Cite this paper:
LIANG Sijie, ZHANG Zhongyi, LIU Hang, GUO Li, SUN Shiyang, YANG Guanpin. Digging out molecular markers associated with low salinity tolerance of Nannochloropsis oceanica through bulked mutant analysis[J]. Journal of Oceanology and Limnology, 2020, 38(6): 1867-1879

Digging out molecular markers associated with low salinity tolerance of Nannochloropsis oceanica through bulked mutant analysis

LIANG Sijie1,2, ZHANG Zhongyi1,2, LIU Hang1,2, GUO Li1,2, SUN Shiyang3, YANG Guanpin1,2,4
1 College of Marine Life Sciences, Ocean University of China(OUC), Qingdao 266003, China;
2 Key Laboratory of Marine Genetics and Breeding of Ministry of Education, OUC, Qingdao 266003, China;
3 College of Fisheries, OUC, Qingdao 266003, China;
4 Institute of Evolution and Marine Biodiversity, OUC, Qingdao 266003, China
Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance. It grows fast, contains rich oils, reproduces asexually, holds a small and haploidy genome, and is easy to be modified genetically. However, the genetic study of N. oceanica is scarce. Very less genetic bases of its traits have been deciphered, and no gene has been isolated from it with the function verified simultaneously via either genetic or reverse genetic approaches or both (de novo cloned). Changing medium salinity may aid to control harmful organisms met during large scale cultivation. As a stress, it may also facilitate the accumulation of desirable chemicals including fatty acids. In order to decipher the genetic basis of the low salinity tolerance of N. oceanica, we mutated N. oceanica with Zeocin. In total, five mutant bulks were constructed at equal number of cells, 100 mutants each, which were tolerant to a discontinuous serial of salinities from that of 100% of f/2 to that of a mixture of 4% of f/2 and 94% of BG11. The bulks were genotyped through whole genome re-sequencing and analyzed with bulked mutant analysis (BMA) newly modified from bulked segregant analysis (BSA). In total, 47 SNPs and 112 InDels were found to associate with the low salinity tolerance, and around them a set of low salinity tolerance associating genes were identified. A set of annotatable genes commonly found between control and different salinities indicated that the genes functioning in gene expression, energy metabolism and cellular structure may be involved in the low salinity tolerance. These associating molecular markers and genes around them were not enough for outlining the physiological mechanism underlining the tolerance; however they should aid to improve N. oceanica genetically.
Key words:    Nannochloropsis oceanica|bulked mutant analysis (BMA)|low salinity tolerance   
Received: 2019-07-22   Revised: 2019-09-09
PDF (536 KB) Free
Print this page
Add to favorites
Email this article to others
Articles by LIANG Sijie
Articles by ZHANG Zhongyi
Articles by LIU Hang
Articles by GUO Li
Articles by SUN Shiyang
Articles by YANG Guanpin
Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, Innan H, Cano L, Kamoun S, Terauchi R. 2012. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 30(2):174-178.
Baek K, Kim D H, Jeong J, Sim S J, Melis A, Kim J S, Jin F, Bae S. 2016. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Scientific Reports, 6:30620.
Belton J M, Mccord R P, Gibcus J H, Naumova N, Zhan Y, Dekker J. 2012. Hi-C:a comprehensive technique to capture the conformation of genomes. Methods, 58(3):268-276.
Chaturvedi R, Fujita Y. 2010. Isolation of enhanced eicosapentaenoic acid producing mutants of Nannochloropsis oculata ST-6 using ethyl methane sulfonate induced mutagenesis techniques and their characterization at mRNA transcript level. Phycological Research, 54(3):208-219.
Dekker J, Rippe K, Dekker M, Kleckner N. 2002. Capturing chromosome conformation. Science, 295(5558):1 306-1 311.
Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, Yaegashi H, Sharma S, Sharma S, Kanzaki H, Matsumura H, Saitoh H, Mitsuoka C, Utsushi H, Uemura A, Kanzaki E, Kosugi S, Yoshida K, Cano L, Kamoun S, Terauchi R. 2013. MutMap+:Genetic mapping and mutant identification without crossing in rice. PLoS One, 8(7):e68529.
Ferenczi A, Pyott D E, Xipnitoua A, Molnar A. 2017. Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and singlestranded DNA. Proceedings of the National Academy of Sciences of the United States of America, 114(51):13 567-13 572.
Fu Y, Springer N M, Gerhardt D J, Ying K, Yeh C T, Wu W, Swanson-Wagner R, D'Ascenzo M, Millard T, Freeberg L, Aoyama N, Kitzman J, Burgess D, Richmond T, Albert T J, Barbazuk W B, Jeddeloh J A, Schnable P S. 2010. Repeat subtraction-mediated sequence capture from a complex genome. The Plant Journal, 62(5):898-909.
Gibcus J H, Dekker J. 2013. The hierarchy of the 3D genome. Molecular Cell, 49(5):773-782.
Guo L, Liang S J, Zhang Z Y, Liu H, Wang S W, Pan K H, Xu J, Ren X, Pei S R, Yang G P. 2019. Genome assembly of Nannochloropsis oceanica provides evidence of host nucleus overthrow by the symbiont nucleus during speciation. Communications Biology, 2:249.
Guo L, Wang Y M, Liang S J, Lin G M, Chen S L, Yang G P. 2016. Tissue-overlapping response of half-smooth tongue sole (Cynoglossus semilaevis) to thermostressing based on transcriptome profiles. Gene, 586(1):97-104.
Guo L, Yang G P. 2015. The mechanism of the acclimation of Nannochloropsis oceanica to freshwater deduced from its transcriptome profiles. Journal of Ocean University of China, 14(5):922-930.
Head B, Griparic L, Amiri M, Gandre-Babbe S, Van Der Bliek A M. 2009. Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells. The Journal of Cell Biology, 187(7):959-966.
Iskandarov U, Khozin-Goldberg I, Cohen Z. 2011. Selection of a DGLA-producing mutant of the microalga Parietochloris incisa:I. Identification of mutation site and expression of VLC-PUFA biosynthesis genes. Applied Microbiology and Biotechnology, 90(1):249-256.
Klein R J, Zeiss C, Chew E Y, Tsai J Y, Sackler R S, Haynes C, Henning A K, SanGiovanni J P, Mane S M, Mayne S T, Bracken M B, Ferris F L, Ott J, Barnstable C, Hoh J. 2005. Complement factor H polymorphism in age-related macular degeneration. Science, 308(5720):385-389.
Leung D Y C, Wu X, Leung M K H. 2010. A review on biodiesel production using catalyzed transesterification. Applied Energy, 87(4):1 083-1 095.
Li H, Durbin R. 2009a. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25(14):1 754-1 760.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. 2009b. The sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25(16):1 653-1 654.
Liang S J, Guo L, Lin G M, Zhang Z Y, Ding H Y, Wang Y M. Yang G P. 2017. Improvement of Nannochloropsis oceanica growth performance through chemical mutation and characterization of fast growth physiology by transcriptome profiling. Chinese Journal of Oceanology and Limnology, 35(4):792-802.
Liang S J, Zhang Z Y, Liu H, Guo L, Sun S Y, Yang G P. 2019. Identifying the growth associating genes of Nannochloropsis oceanica by bulked mutant analysis(BMA) and RNA sequencing (BMR-seq). Journal of Applied Phycology, 1-14,
Lin G M, Wang Y M, Guo L, Ding H Y, Hu Y M, Liang S J, Zhang Z Y, Yang G P. 2017. Verification of mutagen function of Zeocin in Nannochloropsis oceanica through transcriptome analysis. Journal of Ocean University of China, 16(3):501-508.
Lin G M, Zhang Z Y, Guo L, Ding H Y, Yang G P. 2018. Structural variation analysis of mutated Nannochloropsis oceanica caused by Zeocin through genome resequencing. Journal of Ocean University of China, 17(5):1 225-1 230.
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo M A. 2010. The genome analysis toolkit:A map reduce framework for analyzing next-generation DNA sequencing data. Genome Research, 20(9):1 297-1 303.
Michelmore R W, Paran I, Kesseli P V. 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis:a rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences of United States of America, 88(21):9 828-9 832.
Pan K H, Qin J J, Li S, Dai W K, Zhu B H, Jin Y C, Yu W G, Yang G P, Li D F. 2011. Nuclear monoploidy and asexual propagation of Nannochloropsis oceanica(Eustigmatophyceae) as revealed by its genome sequence. Journal of Phycology, 47(6):1 425-1 432.
Poliner E, Pulman J A, Zienkiewicz K, Childs K, Benning C, Farré E M. 2018b. A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced longchain polyunsaturated fatty acid production. Plant Biotechnology Journal, 16(1):298-309.
Poliner E, Takeuchi T, Du Z Y, Benning C, Farré EM. 2018a. Nontransgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779. ACS Synthetic Biology, 7(4):962-968.
Sandhu K S, Li G L, Sung W K, Ruan Y J. 2011. Chromatin interaction networks and higher order architectures of eukaryotic genomes. Journal of Cellular Biochemistry, 112(9):2 218-2 221.
Steemers F J, Chang W H, Lee G, Barker D L, Shen R, Gunderson K L. 2006. Whole-genome genotyping with the single-base extension assay. Nature Methods, 3(1):31-33.
Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano L M, Kamoun S, Terauchi R. 2013a. QTL-seq:Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The Plant Journal, 74(1):174-183.
Takagi H, Uemura A, Yaegashi H, Tamiru M, Abe A, Mitsuoka C, Utsushi H, Natsume S, Kanzaki H, Matsumura H, Saitoh H, Yoshida K, Cano L M, Kamoun S, Terauchi R. 2013b. MutMap-Gap:whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii. New Phytologist, 200(1):276-283.
Todorova T, Miteva D, Chankova S. 2005. DNA damaging effect of zeocin and methyl methanesulfonate in Saccharomyces Cerevisiae measured by CFGE. Comptes Rendus De L'Academie Bulgare Des Sciences, 68(1):71-78.
Visscher P M, Wray N R, Zhang Q, Sklar P, McCarthy M I, Brown M A, Yang J. 2017.10 years of GWAS discovery:Biology, function, and translation. The American Journal of Human Genetics, 101(1):5-22.
Wang C S, Tang S C, Zhan Q L, Hou Q Q, Zhao Y, Zhao Q, Feng Q, Zhou C C, Lyu D F, Cui L L, Li Y, Miao J S, Zhu C R, Lu Y Q, Wang Y C, Wang Z Q, Zhu J J, Shangguan Y Y, Gong J Y, Yang S H, Wang W Q, Zhang J F, Xie H A, Huang X H, Han B. 2019. Dissecting a heterotic gene through Graded Pool-seq mapping informs a riceimprovement strategy. Nature Communications, 10:2 982.
Wang L K, Feng Z X, Wang X, Wang X W, Zhang X G. 2010. DEGseq:an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics, 26(1):136-138.
Wang Q T, Lu Y D, Xin Y, Wei L, Huang S, Xu J. 2016. Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9. The Plant Journal, 88(6):1 071-1 081.
Wei L, Shen C, El Hajjami M, You W X, Wang Q T, Zhang P, Jia Y T, Hu H H, Hu Q, Poetsch A, Xu J. 2019. Knockdown of carbonate anhydrase elevates Nannochloropsis productivity at high CO2 level. Metabolic Engineering, 54:96-108.
Wei L, Wang Q T, Xin Y, Lu Y D, Xu J. 2017b. Enhancing photosynthetic biomass productivity of industrial oleaginous microalgae by overexpression of RuBisCO activase. Algal Research, 27:366-375.
Wei L, Xin Y, Wang Q T, Yang J, Hu H H, Xu J. 2017a. RNAibased targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica. The Plant Journal, 89(6):1 236-1 250.
Wijffels R H, Barbosa M J. 2010. An outlook on microalgal biofuels. Science, 329(5993):796-799.
Xin Y, Lu Y D, Lee Y Y, Wei L, Jia J, Wang Q T, Wang D M, Bai F L, Hu H H, Hu Q, Liu J, Li Y T, Xu J. 2017. Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases. Molecular Plant, 10(12):1 523-1 539.
Xin Y, Shen C, She Y T, Chen H, Wang C, Wei L, Yoon K, Han D X, Hu Q, Xu J. 2019. Biosynthesis of triacylglycerol molecules with a tailored PUFA profile in industrial microalgae. Molecular Plant, 12(4):474-488.
Yang J L, Jiang H Y, Yeh C T, Yu J M, Jeddeloh J A, Nettleton D, Schnable P S. 2015. Extreme-phenotype genome-wide association study (XP-GWAS):A method for identifying trait-associated variants by sequencing pools of individuals selected from a diversity panel. The Plant Journal, 84(3):587-596.
Copyright © Haiyang Xuebao