Characterization of the Chloroplast Genome Facilitated the Transformation of Parachlorella kessleri-I, A Potential Marine Alga for Biofuel Production

Introduction: The microalga Parachlorella kessleri-I produces high biomass and lipid content that could be suitable for producing economically viable biofuel at a commercial scale. Sequencing the complete chloroplast genome is crucial for the construction of a species-specific chloroplast transformation vector. Methods: In this study, the complete chloroplast genome sequence (cpDNA) of P. kessleri-I was assembled; annotated and genetic transformation of the chloroplast was optimized. For the chloroplast transformation, we have tested two antibiotic resistance makers, aminoglycoside adenine transferase (aadA) gene and Sh-ble gene conferring resistance to spectinomycin and zeocin, respectively. Transgene integration and homoplasty determination were confirmed using PCR, Southern blot and Droplet Digital PCR. Results: The chloroplast genome (109,642 bp) exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region. The genome encodes 116 genes, with 80 protein-coding genes, 32 tRNAs and 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species-specific vector that facilitated the transformation of P. kessleri-I chloroplast. The transgenic algal colonies were retrieved on a TAP medium containing 400 mg. L-1 spectinomycin, but no transgenic was recovered on the zeocin-supplemented medium. PCR and Southern blot analysis ascertained the transgene integration into the chloroplast genome, via homologous recombination. The chloroplast genome copy number in wildtype and transgenic P. kessleri-I was determined using Droplet Digital PCR. Conclusion: The optimization of stable chloroplast transformation in marine alga P. kessleri-I should open a gateway for directly engineering the strain for carbon concentration mechanisms to fix more CO2, improving the photosynthetic efficiency and reducing the overall biofuels production cost.

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Chloroplast genome analysis and genetic transformation of Parachlorella kessleri-I a potential marine alga for biofuel production

10.21203/rs.2.12052/v1 ◽

2019 ◽

Author(s):

Prachi Nawkarkar ◽

Sagrika Chugh ◽

Amita Tanwar ◽

Surbhi Sharma ◽

Charli Kaushal ◽

...

Keyword(s):

Homologous Recombination ◽

Chloroplast Genome ◽

Marine Alga ◽

Chloroplast Transformation ◽

Single Copy ◽

Biofuel Production ◽

Essential Information ◽

Complete Chloroplast Genome ◽

Parachlorella Kessleri ◽

Species Specific

Abstract Background: Parachlorella kessleri-I produces higher biomass and lipid content suitable for commercial production of biofuels. Sequencing complete chloroplast genome will be instrumental in the constructing species specific chloroplast transformation vectors and generating chloroplast transgenic microalga with the desired traits and greater productivity, essential for commercial sustainability of microalgae based biofuel production. Results: Complete chloroplast genome sequence (cpDNA) of P. kessleri-I was annotated. The 109,642 bp chloroplast genome exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC) and a small single copy (SSC) region. The genome encodes 117 unique genes, with 70 predicted protein coding genes, 35 tRNAs, 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species specific chloroplast transformation vector that facilitated chloroplast transformation of P. kessleri-I. To optimize chloroplast transformation, two antibiotic resistance makers aminoglycoside adenine transferase (aadA) conferring resistance to spectinomycin and Sh-ble gene from bacteria that conferred resistance to zeocin were tested. Using a aadA gene, transgenic colonies were retrieved on TAP medium containing 400 mg/l spectinomycin. However, no transgenic colonies were recovered in the zeocin supplemented medium. The spectinomycin resistant algal cell lines were analyzed by PCR. Southern blotting confirmed the stable transgenes integration into the chloroplast genome of P. kessleri-I via homologous recombination. Conclusion: The complete chloroplast genome analysis may provide valuable resources for population and evolutionary studies of Parachlorella species and identifying the related species. The chloroplast genome of P. kessleri-I was assembled as a quadripartite structure of 109,642 bp with defined IR regions. Its complete sequencing has provided essential information like codons, UTRs and flanking sequences to generate the species specific chloroplast transformation vector and obtaining the successful site-specific chloroplast transformation in P. kessleri-I via homologous recombination. The optimized chloroplast transformation in marine alga P. kessleri-I should open a new possibilities like controlling the chain lengths of fatty acids for biofuel application, manipulating the RubisCo enzyme for improving photosynthetic process, introducing carbon concentration mechanism (CCM) for fixing higher CO2 that may be instrumental in producing economically viable biofuel molecules.

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Comparative genome analysis revealed gene inversions, boundary expansion and contraction, and gene loss in Stemona sessilifolia (Miq.) Miq. chloroplast genome

10.1101/2021.02.15.431246 ◽

2021 ◽

Author(s):

Jingting Liu ◽

Mei Jiang ◽

Haimei Chen ◽

Yu Liu ◽

Chang Liu ◽

...

Keyword(s):

Chloroplast Genome ◽

Gene Loss ◽

Herbal Medicines ◽

Single Copy ◽

Asparagus Officinalis ◽

Accurate Identification ◽

Protein Coding ◽

Variable Regions ◽

Complete Chloroplast Genome ◽

Next Generation Sequencing Technology

AbstractStemona sessilifolia (Miq.) Miq., commonly known as Baibu, is one of the most popular herbal medicines in Asia. In Chinese Pharmacopoeia, Baibu has multiple authentic sources, and there are many homonym herbs sold as Baibu in the herbal medicine market. The existence of the counterfeits of Baibu brings challenges to its identification. To assist the accurate identification of Baibu, we sequenced and analyzed the complete chloroplast genome of Stemona sessilifolia using next-generation sequencing technology. The genome was 154,039 bp in length, possessing a typical quadripartite structure consisting of a pair of inverted repeats (IRs: 27,094 bp) separating by a large single copy (LSC: 81,950 bp) and a small single copy (SSC: 17,901 bp). A total of 112 unique genes were identified, including 80 protein-coding, 28 transfer RNA, and four ribosomal RNA genes. Besides, 45 tandem, 27 forward, 23 palindromic, and 72 simple sequence repeats were detected in the genome by repeat analysis. Compared with its counterfeits (Asparagus officinalis and Carludovica palmate), we found that IR expansion and SSC contraction events of Stemona sessilifolia resulted in two copies of the rpl22 gene in the IR regions and partial duplication of the ndhF gene in the SSC region. Secondly, an approximately 3-kb-long inversion was identified in the LSC region, leading to the petA and cemA gene presented in the complementary strand of the chloroplast DNA molecule. Comparative analysis revealed some highly variable regions, including trnF-GAA_ndhJ, atpB_rbcL, rps15_ycf1, trnG-UCC_trnR-UCU, ndhF_rpl32. Finally, gene loss events were investigated in the context of phylogenetic relationships. In summary, the complete plastome of Stemona sessilifolia will provide valuable information for the molecular identification of Baibu and assist in elucidating the evolution of Stemona sessilifolia.

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Complete Chloroplast Genome of Argania spinosa: Structural Organization and Phylogenetic Relationships in Sapotaceae

Plants ◽

10.3390/plants9101354 ◽

2020 ◽

Vol 9 (10) ◽

pp. 1354

Author(s):

Slimane Khayi ◽

Fatima Gaboun ◽

Stacy Pirro ◽

Tatiana Tatusova ◽

Abdelhamid El Mousadik ◽

...

Keyword(s):

Chloroplast Genome ◽

Single Copy ◽

Rrna Genes ◽

Trna Genes ◽

Protein Coding ◽

Important Species ◽

Complete Chloroplast Genome ◽

Argania Spinosa ◽

Protein Coding Genes ◽

Cp Genome

Argania spinosa (Sapotaceae), an important endemic Moroccan oil tree, is a primary source of argan oil, which has numerous dietary and medicinal proprieties. The plant species occupies the mid-western part of Morocco and provides great environmental and socioeconomic benefits. The complete chloroplast (cp) genome of A. spinosa was sequenced, assembled, and analyzed in comparison with those of two Sapotaceae members. The A. spinosa cp genome is 158,848 bp long, with an average GC content of 36.8%. The cp genome exhibits a typical quadripartite and circular structure consisting of a pair of inverted regions (IR) of 25,945 bp in length separating small single-copy (SSC) and large single-copy (LSC) regions of 18,591 and 88,367 bp, respectively. The annotation of A. spinosa cp genome predicted 130 genes, including 85 protein-coding genes (CDS), 8 ribosomal RNA (rRNA) genes, and 37 transfer RNA (tRNA) genes. A total of 44 long repeats and 88 simple sequence repeats (SSR) divided into mononucleotides (76), dinucleotides (7), trinucleotides (3), tetranucleotides (1), and hexanucleotides (1) were identified in the A. spinosa cp genome. Phylogenetic analyses using the maximum likelihood (ML) method were performed based on 69 protein-coding genes from 11 species of Ericales. The results confirmed the close position of A. spinosa to the Sideroxylon genus, supporting the revisiting of its taxonomic status. The complete chloroplast genome sequence will be valuable for further studies on the conservation and breeding of this medicinally and culinary important species and also contribute to clarifying the phylogenetic position of the species within Sapotaceae.

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The complete chloroplast genome of Saxifraga sinomontana (Saxifragaceae) and comparative analysis with other Saxifragaceae species

Revista Brasileira de Botânica ◽

10.1007/s40415-019-00561-y ◽

2019 ◽

Vol 42 (4) ◽

pp. 601-611 ◽

Cited By ~ 1

Author(s):

Yan Li ◽

Liukun Jia ◽

Zhihua Wang ◽

Rui Xing ◽

Xiaofeng Chi ◽

...

Keyword(s):

Comparative Analysis ◽

Chloroplast Genome ◽

Phylogenetic Relationships ◽

De Novo ◽

Single Copy ◽

Bootstrap Support ◽

Protein Coding ◽

Complete Chloroplast Genome ◽

Protein Coding Genes ◽

Chloroplast Genomes

Abstract Saxifraga sinomontana J.-T. Pan & Gornall belongs to Saxifraga sect. Ciliatae subsect. Hirculoideae, a lineage containing ca. 110 species whose phylogenetic relationships are largely unresolved due to recent rapid radiations. Analyses of complete chloroplast genomes have the potential to significantly improve the resolution of phylogenetic relationships in this young plant lineage. The complete chloroplast genome of S. sinomontana was de novo sequenced, assembled and then compared with that of other six Saxifragaceae species. The S. sinomontana chloroplast genome is 147,240 bp in length with a typical quadripartite structure, including a large single-copy region of 79,310 bp and a small single-copy region of 16,874 bp separated by a pair of inverted repeats (IRs) of 25,528 bp each. The chloroplast genome contains 113 unique genes, including 79 protein-coding genes, four rRNAs and 30 tRNAs, with 18 duplicates in the IRs. The gene content and organization are similar to other Saxifragaceae chloroplast genomes. Sixty-one simple sequence repeats were identified in the S. sinomontana chloroplast genome, mostly represented by mononucleotide repeats of polyadenine or polythymine. Comparative analysis revealed 12 highly divergent regions in the intergenic spacers, as well as coding genes of matK, ndhK, accD, cemA, rpoA, rps19, ndhF, ccsA, ndhD and ycf1. Phylogenetic reconstruction of seven Saxifragaceae species based on 66 protein-coding genes received high bootstrap support values for nearly all identified nodes, suggesting a promising opportunity to resolve infrasectional relationships of the most species-rich section Ciliatae of Saxifraga.

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Complete Chloroplast Genome of Paphiopedilum delenatii and Phylogenetic Relationships among Orchidaceae

Plants ◽

10.3390/plants9010061 ◽

2020 ◽

Vol 9 (1) ◽

pp. 61 ◽

Cited By ~ 5

Author(s):

Huyen-Trang Vu ◽

Ngan Tran ◽

Thanh-Diem Nguyen ◽

Quoc-Luan Vu ◽

My-Huyen Bui ◽

...

Keyword(s):

Chloroplast Genome ◽

Inverted Repeat ◽

Gc Content ◽

Single Copy ◽

Rrna Genes ◽

Trna Genes ◽

Complete Chloroplast Genome ◽

Critically Endangered Species ◽

Plastid Genomes ◽

Chloroplast Genomes

Paphiopedilum delenatii is a native orchid of Vietnam with highly attractive floral traits. Unfortunately, it is now listed as a critically endangered species with a few hundred individuals remaining in nature. In this study, we performed next-generation sequencing of P. delenatii and assembled its complete chloroplast genome. The whole chloroplast genome of P. delenatii was 160,955 bp in size, 35.6% of which was GC content, and exhibited typical quadripartite structure of plastid genomes with four distinct regions, including the large and small single-copy regions and a pair of inverted repeat regions. There were, in total, 130 genes annotated in the genome: 77 coding genes, 39 tRNA genes, 8 rRNA genes, and 6 pseudogenes. The loss of ndh genes and variation in inverted repeat (IR) boundaries as well as data of simple sequence repeats (SSRs) and divergent hotspots provided useful information for identification applications and phylogenetic studies of Paphiopedilum species. Whole chloroplast genomes could be used as an effective super barcode for species identification or for developing other identification markers, which subsequently serves the conservation of Paphiopedilum species.

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The complete chloroplast genome of Limonium tetragonum (Plumbaginaceae) isolated in Korea

Korean Journal of Plant Taxonomy ◽

10.11110/kjpt.2021.51.3.337 ◽

2021 ◽

Vol 51 (3) ◽

pp. 337-344

Author(s):

Yongsung KIM ◽

Hong XI ◽

Jongsun PARK

Keyword(s):

Phylogenetic Analysis ◽

Chloroplast Genome ◽

Phylogenetic Trees ◽

Sister Group ◽

Single Copy ◽

Nucleotide Polymorphisms ◽

Large Single Copy ◽

Single Nucleotide ◽

Complete Chloroplast Genome ◽

Cp Genome

The chloroplast genome of Limonium tetragonum (Thunb.) Bullock, a halophytic species, was sequenced to understand genetic differences based on its geographical distribution. The cp genome of L. tetragonum was 154,689 bp long (GC ratio is 37.0%) and has four subregions: 84,572 bp of large single-copy (35.3%) and 12,813 bp of small singlecopy (31.5%) regions were separated by 28,562 bp of inverted repeat (40.9%) regions. It contained 128 genes (83 proteincoding genes, eight rRNAs, and 37 tRNAs). Thirty-five single-nucleotide polymorphisms and 33 INDEL regions (88 bp in length) were identified. Maximum-likelihood and Bayesian inference phylogenetic trees showed that L. tetragonum formed a sister group with L. aureum, which is incongruent with certain previous studies, including a phylogenetic analysis.

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The Complete Chloroplast Genome Sequences of Fritillaria ussuriensis Maxim. and Fritillaria cirrhosa D. Don, and Comparative Analysis with Other Fritillaria Species

10.20944/preprints201703.0139.v1 ◽

2017 ◽

Cited By ~ 1

Author(s):

Inkyu Park ◽

Wook-Jin Kim ◽

Sang-Min Yeo ◽

Goya Choi ◽

Young-Min Kang ◽

...

Keyword(s):

Chloroplast Genome ◽

General Structure ◽

Single Copy ◽

Rrna Genes ◽

Trna Genes ◽

Genome Sequences ◽

Complete Chloroplast Genome ◽

Phylogenic Analysis ◽

Fritillaria Species ◽

Fritillaria Cirrhosa

The genus Fritillaria belongs to the widely distributed family Liliaceae. The bulbs of Fritillaria ussuriensis and Fritillaria cirrhosa are valuable herbaceous medicinal ingredients. However, they are still used indiscriminately in herbal medicine. Identification and molecular phylogenic analysis of Fritillaria species is therefore required. Here, we report the complete chloroplast (cp) genome sequences of F. ussuriensis and F. cirrhosa. The two Fritillaria cp genomes were 151,524 and 151,083 bp in length, respectively, including a pair of inverted repeat regions (52,678 and 52,156 bp) separated by a large single copy region (81,732 and 81,390 bp) and small single copy region (17,114 and 17,537 bp). A total of 111 genes in F. ussuriensis and 112 in F. cirrhosa comprised 77 protein-coding genes in F. ussuriensis and 78 in F. cirrhosa, 30 tRNA genes, and four rRNA genes. The gene order, content, and orientation of the two Fritillaria cp genomes exhibited the general structure of flowering plants, and were similar to those of other Fritillaria species. Comparison of the six Fritillaria species’ cp genomes indicated seven highly divergent regions in intergenic spacers and in the matK, rpoC1, rpoC2, ycf1, ycf2, ndhD, and ndhF coding regions. We established the position of the six species through phylogenic analysis. The complete chloroplast genome sequences of two Fritillaria species will be useful genomics resources for identification of Fritillaria species and for studying the phylogenetic relationship among Fritillaria species within the Liliaceae family.

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The complete chloroplast genome of the Jerusalem artichoke (Helianthus tuberosus L.) and an adaptive evolutionary analysis of the ycf2 gene

10.7287/peerj.preprints.27796 ◽

2019 ◽

Author(s):

Qiwen Zhong ◽

Shipeng Yang ◽

Xuemei Sun ◽

Lihui Wang ◽

Yi Li

Keyword(s):

Chloroplast Genome ◽

Adaptive Evolution ◽

Jerusalem Artichoke ◽

Single Copy ◽

Reverse Direction ◽

Evolutionary Analysis ◽

Helianthus Tuberosus ◽

Genome Sequences ◽

Complete Chloroplast Genome ◽

Coding Regions

Jerusalem artichoke (Helianthus tuberosus L.) is widely cultivated in Northwest China which has become an emerging economic crop with rapid development. Because of its elevated inulin content and high resistance, it is widely used in functional food, inulin processing, feed, and ecological management. In this study, Illumina sequencing technology was utilized to assemble and annotate the complete chloroplast genome sequences of Jerusalem artichoke. The total length was 151,431 bp, including four conserved regions: A pair of reverse repeat regions (IRa 24,568 bp and IRb 24,603 bp), a large single-copy region (LSC, 83,981 bp), and a small single-copy region (SSC, 18,279 bp). The genome had a total of 115 genes, with 19 present in the reverse direction in the IR region. 36 simple sequence repeats (SSRs) were identified in the coding and non-coding regions, most of which were biased towards A/T bases. 32 SSRs were distributed in the non-coding regions. Comparative analysis of the chloroplast genome sequence of Jerusalem artichoke and other species of the composite family revealed the chloroplast genome sequences of plants of the composite family to be highly conserved. Differences were observed in 24 gene loci in the coding region, with the degree of differentiation of the ycf2 gene being the most obvious. Phylogenetic analysis showed Helianthus petiolaris subsp. fallax had the closest relationship with Jerusalem artichoke, both members of the Helianthus genus. Selective locus detection of the ycf2 gene in eight species of the composite family was performed to explore adaptive evolution traits of the ycf2 gene in Jerusalem artichoke. The results show that there are significant and extremely significant positive selection sites at the 1239N and 1518R loci, respectively, indicating that the ycf2 gene has been subject to adaptive evolution and has the potential to be used as a phylogenetic reconstruction locus in the composite family. Insights from our assessment of the complete chloroplast genome sequences of Jerusalem artichoke will aid in the in-depth study of the evolutionary relationship of the composite family, and provide significant sequencing information for the genetic improvement of Jerusalem artichoke.

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The complete chloroplast genome of Diarthron linifolium (Thymelaeaceae), a species found on a limestone outcrop in eastern Asia

Korean Journal of Plant Taxonomy ◽

10.11110/kjpt.2021.51.4.345 ◽

2021 ◽

Vol 51 (4) ◽

pp. 345-352

Author(s):

Sang-Tae KIM ◽

Sang-Hun OH ◽

Jongsun PARK

Keyword(s):

Phylogenetic Analysis ◽

Chloroplast Genome ◽

Single Copy ◽

Evolutionary Status ◽

Complete Chloroplast Genome ◽

Chloroplast Genomes ◽

Potential Marker ◽

Anticancer Treatment ◽

Annual Herb ◽

Unique Species

Diarthron linifolium Turcz. is an annual herb usually found in sandy soil or limestone areas. Plants in the genus Diarthron are known to have toxic chemicals that may, however, be potentially useful as an anticancer treatment. Diarthron linifolium is a unique species among the species of the genus distributed in Korea. Here, we determine the genetic variation of D. linifolium collected in Korea with a full chloroplast genome and investigate its evolutionary status by means of a phylogenetic analysis. The chloroplast genome of Korean D. linifolium has a total length of 172,644 bp with four subregions; 86,158 bp of large single copy and 2,858 bp of small single copy (SSC) regions are separated by 41,814 bp of inverted repeat (IR) regions. We found that the SSC region of D. linifolium is considerably short but that IRs are relatively long in comparison with other chloroplast genomes. Various simple sequence repeats were identified, and our nucleotide diversity analysis suggested potential marker regions near ndhF. The phylogenetic analysis indicated that D. linifolium from Korea is a sister to the group of Daphne species.

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Complete chloroplast genome sequences of Dioscorea: Characterization, genomic resources, and phylogenetic analyses

PeerJ ◽

10.7717/peerj.6032 ◽

2018 ◽

Vol 6 ◽

pp. e6032 ◽

Cited By ~ 12

Author(s):

Zhenyu Zhao ◽

Xin Wang ◽

Yi Yu ◽

Subo Yuan ◽

Dan Jiang ◽

...

Keyword(s):

Chloroplast Genome ◽

Phylogenetic Analyses ◽

Single Copy ◽

Genome Sequences ◽

Complete Chloroplast Genome ◽

Genomic Resources ◽

Chloroplast Genomes ◽

The Family ◽

Small Single Copy

Dioscorea L., the largest genus of the family Dioscoreaceae with over 600 species, is not only an important food but also a medicinal plant. The identification and classification of Dioscorea L. is a rather difficult task. In this study, we sequenced five Dioscorea chloroplast genomes, and analyzed with four other chloroplast genomes of Dioscorea species from GenBank. The Dioscorea chloroplast genomes displayed the typical quadripartite structure of angiosperms, which consisted of a pair of inverted repeats separated by a large single-copy region, and a small single-copy region. The location and distribution of repeat sequences and microsatellites were determined, and the rapidly evolving chloroplast genome regions (trnK-trnQ, trnS-trnG, trnC-petN, trnE-trnT, petG-trnW-trnP, ndhF, trnL-rpl32, and ycf1) were detected. Phylogenetic relationships of Dioscorea inferred from chloroplast genomes obtained high support even in shortest internodes. Thus, chloroplast genome sequences provide potential molecular markers and genomic resources for phylogeny and species identification.

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