http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–7 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.880889
ORIGINAL ARTICLE
Simple sequence repeats in bryophyte mitochondrial genomes Chao-Xian Zhao1, Rui-Liang Zhu1, and Yang Liu2 Department of Biology, School of Life Sciences, East China Normal University, Shanghai, China and 2Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, USA
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Abstract
Keywords
Simple sequence repeats (SSRs) are thought to be common in plant mitochondrial (mt) genomes, but have yet to be fully described for bryophytes. We screened the mt genomes of two liverworts (Marchantia polymorpha and Pleurozia purpurea), two mosses (Physcomitrella patens and Anomodon rugelii) and two hornworts (Phaeoceros laevis and Nothoceros aenigmaticus), and detected 475 SSRs. Some SSRs are found conserved during the evolution, among which except one exists in both liverworts and mosses, all others are shared only by the two liverworts, mosses or hornworts. SSRs are known as DNA tracts having high mutation rates; however, according to our observations, they still can evolve slowly. The conservativeness of these SSRs suggests that they are under strong selection and could play critical roles in maintaining the gene functions.
Bryophytes, evolution, microsatellites, mitochondrial genome
Introduction Simple sequence repeats (SSRs, also called microsatellites) are DNA tracts of tandem-repeated motifs of 1–6 bases that are commonly observed in both prokaryotic and eukaryotic genomes. The type and density of SSRs usually vary among organisms and also among genomic regions (Lawson & Zhang, 2006; Rajendrakumar et al., 2007). SSRs can occur in both genic (exonic and intronic) or intergenic regions, and may play an important role in regulating gene expression and modulating gene function, and further result in potential physiologic or phenotypic consequences (Kashi & King, 2006; Subramanian et al., 2003; Thomas, 2005; Zhang et al., 2004). It has been observed that the distribution of SSRs is not random and their location tends to be conserved within the genome during the diversification of plants (Lawson & Zhang, 2006; Mayer et al., 2010). Due to having high mutation rates, SSRs have been utilized widely as phylogenetic makers in plant evolutionary studies. In bryophytes, they have been intensively used for population studies in Sphagnaceae (Karlin et al., 2008a,b; Shaw et al., 2008). Besides SSR itself, SSR flanking regions were also proposed being useful in inferring phylogenetic relationships among species (Nishikawa et al., 2005). Extant bryophytes, comprising liverworts (Marchantiophyta), mosses (Bryophyta) and hornworts (Anthocerotophyta), compose a grade of plants that mark the transition to land (Qiu et al., 2006). Comparison among species from each of the three major bryophyte lineages, i.e. Marchantia polymorpha, Physcomitrella patens and Nothoceros aenigmaticus indicated that the compositional and distribution pattern of mitochondrial (mt) SSRs among them are rather distinct (Kuntal & Sharma, 2011). Situations within each bryophyte group, however, are not clear. Up to now, seven bryophyte mt genomes have been fully sequenced. Correspondence: Rui-Liang Zhu, Department of Biology, School of Life Sciences, East China Normal University, Shanghai 200062, China. Tel: +86-21-62232458. Fax: +86-21-62233754. E-mail: [email protected]
History Received 14 May 2013 Revised 4 January 2014 Accepted 5 January 2014 Published online 3 February 2014
In the present study, we sampled six of them as exemplars, i.e. two liverworts: M. polymorpha (Oda et al., 1992) and Pleurozia purpurea (Wang et al., 2009), two mosses: P. patens (Terasawa et al., 2007) and Anomodon rugelii (Liu et al., 2011), and two hornworts: Phaeoceros laevis (Xue et al., 2010) and N. aenigmaticus (Li et al., 2009), and intensively described and compared the mt SSRs among these bryophytes. Bryophyte mt genomic structures as a whole are distinct among major lineages, but they are overall conserved within each clade (Liu et al., 2011, 2012). This article aims to compare the distribution of the mt SSRs within the bryophytes, test whether the distribution pattern of mt SSRs are correlated with the conservativeness of the mt genome structures, and explore whether any conserved mt SSRs exist among three major bryophyte lineages.
Materials and methods Sequence data The mt genome sequences were retrieved from National Center for Biotechnology Information’s Genome Data Bank (http:// www.ncbi.nlm.nih.gov/). Six bryophyte species studied in the present work include two liverworts: M. polymorpha (NC_001660.1), P. purpurea (NC_013444.1); two mosses: P. patens (NC_007945.1), A. rugelii (NC_016121.1) and two hornworts: P. laevis (NC_013765.1), N. aenigmaticus (NC_012651.1). Additionally, an alga, Chara vulgaris (NC_005255.1), and two vascular plants, Huperzia squarrosa (NC_017755.1) and Arabidopsis thaliana (NC_001284.2) were also included in the present study. Identification of SSRs We used the Perl script MISA (http://pgrc.ipk-gatersleben.de/ misa/misa.html) to search for mt SSRs. The definition of SSR varies (Gandhi et al., 2010; Kuntal et al., 2012; Lawson & Zhang, 2006; Rajendrakumar et al., 2008; Zhang et al., 2004) and we mainly adopted the concept defined by Gandhi et al. (2010),
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whereby the SSR is410 bp, with a minimum number of repeats of 10 for mononucleotides, 5 for dinucleotides, 4 for trinucleotides and 3 for tetranucleotides, pentanucleotides, and hexanucleotides. The distribution of SSRs in genic and intergenic regions was determined based on the information of genome sequence annotations available from the GenBank.
31 SSR loci in N. aenigmaticus, 18 genes with 24 SSR loci in P. laevis, 16 genes with 31 SSR loci in P. patens, 15 genes with 21 SSR loci in A. rugelii and 13 genes with 19 SSR loci in P. purpurea. No SSR matching the definition applied here was detected in any ribosomal RNA and transfer RNA genes among the six mt genomes.
Statistical analysis
SSRs in liverworts (M. polymorpha and P. purpurea)
The SSR densities were measured in base pairs per kilobase pairs (bp/kbp) and SSRs frequencies were defined as number of SSRs per kilobase pairs (no./kbp). If the distance between two SSRs is 510 bp, we treated them as one SSR locus. The upstream and downstream 50-bp flanking regions of each SSR locus were extracted using the software BioEdit (Hall, 1999), and the extractions were used for similarity comparison using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to evaluate the SSR conservativeness among species. AT content was computed for both SSR and its flanking regions, and the correlation among them was estimated by Pearson’s correlation coefficient in the R programming language (http://www.r-project.org/).
The M. polymorpha mt genome contains a total of 31 SSRs in genic regions and 57 SSRs in intergenic regions. The total length and mean length of SSRs are 1007 and 11.44 bp, respectively. We identified 20 SSRs in genic regions and 49 SSRs in intergenic regions respectively in the P. purpurea mt genome. The total length and mean length are 868 and 12.58 bp, respectively. The average SSR density of P. purpurea is less than M. polymorpha (5.15 versus 5.40 bp/kb). The most abundant SSR motif is mononucleotides: M. polymorpha (46.59%) and P. purpurea (43.48%). For a certain type of SSR, AT are the most frequent motif in both M. polymorpha (27.27%) and P. purpurea (26.09%), follow by the T repeats in M. polymorpha (19.32%) and P. purpurea (13.04%).
Results
SSRs in mosses (P. patens and A. rugelii)
SSRs in bryophyte mt genomes
Eighty-three SSRs (32 in genic regions and 51 in intergenic regions) occur in the P. patens mt genome, composing 0.90% of its total content. The total and mean length are 974 and 11.41 bp, respectively, and the total length of SSRs in genic regions is 356 bp compared with 591 bp in intergenic regions. Approximately 0.63% of the mt genome of A. rugelii is composed of SSRs. We detected 59 mt SSRs, comprising 23 in genic and 36 in intergenic regions. Mononucleotides are the most frequent repeat class in both P. patens (48.19% of total SSRs) and A. rugelii (52.54% of total SSRs).
SSRs are more common in intergenic regions than in genic regions in all six bryophyte mt genomes (Figure 1). The proportion of the six repeat classes (mono-, di-, tri-, tetra-, penta- and hexanucleotide) varies among different species (Figures 2 and 3). Some features, such as total number, total length, mean length, frequency, and density of mt SSRs are presented in Table 1. The four most abundant repeat motifs are always mononucleotides and/or dinucleotides (Table 2). However, tetranucleotides are one of the four most abundant repeat motifs in C. vulgaris and A. thaliana. Comparative analyses also display that SSRs distributed in nine mt genomes vary in frequency and density of SSRs. In total, 156 SSR loci from genic regions and 319 SSR loci from intergenic regions were identified. Twenty-one genes with a total of 30 SSR loci occurred in M. polymorpha, 18 genes with Figure 1. Variation of the number of each type of SSR (mono- to hexanucleotide) in genic and intergenic regions of six bryophyte mt genomes. The vertical axis shows the number of different types of SSRs of each species.
SSRs in hornworts (P. laevis and N. aenigmaticus) SSRs form 0.32% of the total mt genome in P. laevis with a frequency (no./kb) of 0.26. Twenty-six SSRs occur in genic regions and 29 SSRs in intergenic regions. About 0.63% of the mt genome of N. aenigmaticus is composed of SSRs. The whole
SSRs in bryophyte mitochondrial genomes
DOI: 10.3109/19401736.2014.880889
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Figure 2. Variation of density (bp/kbp) of each SSR type (mono- to hexanucleotide) in six bryophyte mt genomes. The vertical axis shows the percent of different types of SSRs of each species.
Figure 3. Variation of frequency (no./kbp) of each SSR type (mono- to hexanucleotide) in six bryophyte mt genomes. The vertical axis shows the percent of different types of SSRs of each species.
Table 1. Characteristics of SSRs in each mt genome. Characteristics Total no. Total length (bp) Mean length (bp) Frequency (no./kb) Density (bp/kb) Mt genome length (bp)
C. vulgaris M. polymorpha P. purpurea P. patens A. rugelii P. laevis N. aenigmaticus H. squarrosa A. thaliana 20 232 11.60 0.30 3.43 67,737
88 1007 11.44 0.47 5.40 186,609
69 868 12.58 0.41 5.15 168,526
83 947 11.41 0.79 8.99 105,340
genome contains a total of 69 SSRs (31 in genic and 38 in intergenic regions) with mean length and total length 16.75 and 1156 bp, respectively. Although the mt genome of N. aenigmaticus also has a high prevalence of mononucleotides repeats (46.38%), the percentage of tetranucleotide SSRs (41.82%) in P. laevis is higher than other repeat motifs, followed by
59 657 11.14 0.57 6.30 104,239
55 673 12.24 0.26 3.21 209,482
69 1156 16.75 0.37 6.25 184,908
193 2378 12.32 0.47 5.75 413,530
88 1053 11.97 0.24 2.87 366,924
mononucleotides (32.73%). Four tetranucleotide SSRs (TATG, GAAG, TAGA and TCTA) in N. aenigmaticus are longer than others, and the numbers of repeats run up to 31, 29, 20 and 14, respectively. The longest mononucleotide repeat is G in P. laevis, with as many as 37 repeats, the highest number of the six mt genomes investigated.
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Table 2. Four most abundant SSR motifs and their proportions (number of SSR motifs/total number) in each mt genome. Species C. vulgaris M. polymorpha P. purpurea P. patens A. rugelii P. laevis N. aenigmaticus H. squarrosa A. thaliana
First (%) Second (%) A (25.00) AT (27.27) AT (26.09) T (28.92) T (27.12) G (10.91) G (14.49) A (21.76) A (13.64)
Third (%)
Fourth (%)
T (15.00) AAAT (10.00) ATTG (10.00) T (19.32) A (18.18) TA (6.82) T (13.04) C (11.59) G (10.14) AT (28.92) A (16.87) TA (4.82) A (23.73) AT (20.34) TA (6.78) A (9.09) T (7.27) C (5.45) T (11.59) A (10.14) C (10.14) T (20.73) G (16.06) C (11.92) C (5.69) T (4.55) GAAA (4.55)
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Table 3. AT content of the entire mt genome of SSRs and of their flanking regions (50-bp upstream and downstream) in six species of bryophytes.
Species M. polymorpha P. purpurea A. rugelii P. patens N. aenigmaticus P. laevis
Mt genome AT (%)
SSR AT (%)
SSR flanking region AT (%)
57.60 54.60 58.80 59.40 54.00 55.40
86.20 73.50 93.76 94.61 53.03 45.77
61.82 56.62 60.78 61.08 51.75 51.60
AT content of SSRs and their flanking regions The AT content of mt SSRs varies among major bryophyte lineages. Mosses have higher AT content comparing to liverworts and hornworts (Table 3). Similarly, the AT content in SSR and the SSR flanking regions is also higher in mosses. Among these six bryophytes, the AT content differs little at the entire genome level (range 5%), more among SSR flanking regions (range ca. 10%), and even more among SSRs (range ca. 20%). The SSRs in hornworts have rather low AT content compared to those of mosses and liverworts. In addition, the AT content of SSR is correlated to that of their flanking regions (correlation coefficient: 0.97, p50.005), and to that of the whole-mt genome (correlation coefficient: 0.85, p50.05). Highly conserved SSR loci in bryophyte mt genomes Conserved SSR motifs, usually with the same repeat number, were found within all three bryophyte lineages. We identified three conserved SSRs in three hornwort mt genes, 10 in 9 liverwort mt genes and 14 in 10 moss mt genes. These SSRs distribute in both exonic and intronic regions (Table 4). The BLAST algorithm found a (CAAG)3 SSR from rps1-coding region conserved among liverworts and mosses (Figure 4). The flanking region of this SSR is highly conserved among the three liverworts, but with some mutations comparing to mosses, and this is the only one found conserved across bryophyte major lineages. Besides, conserved SSRs were also found from intergenic regions of liverwort or moss mt genomes. Most of these intergenic mt SSRs are dinucleotide AT/TA repeats, with similarity in flanking regions ranging from 87–98% (Table 5). Intergenic regions are incomparable between bryophytes and other plant lineages, as the mt genome gene orders are so different among them. However, conserved SSRs do have been found in exonic and intronic regions of algae, bryophytes and vascular plants. For example, the (T)10 SSR in liverwort nad1 genes occurs in Chara and Huperzia; the (G)10 SSR in moss mt intron atp9i95 also exist in Huperzia atp9 intron.
Discussion SSR distribution in genomes of plants varies greatly between different species (Kuntal & Sharma, 2011; Lawson & Zhang, 2006). SSR compositional patterns (representation of mono-, di-, tri-, tetra-, penta- and hexanucleotide) showed that liverworts and mosses are relatively more similar to each other than to hornworts. In addition, mosses or liverworts have proportionately more SSRs in intergenic regions than hornworts. The currently, most widely accepted early land plant phylogeny suggests that liverworts are sister to the rest of land plants, and mosses diverged from the common ancestor shared by hornworts and vascular plants (Qiu et al., 2006, 2007). Liverworts and mosses with a much more similar pattern, however, indicate that the evolutionary gap of the SSRs in mt genomes between liverworts and mosses is smaller comparing to hornwort possibly because of broader extinctions between mosses and hornworts, or different evolutionary mechanism acting on the mt genome evolution of hornworts. The AT content is an important genomic feature. The proportion of AT correlates with genome size and genome repeats in Anguilliformes (Ronchetti et al., 1995; Ussery & Hallin, 2004). Also, it has been shown that the selective pressure tends to be higher in AT-rich genomes in Mollicutes, which may have led to the more frequent occurrence of a UGA codon over UGG (Westberg et al., 2004). In liverwort and moss mt genomes, AT contents always increase from whole genome, to flanking regions, to SSRs, and are much higher in SSRs. According to the correlation analysis, the AT content in whole-mt genome, SSR and their flanking regions maybe present a positive correlation within bryophytes. However, the determination of this correlation still needs broader taxon sampling and further study. Although the specific function of SSRs within genomes remains unclear, some evidence suggests that they may be under evolutionary pressure and play a significant role in efficient adaptive evolution (King & Kashi, 2009; Li et al., 2004; Lin & Kussell, 2012). Considering the phylogenetic distance between the samples within each bryophyte lineage and the high conservativeness of some SSR loci detected in this study (Tables 4 and 5), we expect that these SSRs may be subjected to selective pressure and have significant function. It should be noted, in intergenic spacers, some conserved SSRs are right (10–30 bp) upstream on the functional genes, within a range of the presence of promoters (Table 5). These SSR nearby genes might be a part of promoters or enhancers. Also, comparative analysis of mt SSRs and their flanking regions shows conservation and variation in these DNA sequences, which could be used to generate new insights into phylogenetic relationships among species at least. The best example of this information utilized for phylogenetic relationships is the phylogenetic analysis of Oryza species (Nishikawa et al., 2005). In addition, these mt genomic studies allow the development of new SSR markers. Mt SSR primers have already been developed in N. aenigmaticus (Villarreal et al., 2012), Orthotrichum (Sawicki et al., 2012) and Cacao (Yang et al., 2011). These SSRs may be useful to assess the genetic diversity of species for population genetic studies, and even for reconstructing phylogenetic relationships among species (Arroyo-Garcia et al., 2002; Magain et al., 2010; Rajendrakumar et al., 2008). In summary, this study has identified the distribution and characteristic of SSRs in different bryophyte mt genomes, revealing a significant variation pattern, which seems roughly similar in the liverworts and mosses, while the hornworts are more different to one another than to either the liverworts or mosses. In addition, considering the presence of strong positive correlation between AT content and conserved SSR loci, SSRs
SSRs in bryophyte mitochondrial genomes
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Table 4. Conserved SSR motifs within mt protein-coding genes of liverworts, mosses and hornworts.
SSR
Species
Repeat number
Start location
End location
atp8
A
cox1
C
cox2
AGGA
nad1
T
nad2
AT
nad3
ATa
M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea
11 11 10 13 3 3 10 10 5 5 5 and 7 5 and 6 12 12 12 11 3 3 11 11
72,906 64,920 133,905 124,302 83,824 75,520 95,376 86,249 10,585 10,070 40,940 34,758 78,701 70,459 79,210 70,953 71,291 63,309 63,101 55,566
72,916 64,930 133,914 124,314 83,835 75,531 95,385 86,258 10,594 10,079 40,967 34,783 78,712 70,470 79,233 70,974 71,302 63,320 63,111 55,576
A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens
10 11 11 10 3 3 10 11 4 4 3 3 5 7 10 10 10 10 3 3 10 11 10 10 10 10 5 6 and 5
100,701 101,355 103,691 104,813 15,538 15,928 65,014 65,053 66,183 66,228 72,049 72,199 45,223 44,583 47,343 46,546 47,454 46,657 91,081 91,623 92,128 92,663 98,795 99,595 93,493 94,028 30,040 29,777
100,710 101,365 103,701 104,822 15,549 15,939 65,023 65,063 66,194 66,239 72,060 72,210 45,232 44,596 47,352 46,555 47,463 46,666 91,092 91,634 92,137 92,673 98,804 99,604 93,502 94,037 30,049 29,807
5 5 12 10 3 3
172,257 43,907 168,663 36,131 92,756 160,686
172,266 43,916 168,674 36,140 92,767 160,697
Taxon
Gene
Liverwort
Moss
Hornwort
nad4L
T
nad4L
AT
rps1
CAAG
rps3
A
atp9
T
atp9
G
ccmC
TTTA
cox2
A
cox2
GAA
nad1
ATTT
nad5
AT
nad5
A
nad5
T
nad7
TAGA
rpl2
T
rpl6
A
rps19
A
sdh3
AT*
cob
AT
cox2
A
nad1
AAAT
N. aenigmaticus P. laevis N. aenigmaticus P. laevis N. aenigmaticus P. laevis
Similarityb (%)
Region
Directionc
90
Exonic
+
–
Intronic
94
Intronic
+
98
Exonic
+
97
Intronic
+
90
Intronic
+
88
Intronic
+
96
Intronic
+
99
Exonic
+
95
Exonic
+
97
Intronic
+
90
Intronic
+
96
Exonic
+
89
Intronic
+
97
Intronic
+
97
Exonic
+
–
Intronic
+
94
Intronic
+
93
Intronic
+
94
Exonic
+
92
Exonic
+
99
Exonic
+
96
Exonic
+
90
Intronic
97
Intronic
+
–
Intronic
+
98
Exonic
a
Compound SSR, ‘‘–’’ means similarity 580%. bThe proportion of identical bases in the aligned SSR flanking regions (50-bp upstream and downstream). cIn direction, ‘‘+’’ means direct and ‘‘ ’’ means inverted.
Figure 4. The BLAST alignment of the conserved SSR (CAAG)3 in rps1-coding regions among liverworts and mosses. The rectangle shows DNA sequence of SSR locus (CAAG)3. The circles show mutations in flanking regions.
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Table 5. Conserved SSR motifs within mt intergenic spacers of liverworts and mosses.
Taxon Liverwort
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End location
Similaritya (%) 98
SSR
Species
rps1-atp8
A
nad1-cob
AT
nad5-nad4
AT
M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea
10 10 5 5 6 6 4 4
72,386 64,399 95,704 86,577 6,421 5,945 172,666 156,472
72,395 64,408 95,713 86,586 6,432 5,956 172,677 156,483
5 5 5 5 6 6 7 and 10 7 and 12 3 3 5 5 6 6
33,614 33,220 104,813 59,847 59,020 55,677 54,742 58,153 57,330 54,914 54,000 28,989 28,760 99,373
33,623 33,229 104,822 59,856 59,029 55,688 54,753 58,182 57,361 54,925 54,011 28,998 28,769 99,384
Spacer
trnCgca-trnFgaa Moss
Start location
Repeat number
ATT
trnMugg-rrn5
AT
atp6-nad6
AT
trnRacg-trnEuuc
AT
rps7-atp6 nad2-trnGgcc
(AT) (T) ATAA
sdh4-sdh3
TA
rps13-rps11
TA
A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens
88 97 89 97 95 94 95 97 87 90
Distance to upstream gene (bp)
Distance to downstream gene (bp)
452 483 29 29 1285 878 590 581
10 11 6048 5768 36 37 1597 1531
116 116 1498 1493 132 132 1130 1258 2450 2444 488 488 561 561
990 520 1103 1109 561 543 165 164 455 434 31 26 249 237
a
The proportion of identical bases in the aligned SSR flanking regions (50-bp upstream and downstream).
may play a significant role in mt genome evolution and may have different levels of evolutionary importance in different species. Also, the results show that some of the conserved SSR loci have great potentiality for SSR markers that could be useful in phylogenetic studies and population genetics.
Acknowledgements We sincerely thank Bernard Goffinet (University of Connecticut) and Laura L. Forrest (Royal Botanic Garden Edinburgh, UK) for suggestions and linguistic correction of this article.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research was financially supported by the National Natural Science Foundation of China (31170190 and 31370238) and ‘‘Project 211’’ for the East China Normal University. Y.L. is supported by NSF grant (DEB-1240045 to Bernard Goffinet; UCONN) for the postdoctoral position.
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Rajendrakumar P, Biswal AK, Balachandran SM, Srinivasarao K, Sundaram RM. (2007). Simple sequence repeats in organellar genomes of rice: Frequency and distribution in genic and intergenic regions. Bioinformatics 23:1–4. Rajendrakumar P, Biswal AK, Balachandran SM, Sundaram RM. (2008). In silico analysis of microsatellites in organellar genomes of major cereals for understanding their phylogenetic relationships. In Silico Biol 8:87–104. Ronchetti E, Salvadori S, Deiana AM. (1995). Genome size and AT content in Anguilliformes. Eur J Histochem 39:259–64. Sawicki J, Kwasniewski M, Szczecinska M, Chwialkowska K, Milewicz M, Plasek V. (2012). Isolation and characterization of simple sequence repeats (SSR) markers from the moss genus Orthotrichum using a small throughput pyrosequencing machine. Int J Mol Sci 13:7586–93. Shaw AJ, Cao T, Wang LS, Flatberg KI, Flatberg B, Shaw B, Zhou P, et al. (2008). Genetic variation in three Chinese peat mosses (Sphagnum) based on microsatellite markers, with primer information and analysis of ascertainment bias. Bryologist 111: 271–81. Subramanian S, Madgula VM, George R, Kumar S, Pandit MW, Singh L. (2003). SSRD: Simple sequence repeats database of the human genome. Comp Funct Genomics 4:342–5. Terasawa K, Odahara M, Kabeya Y, Kikugawa T, Sekine Y, Fujiwara M, Sato N. (2007). The mitochondrial genome of the moss Physcomitrella patens sheds new light on mitochondrial evolution in land plants. Mol Biol Evol 24:699–709.
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Thomas EE. (2005). Short, local duplications in eukaryotic genomes. Curr Opin Genet Dev 15:640–4. Ussery DW, Hallin PF. (2004). Genome update: AT content in sequenced prokaryotic genomes. Microbiology 150:749–52. Villarreal JC, Forrest LL, McFarland K, Goffinet B. (2012). Chloroplast, mitochondrial, and nuclear microsatellites from the Southern Appalachian hornwort, Nothoceros aenigmaticus (Dendrocerotaceae). Am J Bot 99:e88–90. Wang B, Xue JY, Li L, Liu L, Qiu YL. (2009). The complete mitochondrial genome sequence of the liverwort Pleurozia purpurea reveals extremely conservative mitochondrial genome evolution in liverworts. Curr Genet 55:601–9. Westberg J, Persson A, Holmberg A, Goesmann A, Lundeberg J, Johansson KE, Pettersson B, Uhlen M. (2004). The genome sequence of Mgcoplasma mycoides subsp mycoides SC type strain PG1(T), the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res 14:221–7. Xue JY, Liu Y, Li L, Wang B, Qiu YL. (2010). The complete mitochondrial genome sequence of the hornwort Phaeoceros laevis: Retention of many ancient pseudogenes and conservative evolution of mitochondrial genomes in hornworts. Curr Genet 56:53–61. Yang JY, Motilal LA, Dempewolf H, Maharaj K, Cronk QCB. (2011). Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae. Am J Bot 98:E372–4. Zhang L, Yuan D, Yu S, Li Z, Cao Y, Miao Z, Qian H, Tang K. (2004). Preference of simple sequence repeats in coding and non-coding regions of Arabidopsis thaliana. Bioinformatics 20:1081–6.
ORIGINAL ARTICLE
Simple sequence repeats in bryophyte mitochondrial genomes Chao-Xian Zhao1, Rui-Liang Zhu1, and Yang Liu2 Department of Biology, School of Life Sciences, East China Normal University, Shanghai, China and 2Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, USA
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Abstract
Keywords
Simple sequence repeats (SSRs) are thought to be common in plant mitochondrial (mt) genomes, but have yet to be fully described for bryophytes. We screened the mt genomes of two liverworts (Marchantia polymorpha and Pleurozia purpurea), two mosses (Physcomitrella patens and Anomodon rugelii) and two hornworts (Phaeoceros laevis and Nothoceros aenigmaticus), and detected 475 SSRs. Some SSRs are found conserved during the evolution, among which except one exists in both liverworts and mosses, all others are shared only by the two liverworts, mosses or hornworts. SSRs are known as DNA tracts having high mutation rates; however, according to our observations, they still can evolve slowly. The conservativeness of these SSRs suggests that they are under strong selection and could play critical roles in maintaining the gene functions.
Bryophytes, evolution, microsatellites, mitochondrial genome
Introduction Simple sequence repeats (SSRs, also called microsatellites) are DNA tracts of tandem-repeated motifs of 1–6 bases that are commonly observed in both prokaryotic and eukaryotic genomes. The type and density of SSRs usually vary among organisms and also among genomic regions (Lawson & Zhang, 2006; Rajendrakumar et al., 2007). SSRs can occur in both genic (exonic and intronic) or intergenic regions, and may play an important role in regulating gene expression and modulating gene function, and further result in potential physiologic or phenotypic consequences (Kashi & King, 2006; Subramanian et al., 2003; Thomas, 2005; Zhang et al., 2004). It has been observed that the distribution of SSRs is not random and their location tends to be conserved within the genome during the diversification of plants (Lawson & Zhang, 2006; Mayer et al., 2010). Due to having high mutation rates, SSRs have been utilized widely as phylogenetic makers in plant evolutionary studies. In bryophytes, they have been intensively used for population studies in Sphagnaceae (Karlin et al., 2008a,b; Shaw et al., 2008). Besides SSR itself, SSR flanking regions were also proposed being useful in inferring phylogenetic relationships among species (Nishikawa et al., 2005). Extant bryophytes, comprising liverworts (Marchantiophyta), mosses (Bryophyta) and hornworts (Anthocerotophyta), compose a grade of plants that mark the transition to land (Qiu et al., 2006). Comparison among species from each of the three major bryophyte lineages, i.e. Marchantia polymorpha, Physcomitrella patens and Nothoceros aenigmaticus indicated that the compositional and distribution pattern of mitochondrial (mt) SSRs among them are rather distinct (Kuntal & Sharma, 2011). Situations within each bryophyte group, however, are not clear. Up to now, seven bryophyte mt genomes have been fully sequenced. Correspondence: Rui-Liang Zhu, Department of Biology, School of Life Sciences, East China Normal University, Shanghai 200062, China. Tel: +86-21-62232458. Fax: +86-21-62233754. E-mail: [email protected]
History Received 14 May 2013 Revised 4 January 2014 Accepted 5 January 2014 Published online 3 February 2014
In the present study, we sampled six of them as exemplars, i.e. two liverworts: M. polymorpha (Oda et al., 1992) and Pleurozia purpurea (Wang et al., 2009), two mosses: P. patens (Terasawa et al., 2007) and Anomodon rugelii (Liu et al., 2011), and two hornworts: Phaeoceros laevis (Xue et al., 2010) and N. aenigmaticus (Li et al., 2009), and intensively described and compared the mt SSRs among these bryophytes. Bryophyte mt genomic structures as a whole are distinct among major lineages, but they are overall conserved within each clade (Liu et al., 2011, 2012). This article aims to compare the distribution of the mt SSRs within the bryophytes, test whether the distribution pattern of mt SSRs are correlated with the conservativeness of the mt genome structures, and explore whether any conserved mt SSRs exist among three major bryophyte lineages.
Materials and methods Sequence data The mt genome sequences were retrieved from National Center for Biotechnology Information’s Genome Data Bank (http:// www.ncbi.nlm.nih.gov/). Six bryophyte species studied in the present work include two liverworts: M. polymorpha (NC_001660.1), P. purpurea (NC_013444.1); two mosses: P. patens (NC_007945.1), A. rugelii (NC_016121.1) and two hornworts: P. laevis (NC_013765.1), N. aenigmaticus (NC_012651.1). Additionally, an alga, Chara vulgaris (NC_005255.1), and two vascular plants, Huperzia squarrosa (NC_017755.1) and Arabidopsis thaliana (NC_001284.2) were also included in the present study. Identification of SSRs We used the Perl script MISA (http://pgrc.ipk-gatersleben.de/ misa/misa.html) to search for mt SSRs. The definition of SSR varies (Gandhi et al., 2010; Kuntal et al., 2012; Lawson & Zhang, 2006; Rajendrakumar et al., 2008; Zhang et al., 2004) and we mainly adopted the concept defined by Gandhi et al. (2010),
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whereby the SSR is410 bp, with a minimum number of repeats of 10 for mononucleotides, 5 for dinucleotides, 4 for trinucleotides and 3 for tetranucleotides, pentanucleotides, and hexanucleotides. The distribution of SSRs in genic and intergenic regions was determined based on the information of genome sequence annotations available from the GenBank.
31 SSR loci in N. aenigmaticus, 18 genes with 24 SSR loci in P. laevis, 16 genes with 31 SSR loci in P. patens, 15 genes with 21 SSR loci in A. rugelii and 13 genes with 19 SSR loci in P. purpurea. No SSR matching the definition applied here was detected in any ribosomal RNA and transfer RNA genes among the six mt genomes.
Statistical analysis
SSRs in liverworts (M. polymorpha and P. purpurea)
The SSR densities were measured in base pairs per kilobase pairs (bp/kbp) and SSRs frequencies were defined as number of SSRs per kilobase pairs (no./kbp). If the distance between two SSRs is 510 bp, we treated them as one SSR locus. The upstream and downstream 50-bp flanking regions of each SSR locus were extracted using the software BioEdit (Hall, 1999), and the extractions were used for similarity comparison using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to evaluate the SSR conservativeness among species. AT content was computed for both SSR and its flanking regions, and the correlation among them was estimated by Pearson’s correlation coefficient in the R programming language (http://www.r-project.org/).
The M. polymorpha mt genome contains a total of 31 SSRs in genic regions and 57 SSRs in intergenic regions. The total length and mean length of SSRs are 1007 and 11.44 bp, respectively. We identified 20 SSRs in genic regions and 49 SSRs in intergenic regions respectively in the P. purpurea mt genome. The total length and mean length are 868 and 12.58 bp, respectively. The average SSR density of P. purpurea is less than M. polymorpha (5.15 versus 5.40 bp/kb). The most abundant SSR motif is mononucleotides: M. polymorpha (46.59%) and P. purpurea (43.48%). For a certain type of SSR, AT are the most frequent motif in both M. polymorpha (27.27%) and P. purpurea (26.09%), follow by the T repeats in M. polymorpha (19.32%) and P. purpurea (13.04%).
Results
SSRs in mosses (P. patens and A. rugelii)
SSRs in bryophyte mt genomes
Eighty-three SSRs (32 in genic regions and 51 in intergenic regions) occur in the P. patens mt genome, composing 0.90% of its total content. The total and mean length are 974 and 11.41 bp, respectively, and the total length of SSRs in genic regions is 356 bp compared with 591 bp in intergenic regions. Approximately 0.63% of the mt genome of A. rugelii is composed of SSRs. We detected 59 mt SSRs, comprising 23 in genic and 36 in intergenic regions. Mononucleotides are the most frequent repeat class in both P. patens (48.19% of total SSRs) and A. rugelii (52.54% of total SSRs).
SSRs are more common in intergenic regions than in genic regions in all six bryophyte mt genomes (Figure 1). The proportion of the six repeat classes (mono-, di-, tri-, tetra-, penta- and hexanucleotide) varies among different species (Figures 2 and 3). Some features, such as total number, total length, mean length, frequency, and density of mt SSRs are presented in Table 1. The four most abundant repeat motifs are always mononucleotides and/or dinucleotides (Table 2). However, tetranucleotides are one of the four most abundant repeat motifs in C. vulgaris and A. thaliana. Comparative analyses also display that SSRs distributed in nine mt genomes vary in frequency and density of SSRs. In total, 156 SSR loci from genic regions and 319 SSR loci from intergenic regions were identified. Twenty-one genes with a total of 30 SSR loci occurred in M. polymorpha, 18 genes with Figure 1. Variation of the number of each type of SSR (mono- to hexanucleotide) in genic and intergenic regions of six bryophyte mt genomes. The vertical axis shows the number of different types of SSRs of each species.
SSRs in hornworts (P. laevis and N. aenigmaticus) SSRs form 0.32% of the total mt genome in P. laevis with a frequency (no./kb) of 0.26. Twenty-six SSRs occur in genic regions and 29 SSRs in intergenic regions. About 0.63% of the mt genome of N. aenigmaticus is composed of SSRs. The whole
SSRs in bryophyte mitochondrial genomes
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Figure 2. Variation of density (bp/kbp) of each SSR type (mono- to hexanucleotide) in six bryophyte mt genomes. The vertical axis shows the percent of different types of SSRs of each species.
Figure 3. Variation of frequency (no./kbp) of each SSR type (mono- to hexanucleotide) in six bryophyte mt genomes. The vertical axis shows the percent of different types of SSRs of each species.
Table 1. Characteristics of SSRs in each mt genome. Characteristics Total no. Total length (bp) Mean length (bp) Frequency (no./kb) Density (bp/kb) Mt genome length (bp)
C. vulgaris M. polymorpha P. purpurea P. patens A. rugelii P. laevis N. aenigmaticus H. squarrosa A. thaliana 20 232 11.60 0.30 3.43 67,737
88 1007 11.44 0.47 5.40 186,609
69 868 12.58 0.41 5.15 168,526
83 947 11.41 0.79 8.99 105,340
genome contains a total of 69 SSRs (31 in genic and 38 in intergenic regions) with mean length and total length 16.75 and 1156 bp, respectively. Although the mt genome of N. aenigmaticus also has a high prevalence of mononucleotides repeats (46.38%), the percentage of tetranucleotide SSRs (41.82%) in P. laevis is higher than other repeat motifs, followed by
59 657 11.14 0.57 6.30 104,239
55 673 12.24 0.26 3.21 209,482
69 1156 16.75 0.37 6.25 184,908
193 2378 12.32 0.47 5.75 413,530
88 1053 11.97 0.24 2.87 366,924
mononucleotides (32.73%). Four tetranucleotide SSRs (TATG, GAAG, TAGA and TCTA) in N. aenigmaticus are longer than others, and the numbers of repeats run up to 31, 29, 20 and 14, respectively. The longest mononucleotide repeat is G in P. laevis, with as many as 37 repeats, the highest number of the six mt genomes investigated.
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Table 2. Four most abundant SSR motifs and their proportions (number of SSR motifs/total number) in each mt genome. Species C. vulgaris M. polymorpha P. purpurea P. patens A. rugelii P. laevis N. aenigmaticus H. squarrosa A. thaliana
First (%) Second (%) A (25.00) AT (27.27) AT (26.09) T (28.92) T (27.12) G (10.91) G (14.49) A (21.76) A (13.64)
Third (%)
Fourth (%)
T (15.00) AAAT (10.00) ATTG (10.00) T (19.32) A (18.18) TA (6.82) T (13.04) C (11.59) G (10.14) AT (28.92) A (16.87) TA (4.82) A (23.73) AT (20.34) TA (6.78) A (9.09) T (7.27) C (5.45) T (11.59) A (10.14) C (10.14) T (20.73) G (16.06) C (11.92) C (5.69) T (4.55) GAAA (4.55)
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Table 3. AT content of the entire mt genome of SSRs and of their flanking regions (50-bp upstream and downstream) in six species of bryophytes.
Species M. polymorpha P. purpurea A. rugelii P. patens N. aenigmaticus P. laevis
Mt genome AT (%)
SSR AT (%)
SSR flanking region AT (%)
57.60 54.60 58.80 59.40 54.00 55.40
86.20 73.50 93.76 94.61 53.03 45.77
61.82 56.62 60.78 61.08 51.75 51.60
AT content of SSRs and their flanking regions The AT content of mt SSRs varies among major bryophyte lineages. Mosses have higher AT content comparing to liverworts and hornworts (Table 3). Similarly, the AT content in SSR and the SSR flanking regions is also higher in mosses. Among these six bryophytes, the AT content differs little at the entire genome level (range 5%), more among SSR flanking regions (range ca. 10%), and even more among SSRs (range ca. 20%). The SSRs in hornworts have rather low AT content compared to those of mosses and liverworts. In addition, the AT content of SSR is correlated to that of their flanking regions (correlation coefficient: 0.97, p50.005), and to that of the whole-mt genome (correlation coefficient: 0.85, p50.05). Highly conserved SSR loci in bryophyte mt genomes Conserved SSR motifs, usually with the same repeat number, were found within all three bryophyte lineages. We identified three conserved SSRs in three hornwort mt genes, 10 in 9 liverwort mt genes and 14 in 10 moss mt genes. These SSRs distribute in both exonic and intronic regions (Table 4). The BLAST algorithm found a (CAAG)3 SSR from rps1-coding region conserved among liverworts and mosses (Figure 4). The flanking region of this SSR is highly conserved among the three liverworts, but with some mutations comparing to mosses, and this is the only one found conserved across bryophyte major lineages. Besides, conserved SSRs were also found from intergenic regions of liverwort or moss mt genomes. Most of these intergenic mt SSRs are dinucleotide AT/TA repeats, with similarity in flanking regions ranging from 87–98% (Table 5). Intergenic regions are incomparable between bryophytes and other plant lineages, as the mt genome gene orders are so different among them. However, conserved SSRs do have been found in exonic and intronic regions of algae, bryophytes and vascular plants. For example, the (T)10 SSR in liverwort nad1 genes occurs in Chara and Huperzia; the (G)10 SSR in moss mt intron atp9i95 also exist in Huperzia atp9 intron.
Discussion SSR distribution in genomes of plants varies greatly between different species (Kuntal & Sharma, 2011; Lawson & Zhang, 2006). SSR compositional patterns (representation of mono-, di-, tri-, tetra-, penta- and hexanucleotide) showed that liverworts and mosses are relatively more similar to each other than to hornworts. In addition, mosses or liverworts have proportionately more SSRs in intergenic regions than hornworts. The currently, most widely accepted early land plant phylogeny suggests that liverworts are sister to the rest of land plants, and mosses diverged from the common ancestor shared by hornworts and vascular plants (Qiu et al., 2006, 2007). Liverworts and mosses with a much more similar pattern, however, indicate that the evolutionary gap of the SSRs in mt genomes between liverworts and mosses is smaller comparing to hornwort possibly because of broader extinctions between mosses and hornworts, or different evolutionary mechanism acting on the mt genome evolution of hornworts. The AT content is an important genomic feature. The proportion of AT correlates with genome size and genome repeats in Anguilliformes (Ronchetti et al., 1995; Ussery & Hallin, 2004). Also, it has been shown that the selective pressure tends to be higher in AT-rich genomes in Mollicutes, which may have led to the more frequent occurrence of a UGA codon over UGG (Westberg et al., 2004). In liverwort and moss mt genomes, AT contents always increase from whole genome, to flanking regions, to SSRs, and are much higher in SSRs. According to the correlation analysis, the AT content in whole-mt genome, SSR and their flanking regions maybe present a positive correlation within bryophytes. However, the determination of this correlation still needs broader taxon sampling and further study. Although the specific function of SSRs within genomes remains unclear, some evidence suggests that they may be under evolutionary pressure and play a significant role in efficient adaptive evolution (King & Kashi, 2009; Li et al., 2004; Lin & Kussell, 2012). Considering the phylogenetic distance between the samples within each bryophyte lineage and the high conservativeness of some SSR loci detected in this study (Tables 4 and 5), we expect that these SSRs may be subjected to selective pressure and have significant function. It should be noted, in intergenic spacers, some conserved SSRs are right (10–30 bp) upstream on the functional genes, within a range of the presence of promoters (Table 5). These SSR nearby genes might be a part of promoters or enhancers. Also, comparative analysis of mt SSRs and their flanking regions shows conservation and variation in these DNA sequences, which could be used to generate new insights into phylogenetic relationships among species at least. The best example of this information utilized for phylogenetic relationships is the phylogenetic analysis of Oryza species (Nishikawa et al., 2005). In addition, these mt genomic studies allow the development of new SSR markers. Mt SSR primers have already been developed in N. aenigmaticus (Villarreal et al., 2012), Orthotrichum (Sawicki et al., 2012) and Cacao (Yang et al., 2011). These SSRs may be useful to assess the genetic diversity of species for population genetic studies, and even for reconstructing phylogenetic relationships among species (Arroyo-Garcia et al., 2002; Magain et al., 2010; Rajendrakumar et al., 2008). In summary, this study has identified the distribution and characteristic of SSRs in different bryophyte mt genomes, revealing a significant variation pattern, which seems roughly similar in the liverworts and mosses, while the hornworts are more different to one another than to either the liverworts or mosses. In addition, considering the presence of strong positive correlation between AT content and conserved SSR loci, SSRs
SSRs in bryophyte mitochondrial genomes
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Table 4. Conserved SSR motifs within mt protein-coding genes of liverworts, mosses and hornworts.
SSR
Species
Repeat number
Start location
End location
atp8
A
cox1
C
cox2
AGGA
nad1
T
nad2
AT
nad3
ATa
M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea
11 11 10 13 3 3 10 10 5 5 5 and 7 5 and 6 12 12 12 11 3 3 11 11
72,906 64,920 133,905 124,302 83,824 75,520 95,376 86,249 10,585 10,070 40,940 34,758 78,701 70,459 79,210 70,953 71,291 63,309 63,101 55,566
72,916 64,930 133,914 124,314 83,835 75,531 95,385 86,258 10,594 10,079 40,967 34,783 78,712 70,470 79,233 70,974 71,302 63,320 63,111 55,576
A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens
10 11 11 10 3 3 10 11 4 4 3 3 5 7 10 10 10 10 3 3 10 11 10 10 10 10 5 6 and 5
100,701 101,355 103,691 104,813 15,538 15,928 65,014 65,053 66,183 66,228 72,049 72,199 45,223 44,583 47,343 46,546 47,454 46,657 91,081 91,623 92,128 92,663 98,795 99,595 93,493 94,028 30,040 29,777
100,710 101,365 103,701 104,822 15,549 15,939 65,023 65,063 66,194 66,239 72,060 72,210 45,232 44,596 47,352 46,555 47,463 46,666 91,092 91,634 92,137 92,673 98,804 99,604 93,502 94,037 30,049 29,807
5 5 12 10 3 3
172,257 43,907 168,663 36,131 92,756 160,686
172,266 43,916 168,674 36,140 92,767 160,697
Taxon
Gene
Liverwort
Moss
Hornwort
nad4L
T
nad4L
AT
rps1
CAAG
rps3
A
atp9
T
atp9
G
ccmC
TTTA
cox2
A
cox2
GAA
nad1
ATTT
nad5
AT
nad5
A
nad5
T
nad7
TAGA
rpl2
T
rpl6
A
rps19
A
sdh3
AT*
cob
AT
cox2
A
nad1
AAAT
N. aenigmaticus P. laevis N. aenigmaticus P. laevis N. aenigmaticus P. laevis
Similarityb (%)
Region
Directionc
90
Exonic
+
–
Intronic
94
Intronic
+
98
Exonic
+
97
Intronic
+
90
Intronic
+
88
Intronic
+
96
Intronic
+
99
Exonic
+
95
Exonic
+
97
Intronic
+
90
Intronic
+
96
Exonic
+
89
Intronic
+
97
Intronic
+
97
Exonic
+
–
Intronic
+
94
Intronic
+
93
Intronic
+
94
Exonic
+
92
Exonic
+
99
Exonic
+
96
Exonic
+
90
Intronic
97
Intronic
+
–
Intronic
+
98
Exonic
a
Compound SSR, ‘‘–’’ means similarity 580%. bThe proportion of identical bases in the aligned SSR flanking regions (50-bp upstream and downstream). cIn direction, ‘‘+’’ means direct and ‘‘ ’’ means inverted.
Figure 4. The BLAST alignment of the conserved SSR (CAAG)3 in rps1-coding regions among liverworts and mosses. The rectangle shows DNA sequence of SSR locus (CAAG)3. The circles show mutations in flanking regions.
6
C.-X. Zhao et al.
Mitochondrial DNA, Early Online: 1–7
Table 5. Conserved SSR motifs within mt intergenic spacers of liverworts and mosses.
Taxon Liverwort
Mitochondrial DNA Downloaded from informahealthcare.com by Dalhousie University on 06/17/14 For personal use only.
End location
Similaritya (%) 98
SSR
Species
rps1-atp8
A
nad1-cob
AT
nad5-nad4
AT
M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea M. polymorpha P. purpurea
10 10 5 5 6 6 4 4
72,386 64,399 95,704 86,577 6,421 5,945 172,666 156,472
72,395 64,408 95,713 86,586 6,432 5,956 172,677 156,483
5 5 5 5 6 6 7 and 10 7 and 12 3 3 5 5 6 6
33,614 33,220 104,813 59,847 59,020 55,677 54,742 58,153 57,330 54,914 54,000 28,989 28,760 99,373
33,623 33,229 104,822 59,856 59,029 55,688 54,753 58,182 57,361 54,925 54,011 28,998 28,769 99,384
Spacer
trnCgca-trnFgaa Moss
Start location
Repeat number
ATT
trnMugg-rrn5
AT
atp6-nad6
AT
trnRacg-trnEuuc
AT
rps7-atp6 nad2-trnGgcc
(AT) (T) ATAA
sdh4-sdh3
TA
rps13-rps11
TA
A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens A. rugelii P. patens
88 97 89 97 95 94 95 97 87 90
Distance to upstream gene (bp)
Distance to downstream gene (bp)
452 483 29 29 1285 878 590 581
10 11 6048 5768 36 37 1597 1531
116 116 1498 1493 132 132 1130 1258 2450 2444 488 488 561 561
990 520 1103 1109 561 543 165 164 455 434 31 26 249 237
a
The proportion of identical bases in the aligned SSR flanking regions (50-bp upstream and downstream).
may play a significant role in mt genome evolution and may have different levels of evolutionary importance in different species. Also, the results show that some of the conserved SSR loci have great potentiality for SSR markers that could be useful in phylogenetic studies and population genetics.
Acknowledgements We sincerely thank Bernard Goffinet (University of Connecticut) and Laura L. Forrest (Royal Botanic Garden Edinburgh, UK) for suggestions and linguistic correction of this article.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research was financially supported by the National Natural Science Foundation of China (31170190 and 31370238) and ‘‘Project 211’’ for the East China Normal University. Y.L. is supported by NSF grant (DEB-1240045 to Bernard Goffinet; UCONN) for the postdoctoral position.
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