Integration of digital phenotyping, GWAS, and transcriptomic analysis revealed a key gene for bud size in tea plant (Camellia sinensis)

Abstract

Tea plant (Camellia sinensis) is among the most significant beverage crops globally. The size of tea buds not only directly affects the yield and quality of fresh leaves, but also plays a key role in determining the suitability of different types of tea. Analyzing the genetic regulation mechanism of tea bud size is crucial for enhancing tea cultivars and boosting tea yield. In this study, a digital phenotyping technology was utilized to collected morphological characteristics of the apical buds of 280 tea accessions of representative germplasm at the 'two and a bud' stage. Genetic diversity analysis revealed that the length, width, perimeter, and area of tea buds followed a normal distribution and exhibited considerable variation across natural population of tea plants. Comparative transcriptomic analysis of phenotypic extreme materials revealed a strong negative correlation between the expression levels of four KNOX genes and tea bud size. A key candidate gene, CsKNOX6, was confirmed by further genome-wide association studies (GWAS). Its function was preliminarily characterized by heterologous transformation of Arabidopsis thaliana. Overexpression of CsKNOX6 reduced the leaf area in transgenic plants, which initially determined that it is a key gene negatively regulating bud size. These findings enhance our understanding of the role of KNOX genes in tea plants and provide some references for uncovering the genetic regulatory mechanisms behind tea bud size.

Figure 1

Statistical analysis of bud traits based on image collection and feature extraction in 280 tea accessions. (A) Image collection and feature extraction process. (B) Frequency distribution of bud length, width, perimeter, and area. (C) Cluster analysis of 280 tea accessions based on bud size phenotype and the trait distribution for each group. (D) Grouped statistical chart of the top 15 regions by accession numbers ranking.

Figure 2

RNA-seq analysis of bud size in tea plant. (A) Anatomy diagram of tea bud in eight selected accessions. Black bar = 1 cm. (B) Violin-plot distributions of bud length, width, perimeter, and area in eight selected accessions. (C) Gene screening based on Pearson correlation analysis on four traits. (D) Venn diagram of correlation genes for four traits. (E) GO enrichment analysis of 50 genes associated with bud size.

Figure 3

The expression patterns of candidate genes and qRT-PCR validation. (A) The clustering heat map displayed the expression patterns of 50 DEGs. Six TFs were labeled on the right side of the heat map. (B) The expression levels of KNOX genes were validated using qRT-PCR. Results are expressed as mean ± SEM (n = 3). Significant differences (P < 0.001) are indicated as ^***. nd, not detected.

Figure 4

Identification of KNOX genes in tea plant. (A) Phylogenetic analysis of KNOX genes. The neighbor-joining phylogenetic tree was generated with the 1000 bootstrap values. (B) Chromosomal locations of the KNOX genes based on the genome of tea cultivar ‘Shuchazao’. Four candidate KNOX genes have been annotated on Chr4, Chr8, Chr9, and Chr10.

Figure 5

GWAS analysis of four tea bud traits. (A) Manhattan plot and Quantile–quantile (QQ) plot of the GWAS analysis of bud length, width, perimeter, and area. The dotted lines are the threshold level (−log₁₀P = 6). The positions indicated by the arrows represent the candidate interval containing KNOX gene CSS0014431. (B) Box plot of four bud trait values among the 280 tea accessions at the lead GWAS SNP (Chr10:14322573). Different letters indicate significant difference (P < 0.05) according to Tukey’s HSD test.

Figure 6

Functional validation of CsKNOX6. (A) Subcellular localization of CsKNOX6 protein in transformed Nicotiana benthamiana leaves. The first, second, and third columns indicate the locations of the GFP fusion protein, the nucleus, and the chloroplasts, respectively. The 35S::GFP empty vector was used as controls. White bar = 20 μm. (B) Expression levels of CsKNOX6 in T₃-generation homozygous overexpressing Arabidopsis (OE1, OE2, and OE3) and WT plants. Results are expressed as mean ± SEM (n = 3). nd, not detected. (C) Phenotypes of 7-day-old transgenic Arabidopsis and WT plants. Black bar = 0.5 cm. (D) Phenotypes of 14-day-old transgenic Arabidopsis and WT plants. Black bar = 2 cm. (E) Leaf area of transgenic and WT Arabidopsis after 14 days of growth. Results are expressed as mean ± SEM (n = 4). Significant differences (P < 0.001) are indicated as ^***.