611–2620
Gabrielian E, Fragman-Sapir O (2008) Flowers of the Transcaucasus and adjacent areas: including Armenia, Eastern Turkey, Southern Georgia, Azerbaijan and Northern Iran. A.R.G. Gantner Verlag KG, Ruggell
Gabrielian E, Zohary D (2004) Wild relatives of food crops native to Armenia and Nakhichevan. Flora Mediterranea 14:5–80
Güzel ME, Coşkunçelebi K, Kilian N, Makbul S, Gültepe M (2021) Phylogeny and systematics of the Lactucinae (Asteraceae) focusing on their SW Asian centre of diversity. Plant Syst Evol 307:7
Hartl DL, Clark AG (1997) Principles of population genetics, 3rd edn. Sinauer Associates Inc, Sunderland, Massachusetts
He C, Holme J, Anthony J (2014) SNP Genotyping: The KASP Assay. In: Fleury D, Whitford R (eds) Crop Breeding. Methods in Molecular Biology (Methods and Protocols). Humana Press, New York, NY, vol 1145, pp 75–86
Huson DH, Bryant D (2006) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23(2):254–267
Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics 23:1801–1806
Jemelková M, Kitner M, Křístková E, Beharav A, Lebeda A (2015) Biodiversity of Lactuca aculeata germplasm assessed by SSR and AFLP markers, and resistance variation to Bremia lactucae. Bioc Syst Ecol 61:344–356
Kitner M, Lebeda A, Doležalová I, Maras M, Křístková E, Nevo E, Pavlíček T, Meglič V, Beharav A (2008) AFLP analysis of Lactuca saligna germplasm collections from four European and three Middle East countries. Israel J Plant Sci 56:185–193
Kitner M, Majesky L, Křistková E, Jemelková M, Lebeda A, Beharav A (2015) Genetic structure and diversity in natural populations of three predominantly self-pollinating wild Lactuca species in Israel. Genet Resour Crop Evol 62:991–1008
Koopman WJM, Guetta E, Van De Wiel CCM, Vosman B, Van den Berg RG (1998) Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences. American J Bot 85:1517–1530
Lebeda A, Doležalová I, Feráková V, Astley D (2004) Geographical distribution of wild Lactuca species (Asteraceae, Lactuceae). Bot Rev 70:328–356
Lebeda A, Kitner M, Křístková E, Doležalová I, Beharav A (2012) Genetic polymorphism in Lactuca aculeata populations and occurrence of natural putative hybrids between L. aculeata and L. serriola. Bioc Syst Ecol 42:113–123
Lebeda A, Ryder EJ, Grube R, Doležalová I, Křístková E (2007) Lettuce (Asteraceae Lactuca spp.), In: Singh RJ (ed) Genetic Resources, Chromosome Engineering, and Crop Improvement, Vol. 3, Vegetable Crops. CRC Press, Taylor and Francis Group, Boca Raton, FL, USA, pp 377–472
Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K (1995) Oligonucleotide with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. Genome Res 4:357–362
Mabry TJ, Bohlmann F (1977) Summary of the chemistry of the Compositae. In: Heywood VH, Harborne JB, Turner BL (eds) The biology and Chemistry of the Compositae, vol 2. Academic Press, London, pp 1097–1104
McGuire PE, Ryder EJ, Michelmore RW, Clark RL, Antle R, Emery G, Hannan RM, Kesseli RV, Kurtz EA, Ochoa O, Rubatzky VE, Waycott W (1993) Genetic resources of lettuce and Lactuca species in California. An assessment of the USDA and UC collections and recommendations for long-term security. Report no. 12. University of California, Genetic Resource Conservation Program, Davis, CA
Michalska K, Szneler E, Kisiel W (2010) Lactuca altaica as a rich source of sesquiterpene lactones. Biochem Syst Ecol 38:1246–1249
Michalska K, Beharav A, Kisiel W (2014) Sesquiterpene lactones from roots of Lactuca georgica. Phytochem Lett 10:10–12
Nei M (1973) Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci USA 70:3321–3323
Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590
Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol 6:288–295
Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959
Pritchard JK, Wen X, Falush D (2009) Documentation for structure software: Version 2.3. University of Chicago, Chicago, USA. http://pritch.bsd.uchicago.edu/structure. html
Rosenberg A (2004) DISTRUCT: a program for the graphical display of population structure. Mol Ecol Notes 4:137–138
Semagn K, Babu R, Hearne S, Olsen M (2014) Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breeding 33:1–14
Shikari AB, Najeeb S, Khan G et al (2021) KASP™ based markers reveal a population sub-structure in temperate rice (Oryza sativa L.) germplasm and local landraces grown in the Kashmir valley, north-western Himalayas. Genet Resour Crop Evol 68:821–834
Shulha O, Zidorn C (2019) Sesquiterpene lactones and their precursors as chemosystematic markers in the tribe Cichorieae of the Asteraceae revisited: an update (2008–2017). Phytochemistry 163:149–177
Stojakowska A, Michalska K, Malarz J, Beharav A, Kisiel W (2013) Root tubers of Lactuca tuberosa as a source of antioxidant phenolic compounds and new furofuran lignans. Food Chem 138:1250–1255
van Treuren R, van der Arend AJM, Schut JW (2013) Distribution of downy mildew (Bremia lactucae Regel) resistances in a genebank collection of lettuce and its wild relatives. Plant Genet Resour Char Utiliz 11:15–25
van Treuren R, van Eekelen HDLM, Wehrens R, Wehrens R, de Vos RCH (2018) Metabolite variation in the lettuce gene pool: towards healthier crop varieties and food. Metabolomics 14:146
Wei T, van Treuren R, Liu X et al (2021) Whole-genome resequencing of 445 Lactuca accessions reveals the domestication history of cultivated lettuce. Nat Genet 53:752–760
WFO (2022): World Flora Online. http://worldfloraonline.org/. Accessed on: 14 March 2022
Yeh F C, Yang RC, Boyle T (1999) POPGENE version 1.32: Microsoft Windows–based freeware for population genetic analysis, quick user guide. Center for International Forestry Research, University of Alberta, Edmonton, Alberta, Canada
Zidorn C (2008) Sesquiterpene lactones and their precursors as chemosystematic markers in the tribe Cichorieae of the Asteraceae. Phytochem 69:2270–2296
Zohary D (1991) The wild genetic resources of cultivated lettuce (Lactuca sativa L.) Euphytica 53:31–35
Acknowledgements
AB wishes to thank Rijk Zwaan B.V. for providing greenhouse facilities, support in assessment and cultivation of the sampled Lactuca accessions, as well for providing the KASP data points. Performing the Neighbor-Network and Structure analysis by Dr. Lucie Vaculná and Dr. Miloslav Kitner, Department of Botany, Faculty of Science, Palacký University in Olomouc, Olomouc-Holice, Czech Republic, are gratefully acknowledged. Financial support by the Research Authority and calculating the uncorrected p-distance values by Mrs. Efrat Yaskil of the Statistics Consulting Unit, University of Haifa, Israel, are acknowledged. I would also like to thank Dr. Roi Ben-David, Institute of Plant Sciences, Agriculture Research Organization (ARO), Volcani Center, Bet Dagan, Israel, for his valuable comments on the first draft version of this manuscript.
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Beharav, A. Lactuca georgica Grossh. is a wild species belonging to the secondary lettuce gene pool: additional evidence, obtained by KASP genotyping. Genet Resour Crop Evol 70, 1289–1304 (2023). https://doi.org/10.1007/s10722-022-01502-7
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DOI: https://doi.org/10.1007/s10722-022-01502-7
Keywords
- Conservation
- Gene bank
- Genetic diversity
- Genotyping platform
- Single nucleotide polymorphisms (SNPs)
- Wild Lactuca relatives (WLRs)