Screening and verification of extranuclear genetic markers in green tide algae from the Yellow Sea

Abstract

Over the past decade, Ulva compressa, a cosmopolitan green algal species, has been identified as a component of green tides in the Yellow Sea, China. In the present study, we sequenced and annotated the complete chloroplast genome of U. compressa (alpha-numeric code: RD9023) and focused on the assessment of genome length, homology, gene order and direction, intron size, selection strength, and substitution rate. We compared the chloroplast genome with the mitogenome. The generated phylogenetic tree was analyzed based on single and aligned genes in the chloroplast genome of Ulva compared to mitogenome genes to detect evolutionary trends. U. compressa and U. mutabilis chloroplast genomes had similar gene queues, with individual genes exhibiting high homology levels. Chloroplast genomes were clustered together in the entire phylogenetic tree and shared several forward/palindromic/tandem repetitions, similar to those in U. prolifera and U. linza. However, U. fasciata and U. ohnoi were more divergent, especially in sharing complementary/palindromic repetitions. In addition, phylogenetic analyses of the aligned genes from their chloroplast genomes and mitogenomes confirmed the evolutionary trends of the extranuclear genomes. From phylogenetic analysis, we identified the petA chloroplast genes as potential genetic markers that are similar to the tufA marker. Complementary/forward/palindromic interval repetitions were more abundant in chloroplast genomes than in mitogenomes. Interestingly, a few tandem repetitions were significant for some Ulva subspecies and relatively more evident in mitochondria than in chloroplasts. Finally, the tandem repetition [GAAATATATAATAATA × 3, abbreviated as TRg)] was identified in the mitogenome of U. compressa and the conspecific strain U. mutabilis but not in other algal species of the Yellow Sea. Owing to the high morphological plasticity of U. compressa, the findings of this study have implications for the rapid non-sequencing detection of this species during the occurrence of green tides in the region.

Fig 1. Schematic representation of the U. compressa chloroplast genome using OGDRAW.

The predicted genes are shown, with the colors representing functional classifications at the bottom left. The genes outside the circle were transcribed counterclockwise. The inner-circle shows the GC content.

Fig 2. Length of the chloroplast genomes in genus Ulva.

The total length of intergenic regions varied widely compared to that of the corresponding PCGs, rRNAs, and tRNAs.

Fig 3. Ka/Ks value assessment of 71 PCGs in the chloroplast genomes of seven species.

The value was measured using the KaKs_Calculator Toolbox 2.0, the γ-NG method, and the standard genetic code. Ulva compressa (NCBI #KX595275) was used as a control.

Fig 4. Molecular phylogenetic analysis of Ulva species using chloroplast or mitochondrial genomes and the maximum likelihood method based on the General Time Reversible+G+I model (A) and General Time Reversible+G model (B).

(A) Molecular phylogenetic analysis with whole aligned coding genes of the Ulva chloroplast genomes. The coding genes include accD, atpA, atpB, atpE, atpF, atpH, atpI, ccsA, cemA, chlI, clpP, ftsH, infA, petA, petB, petD, petG, petL, psaA, psaB, psaC, psaI, psaJ, psaM, psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ, rbcL, rpl12, rpl14, rpl16, rpl19, rpl2, rpl20, rpl23, rpl32, rpl36, rpl5, rpoA, rpoB, rpoC1, rpoC2, rps11, rps12, rps14, rps18, rps19, rps2, rps3, rps4, rps7, rps8, rps9, tufA, ycf1, ycf12, ycf20, ycf3, and ycf4. (B) Molecular phylogenetic analysis with whole aligned coding genes of the Ulva mitochondrial genomes. The coding genes include atp1, atp4, atp6, atp8, atp9, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, rpl14, rpl16, rpl5, rps10, rps11, rps12, rps13, rps14, rps19, rps2, rps3, and rps4.

Fig 5. Phylogenetic analysis of petA (A) and tufA (B) from the strains of the green tides in the Yellow Sea, and data from NCBI obtained via the maximum likelihood method in the Tamura 3-parameter+G model.

S2 Table in S1 File contains information on the green tide strains in the Yellow Sea.

Fig 6. Testing 12 primer pairs on four constitutive green tide species in the Yellow Sea.

(A) Electrophoresis results following PCR using U. compressa DNA and the 12 primer pairs. (B) Electrophoresis results following PCR using DNA from four constitutive green tide species in the Yellow Sea and the twelve primer pairs. Either “√” or “x” mean there are bands in the electrophoresis with related primers. Wherein “√” means the consistency of the band sequence with predicted tandem repeats, while “x” means not. “-” means no band; blank means not tested. Each primer pair was tested on two strains of the species in Panel A. Information about 12 pairs of primers was detailed in S3 Table in S1 File.

Fig 7. Application of the m-trnG-trnY primer on U. compressa and U. mutabilis.

(A) Lanes 1–22 correspond to amplification using m-trnG-trnY with 22 green tide samples from the Yellow Sea, China, which were detailed in S4 Table in S1 File. Lanes 3 and 22 show the two brightest bands, characterized by the sequence GAAAATATAAATA (× 3), with a length of approximately 800 bp. In comparison with the DNA marker, the bands in lanes 4, 5, 12, 13,16, 18 and 19 were higher than 800 bp. Subsequent sequencing using the m-trnG-trnY primer resulted in no result, suggesting the occurrence of non-specific amplification. (B) Lanes 23–28 correspond to PCR amplification using the m-trnG-trnY primer with U. compressa progeny, the candidate model U. mutabilis, and U. ohnoi. Two different types of standard genetic markers were used in (A) and (B) because the two experiments were performed independently.

Fig 8. Application of primer m-trnG-trnY on 27 Ulva samples collected in the North and Baltic Seas (Europe).

The standard genetic marker is shown corresponding to the individual gel electrophoresis analysis. The name and number of the sampling site for each Ulva isolate are indicated above each lane. Details of the 27 samples are given in S5 Table in S1 File.