Genome-wide association study of leaf-related traits in tea plant in Guizhou based on genotyping-by-sequencing

Abstract

Background: Studying the genetic characteristics of tea plant (Camellia spp.) leaf traits is essential for improving yield and quality through breeding and selection. Guizhou Plateau, an important part of the original center of tea plants, has rich genetic resources. However, few studies have explored the associations between tea plant leaf traits and single nucleotide polymorphism (SNP) markers in Guizhou.

Results: In this study, we used the genotyping-by-sequencing (GBS) method to identify 100,829 SNP markers from 338 accessions of tea germplasm in Guizhou Plateau, a region with rich genetic resources. We assessed population structure based on high-quality SNPs, constructed phylogenetic relationships, and performed genome-wide association studies (GWASs). Four inferred pure groups (G-I, G-II, G-III, and G-IV) and one inferred admixture group (G-V), were identified by a population structure analysis, and verified by principal component analyses and phylogenetic analyses. Through GWAS, we identified six candidate genes associated with four leaf traits, including mature leaf size, texture, color and shape. Specifically, two candidate genes, located on chromosomes 1 and 9, were significantly associated with mature leaf size, while two genes, located on chromosomes 8 and 11, were significantly associated with mature leaf texture. Additionally, two candidate genes, located on chromosomes 1 and 2 were identified as being associated with mature leaf color and mature leaf shape, respectively. We verified the expression level of two candidate genes was verified using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and designed a derived cleaved amplified polymorphism (dCAPS) marker that co-segregated with mature leaf size, which could be used for marker-assisted selection (MAS) breeding in Camellia sinensis.

Conclusions: In the present study, by using GWAS approaches with the 338 tea accessions population in Guizhou, we revealed a list of SNPs markers and candidate genes that were significantly associated with four leaf traits. This work provides theoretical and practical basis for the genetic breeding of related traits in tea plant leaves.

Keywords: Candidate genes; Genome-wide association study (GWAS); Genotyping-by-sequencing (GBS); Leaf traits; Single nucleotide polymorphism (SNP); Tea plant.

Fig. 1

The frequency distribution of MLZ (in three years) and three qualitative traits (MLC, MLS, MLT), the frequency distribution of the three qualitative traits are mainly displayed by morphological characteristics. A Frequency distribution of MLZ in 2019. B Frequency distribution of MLZ in 2020. C Frequency distribution of MLZ in 2021. D Frequency distribution of MLC. Y.G.: yellow-green; L.G.: light green; G.R.: green; D.G.: dark green. E Frequency distribution of MLS. R.O.: round; O.V.: oval; L.O.: long oval; L.A.: lanceolate. F Frequency distribution of MLT. SO: Soft; ME: Medium; HA: Hard

Fig. 2

Distribution of single nucleotide polymorphisms (SNPs) on 15 chromosomes of the tea plant. The horizontal axis shows the chromosome length

Fig. 3

Population structure analysis, principal component analysis (PCA), and phylogenetic tree establishment of the 338 tea accessions. A The neighbor-joining tree was established by 100,829 high-quality SNPs. B Population structure separates the accessions into four subgroups (K = 4). C PCA of 338 tea accessions. And first and third components of the PCA analyses were shown; each dot represents an individual. D When K = 4, the CV error value was the smallest (0.51127)

Fig. 4

L.D. decay of 338 tea accessions

Fig. 5

Manhattan plots and Q-Q plots of the 3-years (2019, 2020, 2021) cMLM-P + K model for MLZ traits of the tea plant. A MLZ-cMLM-P + K-2019; B MLZ-cMLM-P + K-2020; C MLZ-cMLM-P + K-2021; The red dashed horizontal line indicates the significance threshold (-log₁₀^(P) is about equal to 4.0); The red arrows represent SNPS that have been jointly identified for MLZ traits in three years; “16” represents Contig (A continuous long DNA sequence overlapping fragment formed by joining short DNA fragments in the process of genome sequencing.) in Manhattan plots. Manhattan plots and Q-Q plots of the 1-year optimal model for three qualitative traits (MLC, MLS, MLT). D MLC-GLM-Q; E MLS-cMLM-P + K; (F) MLT-GLM-P; The red dashed horizontal line indicates the significance threshold (-log₁₀^(P) is about equal to 4.0); The red arrows represent the ultimately significant SNPS associated with the three traits; “16” represents Contig (A continuous long DNA sequence overlapping fragment formed by joining short DNA fragments in the process of genome sequencing.) in Manhattan plots

Fig. 6

A The expression level of TEA005350.1 gene, a candidate gene associated with mature leaf size (MLZ), in leaflet, middle, and large leaves. L.F.: leaflet; M.L.: middle leaf; L.L.: large leaf. B Expression level of TEA027527.1 gene associated with mature leaf color (MLC) in yellow-green, light green, green, and dark green leaves. Y.G.: yellow-green; L.G.: light green; G.R.: green; D.G.: dark green

Fig. 7

Design and verification of the dCAPS marker. A Establishment of a dCAPS marker with the NlaIII restriction enzyme (the underlined part is the primer sequence, italics are digestion sites, the yellow bases are the introduced one mismatch into the forward primer, the red base is the SNP site); B PCR products of six tea accessions using the dCAPS primer

Fig. 8

Distribution of 336 materials from 30 counties in Guizhou Province

Fig. 9

A Three morphological characteristics of “Mature leaf size (MLZ)” phenotypic trait. L.F.: leaflet; M.L.: middle leaf; L.L.: large leaf. B Morphological characteristics of four “Mature leaf colors (MLC)”. Y.G.: yellow-green; L.G.: light green; G.R.: green; D.G.: dark green. C Morphological characteristics of four “Mature leaf shapes (MLS)”. R.O.: round; O.V.: oval; L.O.: long oval; L.A.: lanceolate