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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.

Summary The protein–protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.

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.

Tomato spotted wilt virus (TSWV) is an important viral disease affecting pepper production worldwide. A single dominant resistance gene, Tsw, originating from Capsicum chinense has been identified and utilized during the last several decades. However, there have been reports that Tsw resistance can be overcome by new field isolates of TSWV. This has necessitated the identification of a new source of resistance. Here, a set of pepper germplasm collections comprising 487 accessions from six Capsicum species and 30 commercial F1 hybrids was evaluated for resistance to TSWVPap. A new resistance source, C. chinense ‘AC09-207’, was identified and characterized. Genetic analysis showed that the resistance in C. chinense ‘AC09-207’ was conferred by a single dominant gene. The resistance responses of ‘AC09-207’ were compared with other known resistance sources. The timing and number of necrotic response were similar to C. chinense ‘PI152225’, whereas the premature abscission of inoculated cotyledons and leaves were significantly different from other resistance sources, ‘PI152225’ and ‘PI159236’. To compare genome locations between the new resistance gene and Tsw, an allelism test was conducted. No recombinants were found in all F1, F2 and reciprocal backcross populations derived from the new resistance source and three known resistance sources (‘PI152225’, ‘PI159236’, and ‘PI159234’) demonstrating that the new resistance gene may be a unique allele at the Tsw locus or be controlled by a different gene tightly linked to Tsw.

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.

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L 4 gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L 4 - and L 3 -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L 3 , was located on the opposite side of 087H3T7, about 0.7 cM away from L 4 . This supports the idea that L 3 and L 4 may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L 4 to elite germplasm against most aggressive tobamoviruses pathotype P1,2,3 is discussed.

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.

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum . Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated. Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F2 population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 103/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F1 commercial cultivars. Among the markers, Phyto5NBS1 showed about 90 % accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.

The protein–protein interaction between VPg (viral protein genome‐linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad‐spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge‐based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap‐binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap‐binding pockets, and mutated. Yeast two‐hybrid assay and co‐immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E‐knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild‐type were over‐expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T (1) and T (2) transformants demonstrated that the over‐expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge‐based approaches for the engineering of broad‐spectrum resistance in Chinese cabbage.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Cucumber mosaic virus (CMV) is one of the most destructive viruses in the Solanaceae family. Simple inheritance of CMV resistance in peppers has not previously been documented; all previous studies have reported that resistance to this virus is mediated by several partially dominant and recessive genes. In this study, we showed that the Capsicum annuum cultivar 'Bukang' contains a single dominant resistance gene against CMV(Korean) and CMV(FNY) strains. We named this resistance gene Cmr1 (Cucumber mosaic resistance 1). Analysis of the cellular localization of CMV using a CMV green fluorescent protein construct showed that in 'Bukang,' systemic movement of the virus from the epidermal cell layer to mesophyll cells is inhibited. Genetic mapping and FISH analysis revealed that the Cmr1 gene is located at the centromeric region of LG2, a position syntenic to the ToMV resistance locus (Tm-1) in tomatoes. Three SNP markers were developed by comparative genetic mapping: one intron-based marker using a pepper homolog of Tm-1, and two SNP markers using tomato and pepper BAC sequences mapped near Cmr1. We expect that the SNP markers developed in this study will be useful for developing CMV-resistant cultivars and for fine mapping the Cmr1 gene.

Diaphragmatic rupture (DR) is most commonly seen after a blunt trauma. It rarely occurs spontaneously. Many cases of spontaneous DR followed by strenuous sports activity have been reported in the medical literature. However, there has been no previous report on a case of spontaneous DR after a static sport activity. We report the case of a 29-year old woman who presented to the emergency department (ED) with pain in the epigastric area that started 1 day before visiting the ED during deep breathing in Pilates. The radiography and computed tomography of her chest demonstrated a left diaphragmatic rupture complicated with the perforation of viscera. She immediately underwent left thoracotomy. In addition, primary repair of the diaphragm and stomach was performed. On the basis of our findings, we conclude that spontaneous DR may be caused by a static sport activity, such as Pilates, causing a serious life threatening condition.

Background: Blood pressure is clinically used for monitoring shock patients and as a therapeutic indicator for them. Non-invasive blood pressure measurement has weak points such as the use of a cuff and it is a discontinuous measurement. A method of measuring the blood pressure by using the PWTT (pulse wave transit time) has been studied to make up for those weak points. If blood pressure monitoring can be done by using the difference of the PWTT between different points in the body, then this method wil l be a quite useful to monitor the BP of seriously ill patients. This study aimed to verify whether or not the PWTT has a significant correlation with the blood pressure of shock patients who received vasopressor infusion and whether this method is clinically applicable. Methods: The study subjects were 20 shock patients who were hospitalized in intensive care units and they had received vasopressor, and we measured the PWTT and we analyzed its correlation with the SBP (systolic blood pressure) and DBP (diastolic blood pressure), as measured by non-invasive monitoring. We then determined the effects of the PWTT on the SBP and DBP. Results: From the results of correlation analysis between the PWTT and the SBP and DBP, the SBP displayed a statistically significant negative correlation with the PWTT of 18 patients, while no significant correlation between the PWTT and DBP was observed. At the same time, from the results of the regression analysis of the blood pressures and the PWTT of each patient, it was found that the PWTT had a negative effect on the SBP of all the patients, except two. Conclusions: The PWTT has a negative correlation with the SBP of the patien ts who received vasopressor infusion.