Development of eight mitochondrial markers for Funariaceae (Musci) and their amplification success in other mosses†
Freely available online through the AJB Open Access option (http://www.amjbot.org/site/misc/ifora.xhtml#2OpenAccessPolicy).
This research was supported by the National Science Foundation (grant 0919284) to B.G.
Abstract
• Premise of the study: In comparison to the wide use of chloroplast markers, few mitochondrial markers are available for phylogenetic studies in bryophytes. We investigated the phylogenetic suitability of several mtDNA markers within the Funariaceae and across mosses.
• Methods and Results: By comparing mitochondrial genomes of two mosses, eight regions with higher substitution rates were identified and sequenced for three species in the Funariaceae and one outgroup taxon. Variations in the substitution rate of these new loci were compared to previously used markers. Thirty-four samples representing all major moss lineages were targeted to assess the universality of the newly designed primers.
• Conclusions: The new markers provided similar or more sequence variations in Funariaceae compared to previously developed mtDNA markers. Five out of eight loci were amplified in 70% of other taxa, indicating that these markers may be suitable for phylogenetic studies in other moss lineages.
Mitochondrial loci are characterized by low substitution rates and less homoplasy, and are hence suitable for phylogenetic inferences at least above the species rank (8). However, mitochondrial (mt) makers are much less commonly targeted than chloroplast (cp) and nuclear (nu) loci for phylogenetic studies in bryophytes (9). The most often used mtDNA marker is nad5, in particular, its group I intron (nad5i753g1). Other mitochondrial markers, including the coding sequences SSU and cox3, the intergenic spacer nad4-5, and the introns cobi420g1, nad2 (nad2i156g2), and nad7 (nad7i140g2), are only rarely used (9). According to the GenBank record, only around 30 mitochondrial genomes have been fully sequenced for land plants, which is far fewer than the number of chloroplast genomes sequenced (> 200). The mitochondrial genome is characterized by dramatic variation in genome size, gene content, and gene order (5). These characters have prevented not only sequencing of the complete mitochondrial genomes but also using mtDNA sequences as phylogenetic markers. However, recent studies have revealed that mitochondrial genomes are conservative within bryophyte lineages. Comparison of the mt genome of Anomodon rugelii (Müll. Hal.) Keissl. (6) to that of Physcomitrella patens (Hedw.) Bruch & Schimp. (11) indicated that the mtDNA of the two species share the same gene content, and are perfectly collinear. Considering the distant relationship between the two taxa, the mitochondrial genome thus appears to have been highly conserved during the evolutionary history of mosses. The identical gene order allows for designing universal primers targeting the intergenic spacers. The spacers usually show higher mutation rates, which makes them attractive targets for potentially phylogenetically informative characters.
The moss family Funariaceae includes P. patens, a model organism used for studies in hybridization, physiology, genetics, and functional genomics (4). The phylogeny of the family remains poorly understood despite recent progress (7), and in part due to the lack of rapidly evolving markers. We targeted exemplars of three of the 15 genera within the Funariaceae and one outgroup taxon, sequenced all newly selected mitochondrial regions, and compared them with the previously used mtDNA, cpDNA, and nuDNA markers. We then tested the primers in representatives of most of the other main moss lineages.
METHODS AND RESULTS
To visualize the distribution of variable sites, the whole mitochondrial genome of P. patens was downloaded from GenBank (NC_007945) and compared with A. rugelii (6) using the online program VISTA (3; http://genome.lbl.gov/vista/). Regions with many variable sites distributed within 500 to 1000 base pairs, a length suitable for PCR amplification and Sanger sequencing, were identified. External and internal primer pairs were designed for eight mitochondrial regions (Table 1), including five intergenic spacers (atp1-trnW, atp9-trnI, nad2-trnG [one pair of primers only], rpl5-16, and rps7-atp6), two protein coding genes (rpl2 and rps3), and one Group II intron (nad1i287).
Region | Product sizea (bp) | Ta (°C) | Primer nameb | Primer sequences (5′-3′) |
---|---|---|---|---|
atp1-trnW | 759 | 48 | atp1-F | TTTCTGCTATCGTACAAC |
trnW-R | AATGGTAGAACAATGGTC | |||
694 | 50 | atp1-F1* | AGTCAACTGGCTACC | |
trnW-R1* | ACAGGTTAAGGGTTC | |||
atp9-trnI | 638 | 48 | atp9-F | CTATCCAACCTCATTCTG |
trnI-R | CGAACCTACAATATCACC | |||
523 | 50 | atp9-F1* | TGTTTGCCTTAATGATGG | |
trnI-R1* | TTATGAGCGGTGCGTTTG | |||
nad1i287 | 1225 | 50 | nad1-F | GTGGGATTGTTTGGATTG |
nad1-R | TTCTGCTTCTGGTAAATC | |||
742 | 52 | nad1-F1* | TGCTTGGGCTGTTATACC | |
nad1-R1* | GTAGGGGTCGGAGATTTC | |||
nad2-trnG | 703 | 55 | nad2-F | AGGCTGTGGGGCYTACTTAC |
trnG-R | AAGGCTRAGGTTGAGGGTTC | |||
rpl2 | 1184 | 52 | rpl2-F | TTCATCAGGGCGTATTAC |
rpl2-R | CGGATTCATAGCAACACC | |||
813 | 52 | rpl2-F1* | AACACTTCGTCTATTGGC | |
rpl2-R1* | GATACGCAATTTCCTACC | |||
rpl5-16 | 1266 | 52 | rpl16-F | CCAGTAAACCCACCGAAG |
rpl5-R | AGAGTGCTTTGTGCTAGG | |||
890 | 54 | rpl16-F1* | GGATGGTGTGAGTTTGTC | |
rpl5-R1* | CGGAGTCTATTTGGAGTG | |||
rps3 | 1503 | 48 | rps3-F | TCGTAGTTCAGATTCAAG |
rps3-R | CACCTAAAATCCCATAAG | |||
1040 | 50 | rps3-F1* | GCAATACAATCGGTCAAG | |
rps3-R1* | GAGCAATTAGAGAAGCAC | |||
rps7-atp6 | 1178 | 50 | rps7-F | AAGCCTRTTTGTGAAGTGAA |
atp6-R | CTGGTCATAGTTTAGTMAAG | |||
1068 | 52 | rps7-F1* | TATRGCARCAAATCGTCAAG | |
atp6-R1* | TATCTATGGGGGGTATTATG |
- Note: Ta = annealing temperature.
- a The product size of each marker is based on the length in the Physcomitrella mitochondrial genome (GenBank NC_007945).
- b The internal primers are marked with an asterisk (*).
Specimens were obtained from wild-collected or cultured plants (Table 2, Appendix 1). Total genomic DNA was extracted from all materials using a modified cetyltrimethylammonium bromide (CTAB) method (1). PCR amplification was carried out in a 25 μL reaction containing 18.5 μL of nuclease-free water, 2.5 μL of 10× buffer, 1 μL of dNTPs (10 mM), 1 μL of each primer (5 μM), 0.15 μL of GoTaq DNA polymerase (Promega, Madison, Wisconsin, USA), and 1 μL of DNA template. The thermal cycling conditions were as follows: initial denaturation at 94°C for 3 min; 34 cycles of 94°C for 0.5 min, 48–55°C for 1 min, 70°C for 1 min; and a final elongation at 70°C for 10 min. The annealing temperature varied depending on the marker (Table 1). Nested PCR was used in some cases to improve amplification success rate. The annealing temperature of the nested PCR was usually 2°C higher than the first round. PCR products were visualized under ultraviolet light on a 0.5% TBE agarose gel using SYBR Safe DNA gel stain (Invitrogen, Eugene, Oregon, USA). Subsequently, the products were purified using the NucleoSpin PCR purification kit (Macherey & Nagel, Düren, Germany). Sequencing reactions were performed in 10 μL reactions using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA). All loci were sequenced using the internal primer pairs. Sequencing products were purified using Sephadex G-50 (Amersham, Piscataway, New Jersey, USA) gel filters, and then run on an ABI PRISM 3100 Genetic Analyzer. Nucleotide sequences were edited and assembled using Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, Michigan, USA). The new sequences were deposited at GenBank (Appendix 1).
Family | Species | Collection no.a | atp1-trnW | atp9-trnI | nad1i287 | nad2-trnG | rpl2 | rpl5-16 | rps3 | rps7-atp6 |
---|---|---|---|---|---|---|---|---|---|---|
Andreaeobryaceae | Andreaeobryum macrosporum Steere & B. M. Murray | Rupp 07-22-1999 | − | − | − | − | − | − | − | − |
Anomodontaceae | Haplohymenium triste (Ces.) Kindb. | Goffinet 6484 | + | + | + | − | + | + | + | + |
Archidiaceae | Archidium alternifolium (Dicks. ex Hedw.) Mitt. | Anderson & Crum 13505 | + | − | − | − | − | − | − | − |
Bartramiaceae | Bartramia pomiformis Hedw. | Hax CT01 | + | − | − | + | − | + | − | − |
Brachytheciaceae | Brachythecium velutinum (Hedw.) Schimp. | Sotiaux 27346 | + | + | − | + | + | + | − | − |
Bryaceae | Bryum argenteum Hedw. | Goffinet 6765 | + | + | + | + | + | + | − | + |
Buxbaumiaceae | Buxbaumia aphylla Hedw. | Goffinet 8746 | + | + | − | + | + | + | + | + |
Climaciaceae | Climacium americanum Brid. | Liu 102310 | + | + | + | − | + | + | + | + |
Cryphaeaceae | Dendroalsia abietina (Hook.) E. Britton | Goffinet 7764 | + | + | + | + | + | + | + | + |
Dicranaceae | Holodontium strictum (Hook. f. & Wilson) Ochyra | Fife 12115 | + | + | − | + | + | + | − | − |
Diphysciaceae | Diphyscium foliosum (Hedw.) D. Mohr | Goffinet 4595 | + | + | + | + | + | + | + | + |
Fissidentaceae | Fissidens polypodioides Hedw. | Goffinet 4979 | + | + | + | + | + | + | − | − |
Funariaceae | Funaria hygrometrica Hedw. | Goffinet 5576 | + | + | + | + | + | + | + | + |
Funariaceae | Physcomitrium pyriforme (Hedw.) Hampe | Goffinet 4737 | + | + | + | + | + | + | + | + |
Funariaceae | Physcomitrella patens (Hedw.) Bruch & Schimp. | Withehouse 1962 | + | + | + | + | + | + | + | + |
Grimmiaceae | Grimmia montana Bruch & Schimp. | Vanderpoorten F14 | + | + | + | − | + | + | − | + |
Hedwigiaceae | Hedwigia ciliata (Hedw.) P. Beauv. | Goffinet 8955 | + | + | − | + | + | + | − | + |
Hookeriaceae | Hookeria lucens (Hedw.) Sm. | Goffinet 8030 | + | − | − | + | + | + | + | + |
Hypopterygiaceae | Lopidium concinnum (Hook.) Wilson | Holz & Franzaring 00-123 | + | + | + | + | + | + | − | − |
Mniaceae | Mnium hornum Hedw. | Goffinet 8830 | + | + | + | + | − | + | − | + |
Neckeraceae | Circulifolium microdendron (Mont.) S. Olsson, Enroth & D. Quandt | Jordan 08-27-2001 | + | + | + | + | + | + | + | + |
Oedipodiaceae | Oedipodium griffithianum (Dicks.) Schwägr. | Schofield 98670 | + | + | − | + | − | − | − | − |
Orthotrichaceae | Orthotrichum sp. | Goffinet 7055 | + | + | + | + | + | + | − | − |
Pleuroziopsidaceae | Pleuroziopsis ruthenica (Weinm.) Kindb. ex E. Britton | Damman 92045 | + | + | + | − | + | + | + | − |
Polytrichaceae | Atrichum undulatum (Hedw.) P. Beauv. | Goffinet 7605 | + | − | − | + | + | − | − | − |
Pottiaceae | Tortula subulata Hedw. | Satory 1995 | + | + | − | + | + | + | − | − |
Ptychomitriaceae | Ptychomitrium incurvum (Schwägr.) Spruce | Goffinet 4703 | + | + | − | + | − | − | − | − |
Racopilaceae | Racopilum tomentosum (Hedw.) Brid. | Goffinet 6469 | + | + | + | + | + | + | + | + |
Rhizogoniaceae | Pyrrhobryum spiniforme (Hedw.) Mitt. | Goffinet 5107-b | + | − | + | − | + | − | − | − |
Sphagnaceae | Sphagnum sp. | Liu 031511 | + | + | + | − | + | + | + | + |
Splachnaceae | Tetraplodon mnioides (Sw. ex Hedw.) Bruch & Schimp. | Goffinet 10507 | + | + | + | + | + | + | + | + |
Takakiaceae | Takakia lepidozioides S. Hatt. & Inoue | Rupp & Schofield 08-10-1999 | + | − | − | − | − | + | − | − |
Tetraphidaceae | Tetraphis geniculata Girg. ex Milde | Schofield 103022 | + | − | + | + | + | + | − | − |
Timmiaceae | Timmia megapolitana Hedw. | Schofield 97957 | + | + | + | + | + | + | + | + |
Total number | 34 | 33 | 26 | 21 | 25 | 27 | 28 | 15 | 18 |
- Note: + = successful PCR amplification; – = PCR failure.
- a All vouchers have been deposited in George Safford Torrey Herbarium at the University of Connecticut (CONN).
Sequences were aligned with MUSCLE 3.8.31 (2) and imported into MEGA 4 (10) for estimating variable site numbers. The analyses revealed that the nuclear marker presented a higher percentage of variation than all chloroplast markers, and the chloroplast markers in turn presented a higher percentage of variation than all mitochondrial markers (Fig. 1). Among the mitochondrial markers, nad2 (nad2i156g2) has the most variable sites (73 sites), reflecting its much longer size (2220 bp). The newly designed rpl5-16 has the highest percentage of variable characters (7.0%), which is almost three times as many as the marker with the least (nad4-5, 2.6%). The mitochondrial rpl5-16 shows a similar substitution rate as the chloroplast rps4 gene. Five (rpl5-16, atp9-trnI, rpl2, atp1-trnW, and rps7-atp6) out of eight newly designed mtDNA markers have a higher degree of variation compared to the previously used ones (Fig. 1). The five markers showed percentage of variable characters from 4.8% to 7.0%, and should be suitable for phylogenetic inferences within mosses. In addition, multiple indels were observed from the eight mtDNA regions (Fig. 1), of which two are significant in length: an indel located in atp9-trnI is 103 bp and an indel in rps7-atp6 is 304 bp long. These indels might also be important characters in phylogenetic studies.

Comparison of commonly used and newly designed mitochondrial markers. The calculation was made among three Funariaceae (Funaria hygrometrica, Physcomitrium pyriforme, and Physcomitrella patens) and one outgroup (Timmia megapolitana) taxa. Columns represent numbers of variable sites, and lines represent variation rate (number of variable sites/aligned length). Columns in darker gray are newly designed mtDNA markers. Numbers of indels detected in the eight mtDNA regions are shown above the columns.
Screening exemplars of other moss lineages yielded the highest success (33 out of 34) in atp1-trnW, more than 70% success in atp9-trnI, nad2-trnG, rpl2, and rpl5-16, and 40–60% success in nad1i287, rps3, and rps7-atp6 (Table 2). The low PCR success of some loci may be due to mutations at the primer annealing sites, genomic size change in certain taxa, or genomic rearrangement. However, as the strategy of this study is to investigate molecular markers at the family level (generic relationship in a family), those low-success markers are useful in select groups.
CONCLUSIONS
Results show that the newly developed mitochondrial markers presented similar or higher substitution rates within Funariaceae than previously identified mtDNA markers. These new markers should be useful in phylogenetic studies within Funariaceae and likely in other moss lineages because the mitochondrial genome contains independent information in comparison to the chloroplast and nuclear genomes. This study provides alternative choices for molecular marker selection in mosses. As more mitochondrial genomes are sequenced in the future, better universal primers and more mtDNA loci are expected to be developed for mosses.
Appendix 1
Sequences used in this study for primer design and variation comparison.a The newly generated sequences for this study are shown in bold.
A: Sequences generated for newly designed mitochondrial markersb | B: Sequences used for variation comparisonc |
Funaria hygrometrica Hedw.: JN089302, JN584631, JN584634, JN089132, JN089019, JN584637, JN584640, and JN089260; Physcomitrium pyriforme (Hedw.) Hampe: JN089319, JN584632, JN584635, JN089149, JN089035, JN584638, JN584641, and JN089275; Timmia megapolitana Hedw.: JN089323, JN584630, JN584633, JN089153, JN089039, JN584636, JN584639, and JN089279. | Funaria hygrometrica Hedw.: JN089174, DQ397164, JN088948, JN089062, JN088980, FJ870711, AY330463, AJ299534, Z98959, EU095279; Physcomitrium pyriforme (Hedw.) Hampe: JN089190, JN089233, JN088962, JN089079, JN088993, FJ870712, AY330461d, EU095312, AY312876d, EU095280; Timmia megapolitana Hedw.: JN089194, JN089237, JN088966, JN089083, AY908619, FJ870704, AY330475, AJ299532, AY312890, EU095276. |
- a All sequences for Physcomitrella patens (Hedw.) Bruch & Schimp. were extracted from chloroplast (GenBank NC_005087) or mitochondrial (GenBank NC_007945) genomes.
- b GenBank accession numbers for mitochondrial atp1-trnW, atp9-trnI, nad1i287, nad2-trnG, rpl2, rpl5-16, rps3, and rps7-atp6.
- c GenBank accession numbers for ITS, atpB-rbcL, trnL-F, psbA-trnH, rps4, cobi420, nad7 (nad7i140g2), nad2 (nad2i156g2), nad5 (nad5i753g1), and nad4-5.
- d Sequences were from Entosthodon laevis (Mitt.) Fife.