Genome Editing of eIF4E1 in Chinese cabbage Confers Resistance to Turnip Mosaic Virus

Muhammad Irfan Siddique,Ye-Rin Lee,Hye-Eun Lee,Eun Su Lee,Koeun Han,Do-Sun Kim 한국원예학회 2021 한국원예학회 학술발표요지 Vol.2021 No.10

Many of the recessive virus-resistance genes in plants encode eukaryotic translation initiation factors (eIFs), including eIF4E, eIF4G, and related proteins. Particularly, eIF4E and its isoform eIF(iso)4E are crucial for viral infection and act as recessive resistance genes against various potyviruses in a broad range of plants. In the present study, we used Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/ Cas9)-mediated directed mutagenesis to test whether novel sequence-specific mutations at eIF4E1 in Brassica rapa (Chinese cabbage) cv. ‘Seoul’ could confer enhanced resistance to potyviruses. This approach produced mutations in the transgene-free E1 generation. Sequence analysis of eIF4E1 from E0 transgenic plants expressing Cas9 and eIF4E-sgRNA transcripts identified chimeric mutations (insertions/deletions) ranging from 1 to 8 bp. Genotype analysis of the eIF4E1-edited lines in E0 and E1 transgenic Chinese cabbage plants showed that the mutations were transmitted to subsequent generation. When mutant lines were tested for resistance to potyviruses Turnip mosaic virus (TuMV), they exhibited resistance against virus. Further, ELISA confirmation showed no accumulation of viral particles upon infection with (TuMV). The further analysis of mutation heritability in subsequent generations (E2) is going on. These results indicate that site-specific mutation of tomato eIF4E1 successfully conferred enhanced resistance to TuMV. Thus, this study demonstrates the feasibility of the use of CRISPR/Cas9 approach to accelerate breeding for trait improvement in Chinese cabbage plants.

Resistance to Phytophthora capsici, Restorer-of-fertility Genotype for Cytoplasmic Male Sterility and Chemical Quality Components of Breeding Lines Developed for Improvement of Subicho, a Land Race of Pepper in Yeongyang

Muhammad Irfan Siddique,Khin Pa Pa Wai,Hwang-Sung Mo,Hee-Ju Yoo,Kil-Su Jang,Su-Gyeong Jeon,Ji-Eun Hwang,Byung-Soo Kim 한국원예학회 2017 원예과학기술지 Vol.35 No.6

Over the last 25 years, a breeding line of pepper (Capsicum spp.) PVR Subi has been bred by the introduction of the Phytophthora capsici resistance found in pepper line PI201234 and the field resistance to viruses found in pepper accession KC00406 into a small-fruited, earlymaturing, and good-tasting land race, Subicho, in Yeongyang. In 2009, PVR Subi was crossed with a new version of Subi (New Subi) and an accession, KC01522, with mild and good dry fruit quality. This was done to meet growers’ demands for larger-fruited, open-pollinated local cultivars adapted to conditions in the Subi area of Yeongyang. Twenty-nine F5 and 37 F6 lines that developed from the two crosses were evaluated for resistance to P. capsici by pathogen inoculation at the seedling stage. Selected lines were grown to record horticultural characters in F5 and to analyze chemical quality components such as capsaicinoids, sugar and color in F6. Most of the breeding lines carried pathogen resistance comparable with the commercial resistant hybrid control cultivar, ‘Baerota’, and many lines were found to contain higher capsaicinoid and sugar levels, as well as ASTA color value, than the commercial control. All the breeding lines were identified as restorer-of-fertility lines with N(S)RfRf genotype, producing stable male fertile F1 plants in crosses with a cytoplasmic male sterile line (CMS). These breeding lines are promising as basic materials for selection of open-pollinated cultivars adapted to grow in Yeongyang, and as pollen parents for a hybrid seed production system using cytoplasmic male sterility.

Fertility-restoring Genotype Interacting with Male Sterile Cytoplasm of Youngyang Local Breeds of Pepper Resistant to Phytophthora capsici

Muhammad Irfan Siddique,Hwang-sung Mo,Khin Pa Pa Wai,Byoung-Soo Kim 한국원예학회 2014 한국원예학회 학술발표요지 Vol.2014 No.5

Assessment of EMS Mutagenic Effects in Capsicum annuum Population for the Identification of Non-pungent Mutants

Muhammad Irfan Siddique,Koeun Han,Doyeon Hwang,Hee-Jin Jeong,Byoung-Cheorl Kang 한국원예학회 2015 한국원예학회 학술발표요지 Vol.2015 No.10

Development of EMS mutant populations in Capsicum annuum and identification of non-pungent mutants

Muhammad Irfan Siddique,Koeun Han,Doyeon Hwang,Hee-Jin Jeong,Arti Rai,Byoung-Cheorl Kang 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Plant breeding requires genetic diversity of useful traits for crop improvement. EMS-induced mutation is practiced to generate mutations at loci regulating economically important traits and/or to knock out the genes to elucidate their functions. The present study was aimed to induce mutations in a Korean local land race Capsicum annuum ‘Yuwol-cho’. This accession is pungent and also has advantage to mature early. A total of about 1,500 M2 families were screened and three non-pungent mutants were identified and crossed with wild type ‘Yuwol-cho’. After phenotyping of F2 population for pungency, MutMap approach will be used to identify the genes controlling the pungency in mutants. In addition to this, another C. annuum accession “Micro-Pep” was used to develop a mutant population. Micro-Pep is a small, pungent pepper generally used as ornamental purpose. Having compact growth habit, and small size, it has advantage to handle and utilize easily in mutation study and molecular research. On the basis of preliminary experiment 1.3% of mutagen was used for treatment of pepper seeds and 30% less germination percentage was observed in EMS treated seeds in comparison to control seeds. A total of 4,674 M1 plants are grown under greenhouse condition and M2 population will be studied for characterization of phenotypic variation including fruit color and pungency. Newly constructed mutant populations will be valuable assets for identification of functional genes and molecular breeding of pepper.

Characterization and interaction analysis of two QTLs, QTL5-1 and QTL5-2, controlling Phytophthora capsici resistance in Capsicum annuum using near-isogenic lines

Hyeon-Seok Jeong,Muhammad Irfan Siddique,Jeong-Tak An,Ki‑Taek Kim,Gyung Ja Choi,Darush Struss,Byoung-Cheorl Kang 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Phytophthora capsici an Oomycete pathogen is a major challenge to the pepper (Capsicum spp.) production around the world. Control measures are proved ineffective, so breeding resistant cultivars are the most promising strategy against the pathogen. Resistance against P. capsici is governed by quantitative trait loci (QTL). According to previous studies on QTL detection, the QTL on pepper chromosome 5 is a major contributor to resistance. In this study, to exploit the involvement of this QTL and identify its contributing genes, the F2 population derived from a cross between ECW30R and CM334 was inoculated with a medium virulence P. capsici strain JHAI1-7 zoospores at the 6-8 leaf stage. Composite interval mapping revealed two major QTLs; QTL5-1 from 7 days post inoculation (dpi) and QTL5-2 from 16 dpi on chromosome 5. To characterize and detect interactions of the two QTLs, near isogenic lines (NIL) were constructed by crossing Tean and recombinant inbred line (RIL) derived from a cross between YCM334 and Tean. RILs were screened with P. capsici strain MY-1 and resistant lines were selected. Among the resistance RILs most closely related to Tean were selected using AFLP and SSR genotyping data. These RILs were named as YT39-2 and YT143-2. To develop more advanced NILs, two rounds of marker-assisted backcrossing were done using a high-throughput SNP genotyping system (EPI Fluidigm, USA). Among the NILs derived from YT39-2, YT39-2-64 contains only QTL5-1 whereas YT39-2-61 and YT39-2-69 were identified to have both QTLs. On the other hand, YT143-2-55-7 with the highest Tean genetic background contains QTL5-1 only. In the next step, the 3 different NILs having QTL5-1, QTL5-2 individually and both QTLs will be identified. Furthermore, phenotyping and fine mapping will be done for the analysis of individual and interaction effects of QTLs.

Ginsenoside Rg3-enriched red ginseng extract inhibits platelet activation and in vivo thrombus formation

정다혜,Muhammad Irfan Siddique,김성대,김석,오준환,박재규,김현경,이만휘 고려인삼학회 2017 Journal of Ginseng Research Vol.41 No.4

Background: Korean Red Ginseng has been used for several decades to treat many diseases, enhancing both immunity and physical strength. Previous studies have documented the therapeutic effects of ginseng, including its anticancer, antiaging, and anti-inflammatory activities. These activities are mediated by ginsenosides present in the ginseng plant. Ginsenoside Rg3, an effective compound from red ginseng, has been shown to have antiplatelet activity in addition to its anticancer and anti-inflammatory activities. Platelets are important for both primary hemostasis and the repair of the vessels after injury; however, they also play a crucial role in the development of acute coronary diseases. We prepared ginsenoside Rg3-enriched red ginseng extract (Rg3-RGE) to examine its role in platelet physiology. Methods: To examine the effect of Rg3-RGE on platelet activation in vitro, platelet aggregation, granule secretion, intracellular calcium ([Ca2þ]i) mobilization, flow cytometry, and immunoblot analysis were carried out using rat platelets. To examine the effect of Rg3-RGE on platelet activation in vivo, a collagen plus epinephrine-induced acute pulmonary thromboembolism mouse model was used. Results: We found that Rg3-RGE significantly inhibited collagen-induced platelet aggregation and [Ca2þ]i mobilization in a dose-dependent manner in addition to reducing ATP release from collagen-stimulated platelets. Furthermore, using immunoblot analysis, we found that Rg3-RGE markedly suppressed mitogen-activated protein kinase phosphorylation (i.e., extracellular stimuli-responsive kinase, Jun Nterminal kinase, p38) as well as the PI3K (phosphatidylinositol 3 kinase)/Akt pathway. Moreover, Rg3- RGE effectively reduced collagen plus epinephrine-induced mortality in mice. Conclusion: These data suggest that ginsenoside Rg3-RGE could be potentially be used as an antiplatelet therapeutic agent against platelet-mediated cardiovascular disorders.

Pathotypes of Bacterial Spot Pathogen Infecting Capsicum Peppers in Korea

Wai, Khin Pa Pa,Siddique, Muhammad Irfan,Mo, Hwang-Sung,Yoo, Hee Ju,Byeon, Si-Eun,Jegal, Yoonhyuk,Mekuriaw, Alebel A.,Kim, Byung-Soo The Korean Society of Plant Pathology 2015 Plant Pathology Journal Vol.31 No.4

Sixty-seven isolates of bacterial spot pathogen (Xanthomonas spp.) collected from six provinces of Korea were tested for the identification of their pathotypes and determination of their distribution throughout Korea in an effort to genetically manage the disease. Near isogenic lines of Early Calwonder (Capsicum annuum) pepper plants carrying $Bs_1$, $Bs_2$ and $Bs_3$, and PI235047 (C. pubescens) were used as differential hosts. Race P1 was found to be predominant, followed by race P7, and races P3 and P8 were also observed. This is the first report of races P7 and P8 in Korea. The races P7 and P8 were differentiated from the former races P1 and P3, respectively, on the basis of their ability to elicit hypersensitive reactions to PI235047.

Pathotypes of Bacterial Spot Pathogen Infecting Capsicum Peppers in Korea

Khin Pa Pa Wai,Muhammad Irfan Siddique,모황성,유희주,변시은,제갈윤혁,Alebel A. Mekuriaw,김병수 한국식물병리학회 2015 Plant Pathology Journal Vol.31 No.4

Sixty-seven isolates of bacterial spot pathogen (Xanthomonas spp.) collected from six provinces of Korea were tested for the identification of their pathotypes and determination of their distribution throughout Korea in an effort to genetically manage the disease. Near isogenic lines of Early Calwonder (Capsicum annuum) pepper plants carrying Bs1, Bs2 and Bs3, and PI235047 (C. pubescens) were used as differential hosts. Race P1 was found to be predominant, followed by race P7, and races P3 and P8 were also observed. This is the first report of races P7 and P8 in Korea. The races P7 and P8 were differentiated from the former races P1 and P3, respectively, on the basis of their ability to elicit hypersensitive reactions to PI235047.

RESEARCH ARTICLES : Gene-Based Markers for the Tomato Yellow Leaf Curl Virus Resistance Gene Ty-3

( Panpan Dong ),( Koeun Han ),( Muhammad Irfan Siddique ),( Jin Kyung Kwon ),( Meiai Zhao ),( Fu Wang ),( Byoung Cheorl Kang ) 한국육종학회 2016 Plant Breeding and Biotechnology Vol.4 No.1

The viral disease induced by Tomato yellow leaf curl virus (TYLCV) reduces tomato (Solanum lycopersicum) yield significantly in tropical and subtropical regions. A number of loci, including Ty-1 to Ty-5, conferring resistance to TYLCV have been described and introgressed into modern tomato cultivars. The availability of molecular markers linked to these genes would expedite the introgression of TYLCV resistance into commercial cultivars. In the present study, we developed gene-based markers linked to the Ty-3 gene using a segregating population derived from a cross between the TYLCV-resistant line S. lycopersicum ‘A45’ and the susceptible line S. lycopersicum ‘A39’. Agrobacterium-mediated screening was used to test TYLCV resistance of plants in the segregating population, and the resistance was evaluated by a visual scoring method and polymerase chain reaction analysis. By comparing sequences of the Ty-3 genes of the resistant and susceptible lines, two high-resolution melting (HRM) markers (Ty3-HRM1 and Ty3-HRM2) and one sequence characterized amplified region (SCAR) marker (Ty3-SCAR1) were developed. The HRM markers were based on single nucleotide polymorphisms at the 13th exon and the 15th intron, whereas the SCAR marker was based on a 246-bp deletion in the 16th intron. These gene-based markers will be useful tools for marker-assisted selection in breeding programs to improve TYLCV resistance of tomato.