Extensive Structural Variations Between Mitochondrial Genomes of CMS and Normal Peppers (Capsicum Annuum L.) Revealed by Complete Nucleotide Sequencing

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp. Results: We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (psi atp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and psi atp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes. Conclusions: Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.Golden Seed ProjectMinistry of Agriculture, Food and Rural Affairs (MAFRA)Ministry of Oceans and Fisheries (MOF)Rural Development Administration (RDA)Korea Forest Service (KFS)Vegetable Breeding Research Center through the R&D Convergence Center Support Program, Ministry of Agriculture, Food and Rural Affairs (MAFRA) Republic of Korea 710001-07Molecular Bioscience

Fine mapping and identification of candidate genes for the sy-2 locus in a temperature-sensitive chili pepper (Capsicum chinense)

Key message The sy-2 temperature-sensitive gene from Capsicum chinense was fine mapped to a 138.8-kb region at the distal portion of pepper chromosome 1. Based on expression analyses, two putative F-box genes were identified as sy-2 candidate genes. Abstract Seychelles-2 (sy-2) is a temperature-sensitive natural mutant of Capsicum chinense, which exhibits an abnormal leaf phenotype when grown at temperatures below 24 °C. We previously showed that the sy-2 phenotype is controlled by a single recessive gene, sy-2, located on pepper chromosome 1. In this study, a high-resolution genetic and physical map for the sy-2 locus was constructed using two individual F2 mapping populations derived from a cross between C. chinense mutant sy-2 and wild-type No. 3341. The sy-2 gene was fine mapped to a 138.8-kb region between markers SNP 5-5 and SNP 3-8 at the distal portion of chromosome 1, based on comparative genomic analysis and genomic information from pepper. The sy-2 target region was predicted to contain 27 genes. Expression analysis of these predicted genes showed a differential expression pattern for ORF10 and ORF20 between mutant and wild-type plants; with both having significantly lower expression in sy-2 than in wild-type plants. In addition, the coding sequences of both ORF10 and ORF20 contained single nucleotide polymorphisms (SNPs) causing amino acid changes, which may have important functional consequences. ORF10 and ORF20 are predicted to encode F-box proteins, which are components of the SCF complex. Based on the differential expression pattern and the presence of nonsynonymous SNPs, we suggest that these two putative F-box genes are most likely responsible for the temperature-sensitive phenotypes in pepper. Further investigation of these genes may enable a better understanding of the molecular mechanisms of low temperature sensitivity in plants.OAIID:RECH_ACHV_DSTSH_NO:T201625254RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A076900CITE_RATE:3.9FILENAME:TAG(2016) Fine mapping and identification of candidate genes for sy2.pdfDEPT_NM:식물생산과학부EMAIL:[email protected]_YN:YFILEURL:https://srnd.snu.ac.kr/eXrepEIR/fws/file/c773c8d3-fc79-496f-af60-fa1b9b2e214e/linkCONFIRM:

Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein

Key message Using fine mapping techniques, the genomic region co-segregating with Restorer-of-fertility (Rf) in pepper was delimited to a region of 821 kb in length. A PPR gene in this region, CaPPR6, was identified as a strong candidate for Rf based on expression pattern and characteristics of encoding sequence. Abstract Cytoplasmic-genic male sterility (CGMS) has been used for the efficient production of hybrid seeds in peppers (Capsicum annuum L.). Although the mitochondrial candidate genes that might be responsible for cytoplasmic male sterility (CMS) have been identified, the nuclear Restorer-of-fertility (Rf) gene has not been isolated. To identify the genomic region co-segregating with Rf in pepper, we performed fine mapping using an Rf-segregating population consisting of 1068 F2 individuals, based on BSA-AFLP and a comparative mapping approach. Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence. Prediction of expressed genes in this region based on transcription analysis revealed four candidate genes. Among these, CaPPR6 encodes a pentatricopeptide repeat (PPR) protein with PPR motifs that are repeated 14 times. Characterization of the CaPPR6 protein sequence, based on alignment with other homologs, showed that CaPPR6 is a typical Rf-like (RFL) gene reported to have undergone diversifying selection during evolution. A marker developed from a sequence near CaPPR6 showed a higher prediction rate of the Rf phenotype than those of previously developed markers when applied to a panel of breeding lines of diverse origin. These results suggest that CaPPR6 is a strong candidate for the Rf gene in pepper.OAIID:RECH_ACHV_DSTSH_NO:T201625308RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A076900CITE_RATE:3.9FILENAME:TAG(2016) Fine mapping of Rf in pepper.pdfDEPT_NM:식물생산과학부EMAIL:[email protected]_YN:YFILEURL:https://srnd.snu.ac.kr/eXrepEIR/fws/file/8c5822f8-5600-4a60-a386-0529e2f49934/linkCONFIRM:

Frequency and Spectrum of Radiation-Induced Mutations Revealed by Whole-Genome Sequencing Analyses of Plants

Mutation breeding and functional genomics studies of mutant populations have made important contributions to plant research involving the application of radiation. The frequency and spectrum of induced mutations have long been regarded as the crucial determinants of the efficiency of the development and use of mutant populations. Systematic studies regarding the mutation frequency and spectrum, including genetic and genomic analyses, have recently resulted in considerable advances. These studies have consistently shown that the mutation frequency and spectrum are affected by diverse factors, including radiation type, linear energy transfer, and radiation dose, as well as the plant tissue type and condition. Moreover, the whole-genome sequencing of mutant individuals based on next-generation sequencing technologies has enabled the genome-wide quantification of mutation frequencies according to DNA mutation types as well as the elucidation of mutation mechanisms based on sequence characteristics. These studies will contribute to the development of a highly efficient and more controlled mutagenesis method relevant for the customized research of plants. We herein review the characteristics of radiation-induced mutations in plants, mainly focusing on recent whole-genome sequencing analyses as well as factors affecting the mutation frequency and spectrum

Genotype-by-Sequencing Analysis of Mutations and Recombination in Pepper Progeny of Gamma-Irradiated Gametophytes

The irradiation of dry seeds is the most widely-used irradiation method for improving seed-propagated crops; however, the irradiation of other tissues also has useful effects. The irradiation of plant reproductive organs, rather than seeds, for mutation breeding has advantages, such as producing non-chimeric progeny. However, the mutation frequency and spectrum produced using this method have not been analyzed on a genome-wide level. We performed a genotype-by-sequencing analysis to determine the frequencies of single-base substitutions and small (1–2 bp) insertions and deletions in hot pepper (Capsicum annuum L.) plants derived from crosses using gamma-irradiated female or male gametophytes. The progeny of irradiated gametophytes showed similar or higher DNA mutation frequencies, which were dependent on the irradiation dose and irradiated tissue, and less biased single base substitutions than progeny of irradiated seeds. These characteristics were expected to be beneficial for development of mutation population with a high frequency of small DNA mutations and performing reverse-genetics-based mutation screening. We also examined the possible use of this irradiation method in manipulating the meiotic recombination frequency; however, no statistically significant increase was detected. Our results provide useful information for further research and breeding using irradiated gametophytes

Frequency and Spectrum of Radiation-Induced Mutations Revealed by Whole-Genome Sequencing Analyses of Plants

Mutation breeding and functional genomics studies of mutant populations have made important contributions to plant research involving the application of radiation. The frequency and spectrum of induced mutations have long been regarded as the crucial determinants of the efficiency of the development and use of mutant populations. Systematic studies regarding the mutation frequency and spectrum, including genetic and genomic analyses, have recently resulted in considerable advances. These studies have consistently shown that the mutation frequency and spectrum are affected by diverse factors, including radiation type, linear energy transfer, and radiation dose, as well as the plant tissue type and condition. Moreover, the whole-genome sequencing of mutant individuals based on next-generation sequencing technologies has enabled the genome-wide quantification of mutation frequencies according to DNA mutation types as well as the elucidation of mutation mechanisms based on sequence characteristics. These studies will contribute to the development of a highly efficient and more controlled mutagenesis method relevant for the customized research of plants. We herein review the characteristics of radiation-induced mutations in plants, mainly focusing on recent whole-genome sequencing analyses as well as factors affecting the mutation frequency and spectrum

Expression of pepper cytoplasmic male sterility-associated open reading frame, orf507 in transgenic tobacco plants

Cytoplasmic male sterility (CMS) is a mitochondrial inherited trait that prevents a flower from producing normal pollen grains. CMS has widely been used to produce F1 hybrid seed in pepper. Though the orf507 gene is known to be associated with CMS in peppers, definitive and direct evidence that ORF507 induces male sterility is still lacking. In this study, a set of chimeric constructs were developed to confirm the hypothesis that ORF507 protein directly causes male sterility in tobacco. Tapetum-specific promoter TA29, mitochondrial transit sequence yeast coxIV pre-sequence, orf507 and green fluorescent protein (GFP) gene were cloned sequentially and designated as TCOGN (TA29-coxIV pre-sequnce-orf507-GFP-nos terminator). For developing control vectors, orf507 was cloned under TA29 promoter without coxIV pre-sequence (TOGN) or orf507 was cloned with or without coxIV pre-sequence under the constitutive CaMV35S promoter (SCOGN and SOGN). The four constructs (TCOGN, TOGN, SCOGN, and SOGN) were used to transform tobacco using leaf disk transformation mediated by agrobacterium. At flowering stage, transgenic plants will be scored for pollen production and viability. This study is expected to provide evidence that the expression of orf507 gene in the tapetum might be responsible for male sterility in pepper.OAIID:RECH_ACHV_DSTSH_NO:A201625326RECH_ACHV_FG:RR00200003ADJUST_YN:EMP_ID:A076900CITE_RATE:FILENAME:하예성_2016육종학회.pdfDEPT_NM:식물생산과학부EMAIL:[email protected]_YN:FILEURL:https://srnd.snu.ac.kr/eXrepEIR/fws/file/6dd98ec4-fdab-4ecd-a747-0c4e1d86ec6d/linkCONFIRM:

Lettuce Growth Pattern Analysis Using U-Net Pre-Trained with Arabidopsis

To overcome the challenges related to food security, digital farming has been proposed, wherein the status of a plant using various sensors could be determined in real time. The high-throughput phenotyping platform (HTPP) and analysis with deep learning (DL) are increasingly being used but require a lot of resources. For botanists who have no prior knowledge of DL, the image analysis method is relatively easy to use. Hence, we aimed to explore a pre-trained Arabidopsis DL model to extract the projected area (PA) for lettuce growth pattern analysis. The accuracies of the extract PA of the lettuce cultivar “Nul-chung” with a pre-trained model was measured using the Jaccard Index, and the median value was 0.88 and 0.87 in two environments. Moreover, the growth pattern of green lettuce showed reproducible results in the same environment (p < 0.05). The pre-trained model successfully extracted the time-series PA of lettuce under two lighting conditions (p < 0.05), showing the potential application of a pre-trained DL model of target species in the study of traits in non-target species under various environmental conditions. Botanists and farmers would benefit from fewer challenges when applying up-to-date DL in crop analysis when few resources are available for image analysis of a target crop

Crystallographic plane-orientation dependent atomic force microscopy-based local oxidation of silicon carbide

Abstract The effect of crystalline plane orientations of Silicon carbide (SiC) (a-, m-, and c-planes) on the local oxidation on 4H-SiC using atomic force microscopy (AFM) was investigated. It has been found that the AFM-based local oxidation (AFM-LO) rate on SiC is closely correlated to the atomic planar density values of different crystalline planes (a-plane, 7.45 cm-2; c-plane, 12.17 cm-2; and m-plane, 6.44 cm-2). Specifically, at room temperature and under about 40% humidity with a scan speed of 0.5 &#956;m/s, the height of oxides on a- and m-planes 4H-SiC is 6.5 and 13 nm, respectively, whereas the height of oxides on the c-plane increased up to 30 nm. In addition, the results of AFM-LO with thermally grown oxides on the different plane orientations in SiC are compared.</p

Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

Plant breeding explores genetic diversity in useful traits to develop new, high-yielding, and improved cultivars. Ethyl methane sulfonate (EMS) is a chemical widely used to induce mutations at loci that regulate economically essential traits. Additionally, it can knock out genes, facilitating efforts to elucidate gene functions through the analysis of mutant phenotypes. Here, we developed a mutant population using the small and pungent ornamental Capsicum annuum pepper &ldquo;Micro-Pep&rdquo;. This accession is particularly suitable for mutation studies and molecular research due to its compact growth habit and small size. We treated 9500 seeds with 1.3% EMS and harvested 3996 M2 lines. We then selected 1300 (32.5%) independent M2 families and evaluated their phenotypes over four years. The mutants displayed phenotypic variations in plant growth, habit, leaf color and shape, and flower and fruit morphology. An experiment to optimize Targeting Induced Local Lesions IN Genomes (TILLING) in pepper detected nine EMS-induced mutations in the eIF4E gene. The M2 families developed here exhibited broad phenotypic variation and should be valuable genetic resources for functional gene analysis in pepper molecular breeding programs using reverse genetics tools, including TILLING