Selection of a CTAB protocol for high-quality DNA extraction in Oryza sativa L. validated for application in genotyping process based on Illumina sequencing

Pérez-Pazos Jazmin Vanessa,Romero-Ferrer Jorge Luis,Berdugo-Cely Jhon A. 한국작물학회 2023 Journal of crop science and biotechnology Vol.26 No.4

Rice genotypes with possible tolerance to biotic and abiotic stresses are interested in genomic studies for their use in breeding programs. Colombian Plant Germplasm Bank (CPGB) has preserved local rice genotypes without genomic analysis yet. Considering the need to develop genomic studies with these genotypes and minimize costs in the genotyping process, in this research, fve CTAB DNA extraction protocols (P1-P5) were evaluated and compared with a commercial kit. Four local rice accessions from the CPGB were used, and DNA concentration (fuorescence and absorbance), quality (absorbance, integrity, and enzymatic digestion), and cost analysis were determined. A selection index (SI) was established to choose the best DNA protocol. Finally, the selected protocol was validated in 14 local rice genotypes through Illumina sequencing. The six evaluated DNA extraction protocols presented signifcant diferences in quantifcation and quality variables. From the SI established, the protocol selected was the P4 (SI=147.7); this protocol allows obtaining DNA with high concentrations and quality levels at a 50% lower cost than the commercial kit. The sequences generated from the DNA obtained with the P4 protocol presented 7 million reads with high levels of quality (Q30) and length (>50 pb) per evaluated accession; On average, 98.9% of these sequences mapped to the rice genome and identifed up to 15,436 SNP markers without missing data. These results confrm that the DNA from rice local genotypes obtained with the P4 CTAB protocol has the requirements for routine use in genomic studies based on genotyping processes that require Illumina sequencing.

Transcriptomic analysis of developing embryos of apricot (Prunus armeniaca L.)

Yujia Bai,Weicheng Hu,Min Wang,Jing He,Yongxia Tao,Wei Huang,Zuoshan Feng 한국원예학회 2016 Horticulture, Environment, and Biotechnology Vol.57 No.2

Apricot (Prunus armeniaca L.) is an important, nutritionally valuable agricultural crop. A scarcity of transcriptomic and genomic data hinders progress toward understanding the molecular events of apricot embryogenesis, which is the primary determinant of fruit yield and kernel quality. In this study, three cDNA libraries were sequenced using an Illumina/Solexa platform. We obtained 35,928,496, 40,656,940, and 35,970,214 clean reads from endosperm formation (LT-A), embryo development (LT-B), and mature embryo (LT-C) cDNA libraries, respectively. De novo assembly of cDNA sequence data generated 35,614 unigenes with an average length of 973 bp. A total of 29,971 unigenes were matched in Nr, Nt, Swiss-Prot, KEGG, GO, and COG databases. The top matching species in the Nr database was P. persica (88.3% of matched Nr database accessions). A total of 8,327 differentially expressed genes (DEGs) were detected between LT-A and LT-B, of which 3,663 were upregulated and 4,664 were downregulated. Between LT-B and LT-C, 12,673 DEGs were detected, while 13,892 DEGs were uncovered between LT-A and LT-C. This work is the first reported application of next-generation sequencing techniques for transcriptome analysis of apricot embryo development. Besides providing valuable information on apricot embryo development, the comprehensive set of transcript sequences generated in this study may serve as additional molecular resources for the development of apricot functional genomics.

RNA Sequencing, De novo assembly, functional annotation and SSR analysis of the endangered diving beetle Cybister chinensis (= Cybister japonicus) using the Illumina platform

황희주,Bharat Bhusan PATNAIK,강세원,박소영,정종민,상민규,박지은,민혜린,성지연,조용훈,노미영,이종대,정기윤,박홍석,정헌천,이용석 한국곤충학회 2018 Entomological Research Vol.48 No.1

Cybister chinensis Motschulsky, 1854 (synonym Cybister japonicus Sharp, 1873) is a beetle found in ponds and irrigation canals near rice fields regulating the aquatic faunal community through predation. However, due to loss of natural habitats, use of pesticides, and invasion of alien species the beetle is threatened. With lack of understanding at the trophic ecology and genomics level, the conservation study is hindered to a larger extent. In the present study, Illumina HiSeq 4000 platform has been used to unravel the whole‐larval transcriptome of the beetle. A total of 20,129 non‐redundant unigenes were assembled from 67,260,666 clean read sequences. About 18,743 unigenes found a homologous match in any one of the databases like PANM, UniGene, Swiss‐Prot, Clusters of Orthologous Groups (COG), Gene Ontology (GO), KEGG, and InterProScan. While the zinc finger domains topped the unigene hits, about 660 enzymes (2695 sequences) participating in metabolism, environmental information processing, genetic information processing and organismal system pathways were recorded. Furthermore, the HSP70 class, Toll‐like receptors 4, insulin‐receptor substrate, and AMP activated protein kinase showed conspicuous presence in the larval transcriptome. Out of a total of 12,491 unigene sequences examined, 1968 SSRs were detected. Majority of them were dinucleotide repeats with six iterations followed by trinucleotide and tetranucleotide repeats with five and four iterations, respectively. This is the first report of cDNA resources from C. japonicus till date. The data would be crucial for the assessment of the beetle in the wild and making an inventory for utilisation in future genomics and ecological studies.

De novo transcriptome analysis of stressed blood clam (Anadara broughtonii) and identification of genes associated with hemoglobin

Yihang Wang,Shanshan Zhou,Tianyu Liu,Muyan Chen,Xiumei Zhang 한국유전학회 2020 Genes & Genomics Vol.42 No.2

Background Blood clam (Anadara broughtonii) is a commercially important marine bivalve characterised by the red blood. Recently, the clams have been subjected to severe resource recession. Multiple environmental stressors are indispensable for the recession. Objective We aimed to investigate the transcriptome information of blood clam under environmental stressors. Methods Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Several public databases were introduced for gene annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathways analyses. The Open Reading Frame of annotated hemoglobin (Hb) was predicted and validated by DNAMAN 6.0 and NCBI BLASTx, respectively. Results A total of 242,919 transcripts were generated, 116,264 unigenes were subsequently assembled with an average length of 747 base pairs, and 33,776 unigenes were successfully annotated. Gene Ontology (GO) categories indicated that the terms of cellular processes, metabolic processes, cell, cell part, binding, and catalytic activity were dominant. KEGG pathway analyses suggested ribosome, oxytocin, focal adhesion, Ras, and PI3K-Akt were the largest signaling pathway groups, and many presented pathways (Ras, Rap1, and MAPK, etc.) were related to apoptosis, immune and stress response. In addition, a total of 19,306 potential simple sequence repeats (SSRs) were detected in 15,852 sequences. Six hemoglobin-related genes with complete conserved domain sequences were identified and 3 of them were predicted as HbI, HbIIα, and HbIIβ. Conclusion This study provides transcriptome responses to multiple environmental stressors in blood clams and would provide interesting hints for further studies.

Molecular Methods for Studying the Human Microbiota

Choi, Yoon Hee,Chung, Jin,Na, Hee Sam 대한미생물학회 2013 Journal of Bacteriology and Virology Vol.43 No.1

인체에 존재하는 다양한 미생물에 대한 연구는 최근에 발달한 next-generation sequencing 기법의 도입과 함께 눈부시게 발전하고 있다. 가장 대표적인 분석 기반으로는 Roche-454와 Illumina가 있다. Roche-454는 400 bps 내외의 비교적 긴 염기 서열을 단 시간 이내 읽어 들일 수 있는 장점이 있고, Illumina는 100~150 bps의 짧은 염기 서열을 분석하는 반면 방대한 양의 정보를 읽어 들일 수 있는 장점을 갖는다. 이러한 분석의 특징과 장 단점을 비교하고 더불어 최근에 소개되고 있는 PacBio, Oxford Nanopore 등에 대한 소개도 곁들이고자 한다. Vast array of microbes colonize to each anatomical environment of human body. Culture based methods are important in investigating the microbial structure, but they are extremely biased in their evaluation of microbial diversity by selecting particular population of microbiota. Recent advance in molecular technology has allowed sophisticated analysis of complex human microbiota by culture-independent methods. Here, we will discuss features of tools for human microbiota studies including Roche-454 and Illumina platform. We will also briefly discuss features of some strategies that are commonly applied to these platforms including 16S rRNA targeting and shotgun sequencing. New platforms such as PacBio and Oxford Nanopore are also introduced.

Molecular Methods for Studying the Human Microbiota

최윤희,정진,나희삼 대한미생물학회 2013 Journal of Bacteriology and Virology Vol.43 No.1

Vast array of microbes colonize to each anatomical environment of human body. Culture based methods are importantin investigating the microbial structure, but they are extremely biased in their evaluation of microbial diversity byselecting particular population of microbiota. Recent advance in molecular technology has allowed sophisticated analysisof complex human microbiota by culture-independent methods. Here, we will discuss features of tools for humanmicrobiota studies including Roche-454 and Illumina platform. We will also briefly discuss features of some strategiesthat are commonly applied to these platforms including 16S rRNA targeting and shotgun sequencing. New platformssuch as PacBio and Oxford Nanopore are also introduced.

Development and Validation of Microsatellite Markers for the Tiny Dragonfly, Nannophya pygmaea (Odonata: Libellulidae), Which Is Endangered in South Korea

Min Jee Kim,Ah Rha Wang,Sung Soo Kim,Junghwa An,Iksoo Kim 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.04

In this study, we developed 12 microsatellite markers specific to N. pygmaea using Illumina paired-end sequencing. Forty individuals of N. pygmaea collected from three currently known localities in South Korea were genotyped to validate these markers and to preliminarily assess population genetic characteristics. No locus showed significant deviation from the Hardy–Weinberg equilibrium (HWE). Our preliminary data indicate an absence of inbreeding in all populations and an absence of obvious genetic difference. The microsatellite markers developed in this study will be useful for studying the population genetics of N. pygmaea collected from other regions, including additional sites in South Korea.

Diversity and community structure of ectomycorrhizal mycorrhizal fungi in roots and rhizosphere soil of Abies koreana and Taxus cuspidata in Mt. Halla

Lee Ji-Eun,Eom Ahn-Heum 한국균학회 2022 Mycobiology Vol.50 No.6

In this study, the roots and rhizosphere soil of Abies koreana and Taxus cuspidata were col- lected from sites at two different altitudes on Mt. Halla. Ectomycorrhizal fungi (EMF) were identified by Illumina MiSeq sequencing. The proportion of EMF from the roots was 89% in A. koreana and 69% in T. cuspidata. Among EMF in rhizosphere soils, the genus Russula was the most abundant in roots of A. koreana (p < 0.05). The altitude did not affect the biodiver- sity of EMF communities but influenced fungal community composition. However, the host plants had the most significant effect on EMF communities. The result of the EMF commu- nity analysis showed that even if the EMF were isolated from the same altitudes, the EMF communities differed according to the host plant. The community similarity index of EMF in the roots of A. koreana was higher than that of T. cuspidata (p < 0.05). The results show that both altitude and host plants influenced the structure of EMF communities. Conifers inhabit- ing harsh sub-alpine environments rely strongly on symbiotic relationships with EMF. A. kore- ana is an endangered species with a higher host specificity of EMF and climate change vulnerability than T. cuspidata. This study provides insights into the EMF communities, which are symbionts of A. koreana, and our critical findings may be used to restore A. koreana.

Transcriptome Characterization for Non-Model Endangered Lycaenids, <i>Protantigius superans</i> and <i>Spindasis takanosis</i> , Using Illumina HiSeq 2500 Sequencing

Patnaik, Bharat Bhusan,Hwang, Hee-Ju,Kang, Se Won,Park, So Young,Wang, Tae Hun,Park, Eun Bi,Chung, Jong Min,Song, Dae Kwon,Kim, Changmu,Kim, Soonok,Lee, Jae Bong,Jeong, Heon Cheon,Park, Hong Seog,Han, MDPI 2015 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.16 No.12

<P>The Lycaenidae butterflies, <I>Protantigius superans</I> and <I>Spindasis takanosis</I>, are endangered insects in Korea known for their symbiotic association with ants. However, necessary genomic and transcriptomics data are lacking in these species, limiting conservation efforts. In this study, the <I>P. superans</I> and <I>S. takanosis</I> transcriptomes were deciphered using Illumina HiSeq 2500 sequencing. The <I>P. superans</I> and <I>S. takanosis</I> transcriptome data included a total of 254,340,693 and 245,110,582 clean reads assembled into 159,074 and 170,449 contigs and 107,950 and 121,140 unigenes, respectively. BLASTX hits (<I>E</I>-value of 1.0 × 10<SUP>−5</SUP>) against the known protein databases annotated a total of 46,754 and 51,908 transcripts for <I>P. superans</I> and <I>S. takanosis</I>. Approximately 41.25% and 38.68% of the unigenes for <I>P. superans</I> and <I>S. takanosis</I> found homologous sequences in Protostome DB (PANM-DB). BLAST2GO analysis confirmed 18,611 unigenes representing Gene Ontology (GO) terms and a total of 5259 unigenes assigned to 116 pathways for <I>P. superans</I>. For <I>S. takanosis</I>, a total of 6697 unigenes were assigned to 119 pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Additionally, 382,164 and 390,516 Simple Sequence Repeats (SSRs) were compiled from the unigenes of <I>P. superans</I> and <I>S. takanosis</I>, respectively. This is the first report to record new genes and their utilization for conservation of lycaenid species population and as a reference information for closely related species.</P>

일천궁의 연작재배에 따른 토양 이화학성 및 토양세균군집 연구

김기윤,한경민,김현준,전권석,김충우,정충렬 한국환경농학회 2020 한국환경농학회지 Vol.39 No.1

BACKGROUND: The aim of this study was to investigate the soil chemical properties and soil bacterial community of the cropping system for Cnidium officinale Makino. METHODS AND RESULTS: The bacterial community was analyzed for the relative abundance and principal coordinated analysis (PCoA analysis) by using by Illumina Miseq sequencing. The correlation analysis between soil chemical properties and soil bacterial community were analyzed by Spearman’s rank correlation and DISTLM analysis. Soil bacterial community (phylum and class) showed two distinct clusters consisting of cluster 1 (first cropping) and cluster 2 (continuous cropping) from 2 different cultivation methods of Cnidium officinale Makino. PCoA and DISTLM analyses showed that soil pH and Ca significantly affected soil bacterial community in cultivation area of Cnidium officinale Makino. In addition, Spearman’s rank correlation showed significant correlation between relative abundance (Acidobacteria and Actinobacteria) and soil factors (soil pH and Ca). CONCLUSION: The results of this study were considered to be important for determining the correlation between soil properties and soil bacterial community of the cropping method for Cnidium officinale Makino. Furthermore, the results will be helpful to investigate the cause of continuous cropping injury of the Cnidium officinale Makino by examining the changes of soil properties and soil bacterial communities. .