Cauliflower (Brassica oleracea var. botrytis), as an important vegetable crop grown worldwide, is gaining popularity in human diets because of its good flavor, rich nutritional value and anticarcinogenic effects [1]. The production of cauliflower (including broccoli) has increased in recent decades, reaching 36.9 million tons in 2019 [2]. Since the late 1980s, China has become the largest cultivation and production area of cauliflower in the world [3]. The shared progenitor of B. oleracea species was believed to be B. cretica which originated in the eastern Mediterranean region [4, 5]. Cauliflower most likely evolved from broccoli and underwent a strong genetic bottleneck during its differentiation and domestication [6]. To date, reduced genetic variation and limited breeding resources have seriously slowed down the process of cauliflower improvement. Owing to the narrow genetic background and imperfect conservation and protection system, it is inconvenient and difficult for breeders to effectively recognize cauliflower and protect the variety rights [7]. Under these circumstances, an accurate, efficient, and economical method to support breeding efforts and protect plant variety rights is urgently needed for cauliflower.
Over the past two decades, a series of DNA markers, including restriction fragment length polymorphisms (RFLP) [8], random amplified polymorphic DNA (RAPD) [9], amplified fragment length polymorphism (AFLP) [10], inter-simple sequence repeat (ISSR) [11], and simple sequence repeat (SSR) [12] have been developed and widely used in the research fields of genetic diversity analysis, gene mapping, association studies, and molecular-assisted breeding, etc. In recent years, with the rapid development of high-throughput sequencing technologies, Single nucleotide polymorphisms (SNP) markers have gradually become the most popular option because of their relatively low cost, high yield, good stability and reproducibility, uniform distribution across the genome, and ease of documentation [13–18]. Until now, several PCR-based platforms, such as GoldenGate [19], Illumina Infinium [20], TaqMan from Life Technologies [21], and Kompetitive allele-specific PCR (KASP, LGC Bioresearch technologies), have been successfully developed for verification of SNP markers. Of these, the KASP platform, which takes advantages of high accuracy, high throughput, and cost-effectiveness, has been widely used in wheat [22–24], rice [25, 26], cotton [27], cucumber [28], and broccoli [7].
DNA fingerprinting is a powerful approach based on molecular markers or special sequences to identify genetic diversity and distinguish different cultivars in plants [29, 30]. So far, DNA fingerprinting databases have been established in a series of plants such as maize [29, 31], grapevine [32], cigar tobacco [33], and red bayberry [34], demonstrating their technical significance for variety identification and germplasm innovation. In cabbage, 50 core SNPs were selected from 59 varieties and used in efficient identification of seed authenticity and purity [35]. In broccoli, deep sequencing was carried out for 23 representative broccoli lines, resulting in the identification of 100 SNPs for subsequent KASP experiments and 25 core markers for fingerprinting. Further analyses of genetic diversity, genetic relationships, and population structure of 392 broccoli accessions revealed a narrow genetic background in broccoli population [7]. In cauliflower, AFLP markers have been reported to explore the genetic diversity of different cauliflower varieties. The kinships between cauliflower and other subspecies of B. oleracea were revealed [36]. SSR markers were also widely used in revealing the genetic diversity and relationships of cauliflower varieties [37, 38]. However, so far, no SNP-based fingerprinting database has been reported in cauliflower.
In this study, a practical workflow was developed to generate the SNP-based fingerprinting database for cauliflower. First, we screened high-quality SNP markers from the variation database which was previously generated by whole-genome resequencing. We then developed programs to select optimal marker combinations, followed by KASP conversion and fingerprinting of 329 representative cauliflower cultivars mainly collected from the public market. We also performed population genetic analysis and variety identification of these 329 cultivars based on the KASP genotyping results. Our results will provide practical tools for variety authenticity and purity identification and give a new sight of the genetic relationships of current commercial cultivars of cauliflower.