Using bioinformatics technology to explore molecular markers that differ among hybrid vegetable varieties, in order to establish molecular detection technology that can be automated for analysis, assist variety identification, improve control over the purity of first-generation hybrid seeds, and help make seedling sales more advantageous.

Urbanization has gradually reduced the area of farmland, yet the demand to meet food needs keeps rising. Coupled with the severe challenges that extreme climate brings to agriculture, Taiwan's agriculture, relying on its excellent breeding technology, has enabled crops to successfully produce superior varieties that can respond to environmental needs through hybridization under natural conditions, bringing new opportunities for increased agricultural production.

The key to successful breeding lies in seed purity

"Precisely because the success rate of hybridization and the correctness of varieties are extremely important for seedling sales, we began to think about how to establish more accurate and lower-cost detection technology to help seedling operators achieve good quality control," said Associate Professor Hu Kaikang of the Department of Agronomy at National Taiwan University. He also led a research team to carry out research on establishing molecular detection technology to improve the efficiency of hybrid seed purity and combining ability testing.

It turns out that in the past, after seedling operators bred superior first-generation hybrid vegetable varieties, they would entrust professional farmers or seed production companies to produce hybrid seeds. Whether the seed production site was in Taiwan or overseas, seed purity was an important quality control indicator.

Associate Professor Hu Kaikang said, "Seed purity is closely tied to the reputation of seedling operators, including the success rate of hybridization and the purity of the variety, to confirm that there is no foreign pollen contamination or mixture during processing. Strict testing is required before export, but the traditional grow-out test is time-consuming and labor-intensive. In addition to increasing seed production costs, it also often causes continuous disputes between seedling operators and commissioned farmers."

Although crop genomics research and bioinformatics technology have advanced by leaps and bounds in recent years, the seedling industry often has very low acceptance of research and development results. The main reason is that the reproducibility of non-specific molecular marker technology is relatively low, so the technical requirements for experimental operations such as DNA extraction and PCR assembly are relatively high, and the analysis and interpretation process requires the input of a large amount of professional manpower, cannot be automated, and costs rise accordingly.

Full commitment improving detection efficiency

Facing this predicament, after the 99th year Council of Agriculture academic science and technology special project "Establishing molecular-assisted selection technology for important vegetable and fruit crops," the deadlock was finally broken, and high-throughput SNP molecular marker development technology based on next-generation sequencing began to be introduced, developing a technical platform capable of large-scale genotyping analysis and molecular marker development.

Associate Professor Hu Kaikang stated that the first step of this project was to target the most important vegetable crops in Taiwan's seedling industry. After using high-throughput next-generation parallel sequencing (Next Generation Sequencing, NGS) to obtain a large number of fragment sequences near specific restriction enzyme cutting sites, bioinformatics technology was used to explore single nucleotide polymorphism (SNP) molecular markers that differ among individuals in segregating populations. Then, based on the association between the obtained molecular marker genotypes and target traits (for example: disease resistance), molecular markers closely linked to the genes controlling the target traits were obtained and provided to seedling operators as assistance in the breeding selection process. Because selection is directly aimed at loci, compared with traditional selection methods that rely on external traits, this greatly improves the accuracy of breeding selection and shortens the time required for breeding selection.

The SNP molecular markers obtained by the development platform established in the molecular-assisted selection special project have high specificity and reproducibility, and with appropriate instruments can be analyzed automatically. Therefore, after the development platform was established, Associate Professor Hu Kaikang, starting from the 106th year, responded to the needs of the seedling industry and carried out the two-year council science and technology project "Establishing molecular detection technology to improve the efficiency of hybrid seed purity and combining ability testing." For more than 1,000 first-generation hybrid varieties of tomato, cauliflower, watermelon, melon, corn, and pepper provided by seedling operators, at least 5,000 SNP loci were explored for each crop, and from these, core SNP combinations with good primer design characteristics and capable of fully explaining differences among varieties were screened out. Then, based on variety characteristics, the minimal SNP molecular marker combinations suitable for variety identification and hybridization success rate testing were selected as detection standards. These SNP molecular marker combination standards have high reproducibility and can be fully automated, helping the seedling industry improve the purity control capability of the seeds it produces while reducing the burden on manpower and testing costs, thereby enhancing the export competitiveness of the seed industry.

Technology optimization strengthening the competitiveness of seedling operators

"The most important thing, of course, is to make testing both time-saving and money-saving," said Associate Professor Hu Kaikang. Using molecular markers for hybridization success rate testing, compared with using field inspection, can reduce the cost by 4,000 yuan per batch of hybrid seeds.

In addition to significantly reducing the cost of seed testing, molecular detection has high accuracy and can also shorten the testing time to within one month, accelerating the speed at which products can be shipped. It can be said to greatly reduce seed storage and management and marketing costs, fully improving the quality and international competitiveness of seedling operators' products.

Associate Professor Hu Kaikang believes that in the future, whether a credible testing operator receives technology transfer, or seedling operators jointly invest to establish a testing laboratory, both can inject abundant energy into the upgrading of the seedling industry and the optimization of agriculture, embracing a brand-new future.

Related information

To learn more about the details of this project's research and development results, please contact Mr. He of the Agricultural Technology Research Institute, telephone: 03-5185049, email: [email protected]