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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.

A novel multiplexing method, which relies on universal amplification of separated ligation-dependent probes (ASLP), has been developed to genotype single-nucleotide polymorphisms (SNPs). The ASLP technique employs two allele-specific oligonucleotides (ASO), modified with universal forward primer sequences at the 5′-end and a common locus-specific oligonucleotide (LSO) extended with a universal separation (US) sequence at the 3′-end. In the process, allele-specific ligation first takes place when target genomic DNA is hybridized by perfectly matching the ASO together with the LSO. A separation probe, which consists of a universal reverse primer sequence labeled with biotin at the 5′-end and complementary sequence of US at the 3′-end, is then applied to the resulting ligation product. During the extension reaction of the separation probe, the ligated probes dissociate from target genomic DNA in the form of a double-stranded DNA and are separated from the reaction mixture, which includes genomic DNA and unligated probes, by simply using streptavidin-coated magnetic beads. PCR amplification of the separated ligation products is then carried out by using universal primers and the PCR products are hybridized on a DNA microarray using the RecA protein. The advantageous features of the new method were demonstrated by using it to genotype 15 SNP markers for cultivar identification of pepper in a convenient and correct manner.

Two Chinese cabbage (Brassica rapa L. ssp. pekinensis) lines resistant to Turnip mosaic virus (TuMV) CHN5 were identified and found to have broad-spectrum resistance against three other TuMV strains (CHN2, 3, and 4). Genetic analysis indicated that this TuMV resistance is recessive, and a candidate gene approach was used to identify the resistance gene, which we named trs (TuMV resistance discovered at Seoul National University). Based on previous research in Arabidopsis showing that mutations in eIF(iso)4E determine TuMV resistance, the eIF(iso)4E gene was selected as a candidate for the trs gene in Brassica rapa. Three copies of eIF(iso)4E, Braiso4Ea, Braiso4Eb, and Braiso4Ec, were amplified, and polymorphisms between resistant and susceptible lines were analyzed. Sequence polymorphisms were found in Braiso4Ea and Braiso4Eb; in contrast, no sequence differences were found in Braiso4Ec between resistant and susceptible lines. A CAPS marker developed to test the linkage between Braiso4Eb and TuMV resistance displayed no linkage. A SCAR marker, trsSCAR, developed using allele-specific deletions and SNPs in Braiso4Ea, did co-segregate perfectly with trs in three F2 populations. However, the presence or absence of the Braiso4Ea sequence deletion was not consistent between resistant lines and susceptible lines, indicating that Braiso4Ea is not the actual resistance gene. Results from mapping analysis indicated that the trs is located at chromosome A04, between scaffold 000104 and scaffold 040552. This location demonstrated that trs may be another recessive resistance gene tightly linked to retr02 or another allele. The molecular markers developed in this study will be useful for breeding durable resistance.

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.

Tobamovirus is one of most destructive viruses in Capsicum. Accordingly, the L locus, a resistance gene against tobamoviruses, has been used for pepper breeding programs. Previously, the L 3 gene, one of the L alleles, was isolated through map-based cloning, and a L 4 gene candidate was isolated by homology-based PCR methods. Here, the L4segF&R marker was developed based on the leucine-rich repeat (LRR) region of the L 4 candidate, and co-segregation analysis was performed using two L 4 -segregating F2 populations derived from the commercial cultivars Special and Myoung-sung. The L4segF&R marker was located within 0.3 cM of the L 4 gene but did not completely co-segregate with the L 4 gene, indicating that the candidate is not actually L 4 . To confirm the mapping result, L4segF&R genotypes of L 4 -containing breeding lines from three different seed companies were analyzed, resulting in the identification of several recombinants in the breeding lines. Based on these results, we postulate several genetic models that show different introgression histories and genetic structures for the L 4 -containing segment in different breeding lines. All of the models demonstrate that resistance conferred by the L 4 segment could not be explained by the L 4 gene candidate alone. Although the presence of the L 4 gene candidate could not fully explain the L 4 resistance, we were able to develop allele-specific markers for the L locus using the candidate sequence. To develop allele-specific markers for the L locus, HRM analysis was performed using primer pairs based on the LRR sequence of the L 4 gene candidate. When commercial breeding lines homozygous for L 0 , L 1 , L 2 , L 3 or L 4 were analyzed, L4RP-3F/L4RP-3R correctly detected the L allele in 90 out of 91 lines. We believe that the L allele-specific marker developed in the study provides a solution for pepper breeders developing improved resistance lines against tobamoviruses.

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.