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naica® Droplet Chip Digital PCR System Accurately Detects Differences in CITST2 Gene Copy Numbers among Different Watermelon Germplasms
2024-05-07
Watermelon (Citrullus lanatus, 2n=22) is the world's third largest fruit, an important economic crop grown around the world, and a popular fresh fruit that contains compounds that are beneficial to human health, such as sugar, lycopene, and citrulline. Researchers have developed a large number of watermelon varieties through hybridization to meet consumer preferences. However, long-term cultivation and screening for fruit quality traits have shown that cultivated watermelons collected from different geographical regions show low genetic diversity, so more genetic germplasm resources are needed for innovative varieties of sustainable production of watermelon.
Reference genomes are essential for trait and gene discovery. Watermelon genome sequencing work began more than a decade ago. In addition to the first draft of the watermelon genome, three high-quality watermelon reference genomes have been released since 2019. However, each genome is still incomplete and has many gaps. High-quality reference genomes combined with mutant data generated from the same genetic library will help discover and isolate mutants required for genetics and breeding. A gap-free reference genome is the ultimate goal of genome assembly, bringing new opportunities to identify unique genes and structural variations in the "dark matter" region.
Researchers from the Institute of Advanced Agricultural Sciences of Peking University published an article titled "A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding" in Molecular Plant (latest JCR classification Q1, impact factor 21.9496). The researchers used the watermelon elite inbred line G42 to assemble a T2T (telomere-to-telomere) gap-free reference genome, filling all remaining assembly gaps in the currently available reference genomes. Through genome information alignment, a 17.5 kb tandem repeat sequence (sv04611) containing the ClTST2 (vacuole sugar transporter) gene in sweet watermelon germplasm was identified. The study used the naica® droplet chip digital PCR system to detect the copy number of the watermelon CITST2 gene, confirming that there were differences in the copy number of the CITST2 gene between different watermelon germplasms, and the increase in the copy number of the CITST2 gene in the sweet watermelon germplasm may be the reason for the increase in sugar content.
Application highlights:
▶ Accurately quantify the difference in CITST2 gene copy number between sweet and unsweet watermelon germplasms using the naica® droplet chip digital PCR system.
▶ The CITST2 gene in sweet watermelon germplasms has two copies and a single copy, respectively.
▶ Copy number variation of the CITST2 gene may change the sugar content of watermelon.
The G42 genome assembly has higher completeness and accuracy than other reference genome assemblies, providing more data support for more accurate description of gene structural variations (SVs). Digital PCR technology has been proven to be a sensitive and reliable tool for absolute quantitative detection of nucleic acid copy number variation. This study used the naica® droplet chip digital PCR system to accurately quantify the difference in CITST2 gene copy number between different watermelon germplasms and verified the accuracy of the gapless reference genome in identifying SVs.
Research results:
This study successfully assembled the T2T gapless reference genome of G42 watermelon, including information on all 22 telomeres and 11 centromeres. Using the gapless reference genome data, the researchers identified a 17.5 kb tandem repeat sequence (sv04611) containing the ClTST2 gene in the sv04986 structure of sweet watermelon germplasm (97103 and G42). This is not present in the genome of non-sweet watermelon germplasm (PI 595203 and PI 296341-FR). The ClTST2 gene encodes a sugar transporter localized in the vacuole, and its expression is positively correlated with sugar accumulation in watermelon pulp.
▲Figure 1. (A) The sv04986 construct contains the ClTST2 gene in sweet watermelon (97103 and G42) and non-sweet watermelon (PI 595203 and PI 296341-FR) germplasms. A 17.5kb tandem repeat sequence sv04611 containing a 2bp CA insertion mutation is present in the sweet watermelon germplasm. The red arrows indicate the position of primers ClTST2-R8/ClTST2-F3. (B) Results of amplification of four watermelon germplasm DNA samples using ClTST2-R8/ ClTST2 -F3 primers.
The authors then used the naica® Droplet Chip Digital PCR System to accurately quantify the difference in CITST2 gene copy number between sweet and non-sweet watermelon germplasms. The FAM channel only detects the region with the CA insertion mutation. The HEX channel is used to detect the ClTST2 gene. The CY5 channel detects the single-copy reference gene Actin in watermelon. When the CITST2 gene is double-copy, the FAM/HEX/CY5 copy number ratio is about 1:2:1. When the CITST2 gene is single-copy, the FAM/HEX/CY5 copy number ratio is about 0:1:1. The data showed that the two unsweet watermelon germplasms contained only a single copy of the CITST2 gene, while the sweet watermelon germplasm contained two copies of the CITST2 gene.
▲Table 1. Estimation of CITST2 gene copy number in sweet and unsweet watermelon germplasm using dPCR technology
The original link is as follows: https://www.cell.com/molecular-plant/fulltext/S1674-2052(22)00192-7?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1674205222001927%3Fshowall%3Dtrue
Journal introduction:
Molecular Plant is an academic journal sponsored by the Institute of Plant Physiology and Ecology (IPPE) of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and the Chinese Society of Plant Physiology and Plant Molecular Biology (CSPP), and hosted by the Life Science Journal Press of the Shanghai Life Science Information Center of the Chinese Academy of Sciences. It was founded in 2008. The latest JCR classification in 2022 is Q1, with an impact factor of 21.9496.