TEA5K: a high-resolution and liquid-phase multiple-SNP array for molecular breeding in tea plant

Abstract

Background: High-throughput genotyping technology has become an indispensable tool for advancing molecular breeding and genetic research in plants, facilitating large-scale exploration of genomic variation. Genotyping technology based on liquid-phase array utilizes streptavidin-coated nanomagnetic beads to capture biotin-modified probes, thereby capturing the target sequence on the genome, achieving the purpose of genotyping. This study aims to develop a novel liquid-phase for tea plant, which can be used for cultivar identification, genetic map construction, Quantitative Trait Locus (QTL) mapping of key agronomic traits in tea plants, and genetic evolution analysis.

Result: We developed a highly efficient multiple-SNP array, the TEA5K mSNP array, which comprises 5,781 liquid-phase probes based on the Genotyping by Target Sequencing (GBTS) system. Using this array, we genotyped 231 developed tea cultivars, revealing that genetic similarity within the same cultivar ranged from 92.53-97.95%, whereas genetic similarity between different cultivars generally remained below 82.36%. Furthermore, utilizing this array, we constructed a high-density genetic map consisting of 3,274 markers, covering a total genetic distance of 2,225.19 cM, with an average marker interval of 0.76 cM. The high-resolution genetic map facilitated the identification of multiple QTLs linked to eight amino acid components, as well as two molecular markers strongly associated with the albino-leaf trait in the 'Huangjinya' cultivar, both mapped to chromosome 8. Moreover, we applied the array to analyze the population structure and phylogenetic relationships of 519 tea germplasm, classifying them into three major groups: wild accessions, landraces, and modern cultivars. Notably, modern cultivars exhibited lower genetic diversity compared to landraces. Additionally, we observed substantial genetic differentiation between wild resources and modern cultivars, with minimal to no gene flow from wild populations into domesticated cultivars. These findings suggest that modern tea breeding faces an "improvement bottleneck," a challenge similar to that encountered in other perennial crops.

Conclusion: The TEA5K mSNP array is presented as a flexible, cost-effective, and low-maintenance genotyping tool that significantly enhances both genetic research and molecular breeding in tea plants. By providing a robust platform for genome-wide analysis and facilitating the identification of key QTLs, this tool offers valuable insights for improving the genetic diversity and agronomic performance of tea cultivars.

Keywords: Cultivar identification; Gene mapping; Genetic evolution; Genetic maps; Genotyping by target sequencing.

Fig. 1

Marker site analysis of TEA5K mSNP array. (A) Design pipeline of the GenoBaits TEA5K mSNP array; (B) Distribution of 5 K mSNP markers across the 15 chromosomes of tea plant. Marker density is represented by bar color, with each bar corresponding to a 1-Mb genomic window; (C) Statistical analysis of sequencing depth across 519 samples genotyped using the TEA5K mSNP array; (D) Statistical analysis of the missing rate across 519 samples, based on 5,781 SNPs and 36,357 SNPs

Fig. 2

Frequency distribution of TEA5K mSNP across 15 chromosomes. The number of SNPs per amplicon is color-coded to indicate variation in marker coverage across the genome

Fig. 3

Annotation analysis of 36,357 SNP sites. (A) GO enrichment analysis of genes including 36,357 SNP sites; (B) KEGG pathway enrichment analysis of genes including 36,357 SNP sites; (C) Genomic distribution of 36,357 SNP sites developed from different genomic regions; (D) Mutation type classification of 36,357 SNPs, distinguishing between transition and transversion mutations

Fig. 4

Modern cultivar and bud mutant identification analysis. (A) Phenotypic variation among modern cultivars; (B) Vegetative propagation-based phenotypic variation in eight cultivars; (C) Bud mutant phenotype analysis, comparing green-leaf branches and albino mutant branches. (D), E, F. Genetic similarity (GS) analysis of cultivars, vegetative propagation individuals, and bud mutants, evaluated using the 5 K core SNP panel, 36 K SNP panel, and 5 K mSNP panel, respectively. Sample pair counts increase from blue to yellow

Fig. 5

Gene mapping of albino-leaf trait by using TEA5K mSNP array. (A) Parental and offspring phenotypic variation; (B) Genetic map constructed using the TEA5K mSNP array; (C) map-based clone results for the albino-leaf trait; D QTL mapping results for various amino acid components; (E) Result of Bulked Segregant Analysis mapping for albino leaf color

Fig. 6

Distribution and population evolution analysis of 519 tea plant resources. (A) Geographical distribution of 519 tea plant accessions, highlighting the collection regions across multiple Chinese provinces. (B) Principal Component Analysis (PCA) of 519 tea germplasm. (C) Population structure analysis of 519 tea plant accessions, evaluated under K = 2 and K = 3 conditions. (D) Phylogenetic tree of 519 tea resources. The green section represents the majority of modern tea cultivars, the red section corresponds to most landrace resources, and the blue section encompasses almost all wild tea resources. (E) Simulated domestication pathway of tea plants, illustrating the evolutionary transition from wild tea populations to modern cultivars