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Adiantum hispidulum chloroplast genome high-throughput sequencing and comparative analysis of cpDNA inverted repeat regions of Adiantum species Belenikin M.S.
1

1,2,3

, Krinitsina A.A.1, Logacheva M.D.1, Kuptsov S.V.1, Speranskaya A.S.

1

Lomonosov Moscow State University, Lenin Hills 1, 119234 Moscow, Russia
2

Research Center for Children Medical Care, 119620 Moscow, Russia

3

Pirogov Russian National Research Medical University, 117997 Moscow, Russia

Adiantum is a genus of family Pteridaceae that is a large family with a nearly worldwide distribution. Presently 35 genera of family Pteridaceae belongs to four subfamilies. Recent molecular studies supported five major clades within the family: the cryptogrammoids (the CR clade), the ceratopteridoids (the CE clade), the pteridoids (the PT clade), the cheilanthoids (CH clade) and the adiantoids (the AD clade). The AD clade formed by genus Adiantum (tribe Adianteae) together with vittarioid ferns (tribe Vittarieae) [1, 2]. Adiantum (commonly known as maidenhairs ferns) comprising about 200 species and widely distributed mainly in the tropical and subtropical regions. The classifications of Adiantum mainly based on regional studies. Authors pointed out that parallel or convergent evolution probably occurred in the frond architecture and other characters in Adiantum, which may have obscured the relationships among species [2, 3]. Comparative analyses of molecular markers between closely related species may provide clues to understand the intragenus phylogeny and character evolution during their different ecological niche colonization. Analysis of level and pattern of genetic variation using the ISSR fingerprinting method previously demonstrated low level of intrageneric differentiation in four species of the fern genus Adiantum L., originating from South India (A. hispidulum Sw., A. incisum Forrsk., A. raddianum C.Presl, A. zollingeri Mett. ex Kuhn) [4]. Another emerging approach is to use complete chloroplast (cp) DNA sequences but molecular evolutionary and ecology analyses on ferns cpDNA are relatively few in general in comparison with those on seed plants. The complete nucleotide sequence of the chloroplast genome of the fern Adiantum capillus-veneris L. was determined in 2003 [5], but no more Adiantum full genome chloroplast sequences has been published during last decade. This work we present sequencing and assembling the cpDNA of A. hispidulum using Illumina (MiSeq) sequencing technology. For sample preparation the mature alive fern was taken from the collection of the Moscow State University Botanical Garden. Total DNA (partially


enriched by cpDNA) was extracted from fresh fronds and TruSeq (NEBNext reagents) protocol was used for preparing DNA library. Two sequencing runs produced about 2,04M PE(2x300) reads. Trimming procedure resulted to about 2,01M high quality PE reads and cpDNA reached 2.5% of total data. De-novo assembly (Velvet) of cpDNA reads generated 38 contigs with sum length about 140 Kbp. Finalization of the chloroplast genome assembly is in progress. Analysis of five plastid markers (atpA, atpB, rbcL, trnLF and rps4-trnS) was carry out to study of intraspecies variability of cpDNA and searching of relatives of MSU Botanical Garden A. hispidulum specimen. Comparison of our data with previously published sequences demonstrated the undoubted common origin of Russian isolate and cultivated isolate "A79" from USA: Virginia (see table 1). We made comparative analysis of A. hispidulum vs A. capillus-veneris inverted repeats (IR) regions of cpDNA sequences using BLAST algoritm. Adiantum capillus-veneris chloroplast genome consists of 150568 bp [NC_004766.1]. It contains two IR (~23Kbp) that separate large single-copy region (~82 Kbp in A. capillus-veneris) from small single-copy region (~21Kbp in A. capillus-veneris) [1] and comprise 14 genes: transfer RNA genes: trnT-TGT, trnR-ACG, trnH-GTG, trnN-GTT, trnA-TGC, trnI-GAT; ribosomal RNA genes: rrn4.5, rrn5, rrn16, rrn23; ribosomal protein genes: rps7, rps12 (exon 2); photosystem II gene: psbA; hypothetical protein gene: ycf2. Our results demonstrate the same principal organization of IR regions of cpDNA of both Adiantum species. The essential difference between IR regions of A. capillus-veneris and A. hispidulum is the ~180 bp region that is located between ycf2 and trnN-GTT genes. There are only two small differences between two IR regions of A. capillus-veneris cpDNA - at the hypothetical protein gene (ycf2) and on the boundary of IR regions and small single-copy region. Comparative analysis of two IR regions of A. hispidulum cpDNA reveals more differences. This work was supported by grant RFBR 14-04-01852.

1. E. Schuettpelz, K.M. Pryer (2007) Fern phylogeny inferred from 400 leptosporangiate species and three plastid genes, Taxon, 56: 1037-1050. 2. J-M. Lu et al. (2012) Phylogenetic relationships of Chinese Adiantum based on five plastid markers, J. Plant Res, 125: 237-249. 3. R.M. Tryon, A.F. Tryon (1982) In: Ferns and allied plants, with special reference to tropical America. Spinger-Verlag, New York, (cited by [2]) 4. H. Korpelainen et al. (2005) Four tropical, closely related fern species belonging to the genus Adiantum L. are genetically distinct as revealed by ISSR fingerprinting, Genetica, 125: 283-291.


5. P.G. Wolf et al. (2003) Complete Nucleotide Sequence of the Chloroplast Genome from a Leptosporangiate Fern, Adiantum capillus-veneris L., DNA Research, 10: 59-65.

Table 1. Comparison of Russian isolate A. hispidulum with specimens from different geographic locations using five basic plastid markers. marker specifications
Comparison of Russian isolate cp markers to

isolate "A79" USA: Virginia cultivated rbcL atpB atpA rps4trnS trnLF
accession number homology SNP numbers accession number homology SNP numbers accession number homology SNP numbers accession number homology SNP numbers accession number homology SNP numbers JF9353 100% none JF9354 100% none JF9373 99% 1 JF9806 98% 17 JF9806 99% 5 49

isolate "A69" Indonesia: Sulawesi
JF93 99% 6 JF93 99% 2 JF93 99% 2 JF98 97% 31 JF98 99% 7 5341

Nearest homolog of Russian isolate A. hispidulum

31

5423

04

7296

15

0607

isolate "A79", USA data is presented in the same name isolate "A79", USA data is presented in the same name isolate "A79", USA data is presented in the same name isolate "A79", USA data is presented in the same name

column with

column with

column with

column with

94

0686

LC004388 country="Indonesia" collector number: BA706 homology 99% SNP numbers = 1