The development and validation of a genotyping-by-target sequencing chip for fungal population genetic analysis

Abstract

Advanced genotyping technologies for understanding the genetic intricacies of fungal pathogens have broad applications in crop protection. Here, we introduce a novel genotyping-by-target sequencing (GBTS) chip, a versatile tool designed for comprehensive genetic analysis of fungal populations. This technology overcomes key limitations of traditional molecular marker-based approaches by providing a more efficient, economic, and streamlined solution while bypassing the need for labor-intensive pathogen culturing. We demonstrate its utility by applying it to profile Pucciniastriiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. Our analysis involved 225 infected leaves collected from wheat fields in the northwest oversummering region for Pst in China. We delineated three genetic groups and revealed frequent gene flow, with closer connectivity between Qinghai and Gansu than either province with Ningxia, a pattern consistent with wind trajectory models. These findings illustrate a highly connected regional epidemic system and highlight the value of the GBTS chip for genomic epidemiology. The methodology established here provides a scalable framework for population genetic studies in other fungal pathogens, promising to enhance disease monitoring and management across agricultural systems.

Keywords

GBTS / Puccinia striiformis f. sp. tritici / Population genetics / Gene flow / Trajectory tracking

References

[1] Alexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res, 2009, 19(9): 1655-1664

[2] Ali S, Gautier A, Leconte M, Enjalbert J, de Vallavieille-Pope C. A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f. sp. tritici, based on DNA extraction from infected leaf and multiplex PCR genotyping. BMC Res Notes, 2011, 4(1): 240

[3] Ali S, Gladieux P, Leconte M, Gautier A, Justesen AF, Hovmøller MS, Enjalbert J, de Vallavieille-Pope C. Origin, migration routes and worldwide population genetic structure of the wheat yellow rust pathogen Puccinia striiformis f.sp. tritici. PLoS Pathog, 2014, 10(1e1003903

[4] Bai Q, Wan AM, Wang MN, See DR, Chen XM. Molecular characterization of wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) collections from nine countries. Int J Mol Sci, 2021, 22(179457

[5] Bailey J, Karaoglu H, Wellings CR, Park RF. PCR-based simple sequence repeat markers for diagnostic identification of major clonal lineages of Puccinia striiformis f. sp tritici and related stripe rust pathogens in Australia. Austral Plant Pathol, 2015, 44(197-103

[6] Brown JK. Durable resistance of crops to disease: a darwinian perspective. Annu Rev Phytopathol, 2015, 53(1): 513-539

[7] Chen N, Cosgrove EJ, Bowman R, Fitzpatrick JW, Clark AG. Genomic consequences of population decline in the endangered Florida scrub-jay. Curr Biol, 2016, 26(21): 2974-2979

[8] Chen W, Wellings C, Chen XM, Kang ZS, Liu TG. Wheat stripe (yellow) rust caused by Puccinia striiformis f. sp. tritici. Mol Plant Pathol, 2014, 15(5): 433-446

[9] Chen XM. Epidemiology and control of stripe rust [Puccinia striiformis f. sp. tritici] on wheat. Can J Plant Pathol, 2005, 27(3): 314-337

[10] Chen XM, Line RF, Leung H. Relationship between virulence variation and DNA polymorphism in Puccinia striiformis. Phytopathology, 1993, 83(12): 1489-1497

[11] Chen XM, Moore M, Milus EA, Long DL, Line RF, Marshall D, Jackson L. Wheat stripe rust epidemics and races of Puccinia striiformis f. sp. tritici in the United States in 2000. Plant Dis, 2002, 86(1): 39-46

[12] Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:884-890. https://doi.org/10.1093/bioinformatics/bty560

[13] Chen WQ, Kang Z, Ma Z, Xu SC, Jin S, Jiang YY (2013) Integrated management of wheat stripe rust caused by Puccinia striiformis f. sp. tritici in China. Sci Agric Sin 46(20):4254–4262. https://doi.org/10.3864/j.issn.0578-1752.2013.20.008

[14] Chen XM, Kang ZS (2017) Stripe Rust. Springer, Netherlands. https://doi.org/10.1007/978-94-024-1111-9

[15] Cheng P, Chen XM, Xu LS, See D (2012) Development and characterization of expressed sequence tag-derived microsatellite markers for the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. Mol Ecol Resour 12(4):779–781. https://doi.org/10.1094/PHYTO-99-3-0282

[16] Cingolani P, Platts A, Wang leL, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w¹¹¹⁸; iso-2; iso-3. Fly, 2012, 6(2): 80-92

[17] De Vallavieille-Pope C, Ali S, Leconte M, Enjalbert J, Delos M, Rouzet J. Virulence dynamics and regional structuring of Puccinia striiformis f. sp. tritici in France between 1984 and 2009. Plant Dis, 2012, 96(1): 131-140

[18] Ding Y, Cuddy WS, Wellings CR, Zhang P, Thach T, Hovmøller MS, Qutob D, Brar GS, Kutcher HR, Park RF. Incursions of divergent genotypes, evolution of virulence and host jumps shape a continental clonal population of the stripe rust pathogen Puccinia striiformis. Mol Ecol, 2021, 30(24): 6566-6584

[19] Enjalbert J, Duan X, Giraud T, Vautrin D, De Vallavieille-Pope C, Solignac M. Isolation of twelve microsatellite loci, using an enrichment protocol, in the phytopathogenic fungus Puccinia striiformis f.sp. tritici. Mol Ecol Notes, 2002, 2(4): 563-565

[20] Excoffier L, Smouse PE, Quattro JM. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics, 1992, 131(2): 479-491

[21] Guan SY, Li WN, Jin H, Zhang L, Liu GS (2023) Development and validation of a 54K genome-wide liquid SNP chip panel by target sequencing for dairy goat. Genes 14(5):1122. https://doi.org/10.3390/genes14051122

[22] Guo YW, Betzen B, Salcedo A, He F, Bowden RL, Fellers JP, Jordan KW, Akhunova A, Rouse MN, Szabo LJ, Akhunov E. Population genomics of Puccinia graminis f.sp. tritici highlights the role of admixture in the origin of virulent wheat rust races. Nat Commun, 2022, 13(1): 6287

[23] Guo ZF, Wang HW, Tao JJ, Ren YH, Xu C, Wu KS, Zou C, Zhang JN, Xu YB. Development of multiple SNP marker panels affordable to breeders through genotyping by target sequencing (GBTS) in maize. Mol Breed, 2019, 39(337

[24] Hovmøller MS, Sorensen CK, Walter S, Justesen AF. Diversity of Puccinia striiformis on cereals and grasses. Annu Rev Phytopathol, 2011, 49(1197-217

[25] Hubbard A, Lewis CM, Yoshida K, Ramirez-Gonzalez RH, de Vallavieille-Pope C, Thomas J, Kamoun S, Bayles R, Uauy C, Saunders DG. Field pathogenomics reveals the emergence of a diverse wheat yellow rust population. Genome Biol, 2015, 16(1): 23

[26] Jakobsson M, Rosenberg NA. CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics, 2007, 23(141801-1806

[27] Jombart T, Devillard S, Balloux F. Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet, 2010, 11(1): 94

[28] Kamvar ZN, Brooks JC, Grünwald NJ (2015) Novel R tools for analysis of genome-wide population genetic data with emphasis on clonality. Front Genet 6:208. https://doi.org/10.3389/fgene.2015.00208

[29] Kong XY, Ma LJ, Zhou YP, Wei M, Li GS, Lu AJ, Zhao DC, Hu XP (2014) Oversummering of Puccinia striiformis f. sp. tritici status in wheat plant tissue in Gansu province. J of Triticeae Crops 34(8):1141–1145. https://doi.org/10.7606/j.issn.1009-1041.2014.08.19

[30] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009, 25(141754-1760

[31] Li YX, Dai JC, Zhang TX, Wang BT, Zhang SY, Wang CH, Zhang JG, Yao Q, Li MJ, Li CY, Peng YL, Cao SQ, Zhan GM, Tao F, Gao HF, Huang W, Feng XL, Bai YW, Qucuo Z, Shang HS, Huang C, Liu WC, Zhan JS, Xu XM, Chen XM, Kang ZS, Hu XP. Genomic analysis, trajectory tracking, and field surveys reveal sources and long-distance dispersal routes of wheat stripe rust pathogen in China. Plant Commun, 2023, 4(4): 100563

[32] Li YJ, Pootakham W, Ingsriswang S, Cueva FD, Cordez BW, Gafforov Y, Unartngam J, Liu L, Bi GZ, Zhao P, Tsui CKM, Liang JM, Cai L. Biosurveillance of invasive southern corn rust: insights into recent migration patterns and virulencevariation. Mol Plant Pathol, 2025, 26(10e70159

[33] Li ZQ, Zeng SM. Wheat rust in China, 2002, Beijing, China Agriculture Press

[34] Line RF, Qayoum A (1992) Virulence, aggressiveness, evolution and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968-87. Technical Bulletin Number 1788. United States Department of Agriculture, Agricultural Research Service, Washington, DC

[35] Liu SJ, Xiang MJ, Wang XT, Li JQ, Cheng XR, Li HZ, Singh RP, Bhavani S, Huang S, Zheng WJ, Li CL, Yuan FP, Wu JH, Han DJ, Kang ZS, Zeng QD (2025) Development and application of the GenoBaits WheatSNP16K array to accelerate wheat genetic research and breeding. Plant Commun 6(1):101138. https://doi.org/10.1016/j.xplc.2024.101138

[36] Luo HY, Wang XJ, Zhan GM, Wei GR, Zhou XL, Zhao J, Huang LL, Kang ZS. Genome-wide analysis of simple sequence repeats and efficient development of polymorphic SSR markers based on whole genome re-sequencing of multiple isolates of the wheat stripe rust fungus. PLoS ONE, 2015, 10(6e0130362

[37] Malinsky M, Matschiner M, Svardal H. Dsuite-fast D-statistics and related admixture evidence from VCF files. Mol Ecol Resour, 2021, 21(2584-595

[38] Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ. Target-enrichment strategies for next-generation sequencing. Nat Methods, 2010, 7(2): 111-118

[39] Mardis ER. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet, 2008, 9: 387-402

[40] McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. The genome analysis toolkit: a map reduce framework for analyzing next-generation DNA sequencing data. Genome Res, 2010, 20(9): 1297-1303

[41] Meng Y, Yang CB, Jiang SC, Huang LL, Kang ZS, Zhan GM (2020) Development and evaluation of SNP molecular markers of wheat stripe rust based on KASP technology. J Plant Protect 47(1):65–73. https://doi.org/10.13802/j.cnki.zwbhxb.2020.2019047

[42] Nei M. Genetic distance between populations. Am Nat, 1972, 106(949): 283-292

[43] Perrings C, Williamson M, Barbier EB, Delfino D, Dalmazzone S, Shogren J, Simmons P, Watkinson A. Biological invasion risks and the public good: an economic perspective. Conserv Ecol, 2002, 6(1): 1-10stable/26271860

[44] Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics, 2000, 155(2): 945-959

[45] Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet, 2007, 81(3): 559-575

[46] Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci U S A, 1984, 81(24): 8014-8018

[47] Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 1985, 230(4732): 1350-1354

[48] Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A. The global burden of pathogens and pests on major food crops. Nat Ecol Evol, 2019, 3(3): 430-439

[49] Schwessinger B, Jones A, Albekaa M, Hu Y, Mackenzie A, Tam R, Nagar R, Milgate A, Rathjen JP, Periyannan S. A chromosome scale assembly of an Australian Puccinia striiformis f. sp. tritici isolate of the PstS1 lineage. Mol Plant-Microbe Interact, 2022, 35(3): 293-296

[50] Shan WS, Chen SY, Kang ZS, Wu LR, Li ZQ (1998) Genetic diversity in Puccinia striiformis Westend. f.sp. tritici revealed by pathogen genome-specific repetitive sequence. Botany 76(4):587–595. https://doi.org/10.1139/b98-035

[51] Shen Y, Wang J, Shaw RK, Yu H, Sheng X, Zhao Z, Li S, Gu H. Development of GBTS and KASP panels for genetic diversity, population structure, and fingerprinting of a large collection of broccoli (Brassica oleracea L. var. italica) in China. Front Plant Sci, 2021, 12: 655254

[52] Sotiropoulos AG, Arango-Isaza E, Ban T, Barbieri C, Bourras S, Cowger C, Czembor PC, Ben-David R, Dinoor A, Ellwood SR, Graf J, Hatta K, Helguera M, Sanchez-Martin J, McDonald BA, Morgounov AI, Muller MC, Shamanin V, Shimizu KK, Yoshihira T, Zbinden H, Keller B, Wicker T. Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade. Nat Commun, 2022, 13(1): 4315

[53] Takezaki N, Nei M. Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA. Genetics, 1996, 144(1389-399

[54] Tilman D, Balzer C, Hill J, Befort BL. Global food demand and the sustainable intensification of agriculture. Proc Natl Acad Sci U S A, 2011, 108(5020260-20264

[55] Vos P, Hogers R, Bleeker M, Reijans M, Lee T, Hornes M, Friters A, Pot J, Paleman J, Kuiper M, Zabeau M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res, 1995, 23(21): 4407-4414

[56] Wellings CR. Global status of stripe rust: a review of historical and current threats. Euphytica, 2011, 179(1): 129-141

[57] Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res, 1990, 18(22): 6531-6535

[58] Wu J, Yu R, Wang H, Zhou C, Huang S, Jiao H, Yu S, Nie X, Wang Q, Liu S, Weining S, Singh RP, Bhavani S, Kang Z, Han D, Zeng Q. A large-scale genomic association analysis identifies the candidate causal genes conferring stripe rust resistance under multiple field environments. Plant Biotechnol J, 2021, 19(1): 177-191

[59] Xia CJ, Wan AM, Wang MN, Jiwan DA, See DR, Chen XM. Secreted protein gene derived-single nucleotide polymorphisms (SP-SNPs) reveal population diversity and differentiation of Puccinia striiformis f. sp. tritici in the United States. Fungal Biol, 2016, 120(5): 729-744

[60] Xia CJ, Wang MN, Wan AM, Jiwan DA, See DR, Chen XM (2016b) Association analysis of SP-SNPs and avirulence genes in Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen. Am J Plant Sci 7(1):126–137. https://doi.org/10.4236/ajps.2016.71014

[61] Xiang MJ, Liu SJ, Wang XT, Zhang MM, Yan WY, Wu JH, Wang QL, Li CL, Zheng WJ, He YL, Ge YX, Wang CF, Kang ZS, Han DJ, Zeng QD. Development of breeder chip for gene detection and molecular-assisted selection by target sequencing in wheat. Mol Breed, 2023, 43(2): 13

[62] Xu YB, Yang QN, Zheng HJ, Xu YF, Sang ZQ, Guo ZF, Peng H, Zhang C, Lan HF, Wang YB, Wu KS, Tao JJ, Zhang JN. Genotyping by target sequencing (GBTS) and its applications. Sci Agric Sin, 2020, 53(152983-3004

[63] Yao Q, Guo QY, Yan JH, Zhang G, Hou SY, Chen WQ (2014) Survey on overwintering Puccinia striiformis f. sp. tritici at different altitudes in eastern Qinghai. J Plant Protect 41(05):578–583. https://doi.org/10.13802/j.cnki.zwbhxb.2014.05.030

[64] Zeng QD, Zhao J, Wu JH, Zhan GM, Han DJ, Kang ZS (2022) Wheat stripe rust and integration of sustainable control strategies in China. Front Agric Sci Eng 9(1):37–51. https://doi.org/10.15302/j-fase-2021405

[65] Zhan GM, Ji F, Chen XM, Wang JX, Zhang DL, Zhao J, Zeng QD, Yang LJ, Huang LL, Kang ZS. Populations of Puccinia striiformis f. sp. tritici in winter spore production regions spread from southwestern oversummering areas in China. Plant Dis, 2022, 106(11): 2856-2865

[66] Zhu JN, Awais M, Liu MNZ, Li ZJ, Ma JB, Wang L, Zhao J, Kang ZS. Genotyping reveals high genotypic diversity and potential migration pattern of Puccinia striiformis f. sp. tritici populations in Xinjiang and Northwest epidemic regions of China. Phytopathol Res, 2025, 7: 16

Funding

the National Key R&D Program of China(2021YFD1401000)

Major Program of the National Natural Science Foundation of China(32293240)

Innovation Capability Support Program of Shaanxi (No. 2023-CX-TD-56)

the earmarked fund for CARS(CARS-3)

RIGHTS & PERMISSIONS

The Author(s)