Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research

Abstract

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

Keywords: Camellia tetracocca; genetic diversity; intron length polymorphism (ILP); population structure; transferability.

Figure 1

Circle diagrams illustrating the distribution and density of ILP primers developed based on the ‘Shuchazao’ scaffold genome, ‘Longjing 43’ chromosome-level genome, and DASZ chromosome-level genome, respectively. The outermost circle denotes the physical size (Mb) of 1 largest scaffold of ‘Shuchazao’, 15 chromosomes of ‘Longjing 43’ and 15 chromosomes of DASZ, each indicated by different colors. Circles I and II show the distribution positions and densities of designed ILP primers, respectively. Circles III and IV show the distribution density and location of developed ILP Makers, respectively.

Figure 2

Distribution of intron length difference under different ePCR analysis. ePCR1 means 1 band amplified by ePCR, ePCR2 means 2 bands amplified by ePCR, ePCR3 means 3 bands amplified by ePCR, and ePCR > 3 means more than 3 bands amplified by ePCR.

Figure 3

The cross-transferability of tea ILP markers in 6 families based on different reference genomes.

Figure 4

Genetic relationships of 11 plant species as determined by 35 ILP molecular markers. Cr (C. reticulate), Os (Oryza sativa), Ta (Triticum aestivum), Zm (Zea mays), Nt (Nicotiana tabacum), Le (Lycopersicon esculentum), Ca (Capsicum annuum), At (A. thaliana), Bn (Brasscia napus), Ha (Helianthus annus), Cs (Cucumis sativus).

Figure 5

Population clustering map of Puan-cultivated C. tetracocca based on genetic similarity coefficient.

Figure 6

Q value distribution of population structure of cultivated C. tetracocca in Puan. The 176 individuals were divided into subpopulations S1 (red bar graph), S2 (green bar graph), and S3 (blue bar graph), comprising 19, 60, and 97 individuals.

Figure 7

Principal component analysis of cultivated C. tetracocca in Puan.