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Nov.  2019
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An investigation of the possible methods and potential benefits of de novo cloning of Nannochloropsis oceanica genes

  • Corresponding author: Li Guo, qdguoli@139.com
  • Received Date: 2019-07-19
    Accepted Date: 2019-09-27
    Published online: 2019-12-02
  • Edited by Jiamei Li.
  • Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Here we attempt to generate InDel variations in the genome of Nannochloropsis oceanica, and then decipher the genetic basis of its economic and biological traits with bulked mutant analysis modified from bulked segregant analysis. In addition, we describe our efforts to construct site-tagged and gene-traceable mutant libraries to clone its genes through reverse genetic approaches. Currently, more than a half of N. oceanica protein-encoding genes are annotated against databanks. However, no functional gene has been de novo cloned from N. oceanica and no new function has been assigned to any of its annotatable genes. Here, we discuss the possible methods and potential benefits of de novo cloning of N. oceanica genes.
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An investigation of the possible methods and potential benefits of de novo cloning of Nannochloropsis oceanica genes

    Corresponding author: Li Guo, qdguoli@139.com
  • 1. College of Marine Life Sciences, Ocean University of China (OUC), Qingdao 266003, China
  • 2. Institutes of Evolution and Marine Biodiversity, OUC, Qingdao 266003, China
  • 3. Key Laboratory of Marine Genetics and Breeding of Ministry of Education, OUC, Qingdao 266003, China

Abstract: Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Here we attempt to generate InDel variations in the genome of Nannochloropsis oceanica, and then decipher the genetic basis of its economic and biological traits with bulked mutant analysis modified from bulked segregant analysis. In addition, we describe our efforts to construct site-tagged and gene-traceable mutant libraries to clone its genes through reverse genetic approaches. Currently, more than a half of N. oceanica protein-encoding genes are annotated against databanks. However, no functional gene has been de novo cloned from N. oceanica and no new function has been assigned to any of its annotatable genes. Here, we discuss the possible methods and potential benefits of de novo cloning of N. oceanica genes.


Classification and unusual biochemical and ultrastructural characteristics
  • The microalgal genus Nannochloropsis comprises six species that inhabit marine, freshwater, and brackish environments (Fawley and Fawley 2007; Hibberd 1981). Following a phylogenetic analysis of the concatenated 18S ribosomal RNA (rDNA) and rbcL genes, Fawley et al. (2015) described a new species, N. australis and established a new genus, Microchloropsis, comprising M. salina and M. gaditana, both of which were transferred from Nannochloropsis (basionyms N. salina and N. gaditana, respectively). These species are assigned to class Eustigmatophyceae, phylum Ochrophyta, superphylum Heterokonta (or Stramenopiles) (Hibberd 1981). All species of Nannochloropsis and Microchloropsis are small, non-motile, and spherical in shape (Kandilian et al. 2013). Currently, the rbcL amino acid and ribosomal RNA gene sequences are used to identify species in these genera (Andersen et al. 1998; Bailey and Freshwater 1997; Daugbjerg and Andersen 1997).

    Nannochloropsis are able to build up high concentrations of diverse pigments (Lubian et al. 2000) and have chlorophyll a, but completely lack chlorophylls b and c (Manning and Starain 1943). Nannochloropsis spp. also accumulate large quantities of polyunsaturated fatty acids (Boussiba et al. 1987; Sukenik et al. 1989). The ultrastructure of N. oculata is unusual; a nucleus-plastid continuum exists throughout its cell cycle (Murakami and Hashimoto 2009), but the basal body and flagellum observable in green algae (Nishikawa and Tominaga 2001) are absent.

Genomic characteristics and unique speciation process
  • The genomes of two species of the genus Nannochloropsis, N. gaditana (Carpinelli et al. 2014; Radakovits et al. 2012) and N. oceanica (Vieler et al. 2012), have been sequenced on the Illumina platform. Their genomes vary between 28.5 and 29.0 Mb and are characterized by a high density of genes, a low intron content, short spacers between genes (Carpinelli et al. 2014), and low numbers of repeat sequences (Vieler et al. 2012). In addition, a comparison of the genomes of all six species of Nannochloropsis revealed an extreme case of dose expansion of lipid biosynthesis genes (Wang et al. 2014).

    Single molecule real-time (SMRT) sequencing technology has the advantages of long reads and the ability to maintain accuracy by optimizing assembly software using PacBio and Illumina reads (Eid et al. 2009). Furthermore, assembly of pseudochromosomes is possible with the aid of Hi-C technology that can generate the chromosome conformation data and anchor and orient the scaffolds into pseudochromosomes (Burton et al. 2013; Lieberman-Aiden et al. 2009). With these newly developed technologies, the genome of N. oceanica has been resequenced. Its genome is 29.3 Mb and contains 32 chromosomes. Nannochloropsis oceanica may have evolved through the active fusion of a host protist and a photosynthesizing ancient red alga (Guo et al. 2019). During the speciation of N. oceanica through secondary endosymbiosis, the host nucleus was overthrown by the symbiont nucleus. Nannochloropsis oceanica lost its ability to undergo meiosis and to move by means of flagella. The two outermost membranes of its chloroplast may have originated from the endoplasmic reticulum; this may be why it has not evolved a complex protein transport system similar to that of diatoms (see Guo et al. 2019 for supporting information).

Biological restrictions of their evolvement as models
  • Nannochloropsis has shown promising characteristics, allowing convenient genetic improvement, which include, a monoploid nucleus and asexual reproduction, as revealed by the difference between mutation frequencies of nuclear and plastid genomes (Galloway 1990), the single allele of insertion mutated gene (Kilian et al. 2011) and single nucleotide polymorphism (SNP) distribution revealed by preliminary genome sequencing (Pan et al. 2011). Nannochloropsis are easy to be genetically manipulated. Various species of the genus are able to perform homologous recombination (Kilian et al. 2011) and genome editing (Verruto et al. 2018; Wang et al. 2016a, b). Species of Nannochloropsis are widely used as feed for fish larvae and rotifers, as food additives for human nutrition, and as models for industrial applications and biological research (Gee and Niyogi 2017; Weeks 2011).

    In breeding elite varieties (lines, strains) of N. oceanica and studying the genetic basis of its economic and biological traits, we have met three main limitations: i.e., SNP instability; extremely narrow genetic background; and absolute unavailability of segregation populations. The number of N. oceanica cells may double in about 2 days and SNPs may be generated from a N. oceanica cell to a N. oceanica line (Lin et al. 2017, 2018). At the rate of 10-6, a widely believed nucleotide mutation rate during DNA replication, about 30 SNPs will be generated during each cellular division cycle; the genome size of N. oceanica is ~ 30 M (Pan et al. 2011). The SNPs may not be stable among a microalgal population; SNPs may change reversely and other newly generated SNPs may hide the phenotypes controlled by already existing SNPs. We have proposed that a microalgal population is an assemblage of mutants and at least one of them will successfully dominate the population, if the environment changes in its favor (Lin et al. 2017, 2018). It is clear that SNPs themselves and trait-associated SNPs are less applicable for microalgal genetic study and modification. Microalgal strains (lines) are usually clonal (derived from a single cell); thus, the stable variations such as InDel should be very scarce. In addition, the asexual reproduction strategy of Nannochloropsis make their sexual crossing impossible, so segregation populations will never been obtained. This scenario severely limits the opportunity for genetic modification and investigation of N. oceanica.

Finding solutions
  • Various strategies employed in the creation of variety in land crops have been applied to microalgal breeding, including chemical mutation (Anandarajah et al. 2012; Vigeolas et al. 2012), heavy-ion irradiation (Hu et al. 2013), protoplast fusion (Tjahjono et al. 1994), and gene silencing and knockout (Bowler et al. 2008; Vieler et al. 2012). These approaches are less applicable for improving and studying N. oceanica as they are either technologically advanced or practically inconvenient (Wang et al. 2016b).

    We have tried to create stable microalgal variations through Zeocin mutation (Chankova et al. 2007; Lin et al. 2017, 2018) and other chemotherapy drug mutations. Zeocin causes double strand breaks (DSBs) of DNA and introduces InDels during DNA repair. InDels are more stable than SNPs. In addition to mutants for selection, InDels created through Zeocin mutation also provide the convenience of obtaining mutant populations for genetic studies.

    The continuous advancement of DNA sequencing technologies has modernized genotyping by whole-genome resequencing and deciphering the genetic bases of traits by adopting these genotypes through either linkage or association analyses. The genome-wide association study (GWAS) (Klein et al. 2005) has evolved rapidly as an approach to control genes, or the most approximate markers of important traits, of a wide diversity of organisms. The vast majority of genomic variations (including SNPs, InDels, microsatellites, gene copy number, and chromosomal structural variations) can be revealed by whole genome resequencing (WGR) at an affordable cost. Genotyping by WGR is highly productive; however, genotyping a large number of individuals by WGR has been hindering GWAS (Fu et al. 2010; Steemers et al. 2006). Bulked segregant analysis (BSA) was proposed to find the most possible linking molecular markers of segregating traits (Giovannoni et al. 1991; Michelmore et al. 1991). Diverse traditional molecular markers, including randomly amplified polymorphism of DNA (RAPD), restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR or microsatellite), have been used in combination with BSA. With the advancement of DNA sequencing and DNA chip-developing technologies, SNPs were once regarded as the optimal molecular markers and are still widely used even today. BSA has also been extended to bulked segregant RNA sequencing (BSR-seq) to identify the variations among gene transcripts (Liu et al. 2012). BSA has been widely used in studies of rice and other terrestrial crops that have been subjected to continuous modification. Consequently, modified forms of BSA, e.g., MutMap (Abe et al. 2012), QTL-seq (Takagi et al. 2013a), MutMap+ (Fekih et al. 2013), MutMap-Gap (Takagi et al. 2013b), and Graded Pool-seq mapping (GPS) (Wang et al. 2019), have been developed.

    We modified BSA and BSR-seq into bulked mutant analysis (BMA) and bulked mutant RNA sequencing (BMR-seq) to find the association between the genes and the traits of N. oceanica (Pan et al. 2011). We mutated N. oceanica with Zeocin, evaluated the growth performance of a set of mutants, and obtained two extreme mutant bulks (fast growth and slow growth). We then compared the genomic and transcriptomic differences between the two bulks with BMA and BMR-seq methods. We have identified a set of genes that may be associated with the growth performance of N. oceanica (see Liang et al. 2019 for method detail). BMA and BMR-seq genotype the mutants bulk by bulk. Genotyping by resequencing on the second generation of sequencing platforms provides mainly SNPs and InDels, but not the structural variations. Since SNPs are unstable, InDels are recognized as the most appropriate markers. To understand the genetic basis of the growth performance trait, the function of the genes in which the associating markers located should be verified. Unfortunately, we have not progressed to this point yet. It is certain that future studies will be shifted to the real GWAS in which mutants will be genotyped one by one; however, the linkage analysis based on segregation population is not applicable for N. oceanica as it does reproduce sexually.

De novo cloning of N. oceanica genes should not be left out
  • The identification of economic and biological trait-associated markers in N. oceanica is in its infancy (Liang et al. 2019) and cloning genes in this species has not been achieved. Here, the genes were predicted and the functions of these genes were annotated against known databanks in diverse genomic and transcriptomic analyses; however, we are unsure of the functions of the unannotated genes and the novel functions of annotated genes. In other words, no N. oceanica gene has been cloned from scratch (de novo). Such a scenario makes cloning genes of this species a prime area for future work. A set of associated markers and genes in N. oceanica has been identified by bulked mutant analysis (BMA) that was modified from bulked segregant analysis (BSA) (Liang et al. 2019). The identification of trait-associated markers and genes in N. oceanica is pioneering, but preliminary and the functions of the markers and the genes should be verified with tools such as homologous recombination (Kilian et al. 2011) and genome editing (Verruto et al. 2018; Wang et al. 2016a).

Reverse genetics: an alternative approach
  • Mutations generate rich variation. The genes containing these mutations may be identified through GWAS. Traditional methods of cloning genes use a strategy of "from outward appearance (trait) to inner essence (gene)". The greatest achievements of this strategy are the genes cloned by map-based cloning (Jander et al. 2002) and modified map-based cloning (e.g., Wang et al. 2019). A soybean mutant library has been constructed using chemical mutagen ethyl methanesulfonate (EMS) (Tsuda et al. 2015). We have likewise constructed N. oceanica mutant libraries using Zeocin (Lin et al. 2017, 2018) and used this to identify a set of fast growth-associated markers and genes (Liang et al. 2019). The mutants in such libraries may contain more than one mutated site and genetic principles such as linkage and association are observed, when establishing the relationship between mutated sites and traits. In other words, the mutations contained by the mutants in these libraries are neither tagged nor traceable. However, such mutant libraries may aid the user to decipher the genetic bases of diverse economic and biological traits through GWAS and modifications of it; e.g., BMA (Liang et al. 2019).

    With the advancements in molecular genetics and related technologies, it is now feasible to mutate or edit a specific site or a specific gene in the genome providing an alternative approach to clone genes, i.e., a way from gene to trait. This method is called reverse genetics, so named because it works in the opposite in direction of forward genetic screens of classical genetics. It is noteworthy that methods such as RNA interference (RNAi) and homologous recombination (gene knockout) have long been used for verifying the functions of genes.

    The genes in a genome may also be mutated and tagged randomly. This idea emerged very early among bacterial genetic studies, when transposon insertional mutations were applied to clone genes from bacteria (van Opijnen and Camilli 2013). Constructing mutant libraries and tagging the mutated sites making them traceable spontaneously are the most outstanding progress in reverse genetic studies. The mutation site is usually unique within a given genome, either known in advance or being marked by a known sequence (Li et al. 2016, 2019; Meng et al. 2017). In addition, the insertion-mutated sites can be effectively found through whole genome resequencing (Zhang et al. 2014). Once the phenotype of a mutant is described and the mutated site is unique, the gene can be isolated and its function can be described; i.e., the gene can be cloned (not just isolated). We are constructing an insertional mutant library of N. oceanica, trying to clone its genes controlling high lipid content, fast growth, stressing tolerance among others. We suggest that cloning genes is a key direction for future genetic studies of N. oceanica.

  • This study was financially supported by National Key R & D Program of China (2018YFD0900305 and 2018YFD0901506), The Marine S & T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0406-3), and The Fundamental Research Funds for The Central Universities (201762017).

Author contributions
  • GY suggested the general idea of the context of manuscript. ZZ collected and reviewed the papers published early, which report the genetic manipulations of N. oceanica. HL collected and reviewed the papers published that report the strategies and methods either applicable or referable to the genetic studies of N. oceanica. LG drafted the manuscript.

Compliance with ethical standards

    Conflict of interest

  • The authors declare no competing and conflicting interest.

  • Animal and human rights statement

  • The material reviewed here and used in our previous studies are a microalgal species. This species and plants are comparable. The associating statements are not applicable.

Reference (56)



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