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A three-locus model for rind phenotypes in watermelon (Citrullus lanatus) was previously proposed based on genetic analysis. These three loci, S (foreground stripe pattern), D (depth of rind color), and Dgo (background rind color), segregate in a Mendelian manner. Whole genome sequencing of watermelon offers a new strategy for marker development in these rind phenotype-related loci. A genotype analysis using subsets of 188, 273, 287 and 113 probes was performed for the ‘0901’, ‘10909’, ‘109905’ and ‘90509’ rind trait-segregating F2 populations, respectively. A total of 26, 34, 30 and 15 linkage groups with 175, 254, 269 and 79 probes were constructed for the ‘0901’, ‘10909’, ‘109905’ and ‘90509’ populations, respectively. The genetic order of the probes was mostly collinear with the physical order on the reference genome, except for some probes on chromosomes 1, 3 and 11. The three rind-related loci, S, D, and Dgo were anchored near chr6_25767 on chromosome 6, chr8_26061 on chromosome 8 and chr4_150/chr4_249 on chromosome 4, respectively. The three loci are located on different chromosomes, and the three-locus model was therefore verified through molecular genetic analysis. We suggest a rapid and practical marker development strategy that can be used not only for rind traits but also for other agriculturally important traits in watermelon and applied for conventional breeding.

Among the molecular markers used today, single nucleotide polymorphisms (SNP) are the most common type used in genetic diversity analysis due to their abundance. To develop a high-throughput SNP marker set to accelerate genetic background selection in Brassica rapa breeding, we sequenced the transcriptomes of 20 Chinese cabbage accessions representing diversity in traits such as head type, maturity, inner leaf color, and disease resistance. We identified 13,976 SSRs and 380,198 SNPs by aligning their contigs. We chose 189 SNPs that covered the entire B. rapa genome through a filtering process based on criteria such as depth, level of polymorphism, segregation ratio, lack of adjacent SNPs, copy number, and PIC value. To validate the SNP marker set, we genotyped 23 Chinese cabbage accessions and constructed a phylogenetic tree. The results showed that the SNP genotyping data could distinguish the Chinese cabbage accessions according to their phenotypic variations. The 23 accessions were classified into two groups that were characterized by phenotypic traits, especially head type and maturity. In conclusion, the selected SNP marker set is a reliable breeding tool for distribution analysis or selection of different Chinese cabbage accessions and may be applicable for rapid genetic background selection of Chinese cabbages for breeding.

Most agricultural traits are controlled by quantitative trait loci (QTLs); however, there are few studies on QTL mapping of horticultural traits in pepper (Capsicum spp.) due to the lack of high-density molecular maps and the sequence information. In this study, an ultra-high-density map and 120 recombinant inbred lines (RILs) derived from a cross between C. annuum ‘Perennial’ and C. annuum ‘Dempsey’ were used for QTL mapping of horticultural traits. Parental lines and RILs were resequenced at 18× and 1× coverage, respectively. Using a sliding window approach, an ultra-high-density bin map containing 2,578 bins was constructed. The total map length of the map was 1,372 cM, and the average interval between bins was 0.53 cM. A total of 86 significant QTLs controlling 17 horticultural traits were detected. Among these, 32 QTLs controlling 13 traits were major QTLs. Our research shows that the construction of bin maps using low-coverage sequence is a powerful method for QTL mapping, and that the short intervals between bins are helpful for fine-mapping of QTLs. Furthermore, bin maps can be used to improve the quality of reference genomes by elucidating the genetic order of unordered regions and anchoring unassigned scaffolds to linkage groups.

The rind phenotype of watermelon fruits is an important agronomic characteristic in the watermelon market. Inheritance and linkage analyses were performed for three rind-related traits that together determine the rind phenotype: foreground stripe pattern, rind background color, and depth of rind color. The inheritance of the foreground stripe pattern was analyzed using three different F₂ populations, showing that the striped pattern is dominant over the non-striped pattern. The inheritance analysis of the rind background color was performed using F₂ populations of the ‘10909’ and ‘109905’, and the depth of rind color was analyzed using F₂ populations of the ‘90509’ and ‘109905’. Yellow color was found to be dominant over green color, and a deep color was dominant over the standard color. Linkage analysis of the three traits was conducted using three F₂ populations in which two traits were segregating. Each pair of traits was inherited independently, which demonstrated that the three traits are not linked. Therefore, we propose a three-locus model for the determination of rind phenotype, providing novel insight that rind phenotype is determined by the combination of three genetically independent loci.

Modulated electro-hyperthermia (mEHT) has been shown to be effective against various types of human tumours, including hepatocellular carcinoma (HCC). Here we aimed to investigate the molecular mechanism underlying the cytotoxic effects of mEHT to HCC cells.Human liver cancer cell lines, Huh7 and HepG2, were treated with mEHT (42 °C/60 min) three times at 2-day intervals. Growth inhibition and apoptotic induction were evaluated using MTS, microscopic analysis, a clonogenic assay, annexin V/PI staining and a ccK18 ELISA. Global changes in gene expression were examined using RNA sequencing to obtain insights into molecular changes in response to mEHT. For in vivo evaluation of mEHT we used HepG2 HCC xenografts grown in nude mice.mEHT suppressed HCC cell proliferation and long-term colony formation through induction of apoptosis. The growth inhibitory effects are induced through a subset of molecular changes. Notably the expression level of septin 4 (SEPT4) (involved in pro-apoptotic activity and growth suppression) was up-regulated, whereas a key regulator of invasiveness G-Protein coupled receptor 64 (GPR64) was repressed. Subsequent Western blotting confirmed that the common increase in tumour suppressor SEPT4 in both Huh7 and HepG2 cells is accompanied by the restoration of cyclin-dependent kinase (CDK) inhibitor p21 and decrease in pro-caspase 7 and pro-caspase 3, thereby accelerating apoptotic signalling in HCC cells. Additionally, mEHT significantly inhibited the growth of human HCC xenografts in nude mice.These findings suggest that apoptotic cell death induced by mEHT is mediated by the up-regulation of tumour suppressor SEPT4 in human HCC cells.

Molecular markers are essential for breeding major crops today and many molecular marker techniques have been developed. DNA markers are now the most commonly used. This chapter describes the principles of DNA marker techniques and methods to map major genes. DNA markers can be classified into two categories: (1) DNA hybridization-based techniques, including restriction fragment polymorphism and DNA chips, and (2) polymerase chain reaction techniques, including simple sequence repeats, random amplified polymorphic DNA, amplified fragment length polymorphism, and single nucleotide polymorphism. To develop trait-linked markers, segregating populations for the target traits and reliable phenotyping methods are indispensable. With these tools, two approaches can be used to develop trait-linked markers: (1) when there is no biological information for the trait, and (2) when biological information is available. Finally, we describe several case studies for trait-linked marker development.