1 running head: plastid genotyping in cms-t maize...2015/09/02 · 75 plastids, and tested the...
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Running Head: Plastid Genotyping in cms-T Maize 1
Corresponding Author: 2
Pal Maliga, Ph.D. 3
Waksman Institute of Microbiology 4
Rutgers University 5
190 Frelinghuysen Road 6
Piscataway, NJ 08854-8020 7
Office: +1-848-445-5329 8
Fax: +1-732-445-3143 9
Mobile: +1-732-763-1333 10
Email: [email protected] 11
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Research Area: Genes, Development and Evolution 13
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Plant Physiology Preview. Published on September 2, 2015, as DOI:10.1104/pp.15.01147
Copyright 2015 by the American Society of Plant Biologists
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Plastid Genotyping Reveals Uniformity of cms-T Maize Cytoplasms 15
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Massimo Bosacchi†, Csanad Gurdon†, and Pal Maliga 17
Waksman Institute of Microbiology 18
Rutgers, The State University of New Jersey 19
190 Frelinghuysen Road 20
Piscataway, NJ 08854-8020 21
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† These authors contributed equally to this work. 23
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Summary: Genotyping reveals uniformity of plastids in independently isolated cms-T 25
cytoplasms in maize, indicating a single origin and strict maternal co-transmission of plastids and 26
mitochondria to progeny. 27
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Financial sources: M.B. is the recipient of a Charles and Joanna Busch Predoctoral Fellowship 47
from the Waksman Institute of Microbiology, Rutgers University; C.G. was supported by a 48
teaching assistantship from the Division of Life Sciences, Rutgers University. 49
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Corresponding author: Pal Maliga 51
Corresponding author email address: [email protected] 52
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The author(s) responsible for distribution of materials integral to the findings presented in this 54
article in accordance with the Journal policy described in the Instructions for Authors 55
(http://www.plantphysiol.org) is: Pal Maliga [email protected] 56
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Author contribution: P.M. designed and supervised the project; C.G and M.B. assembled and 58
annotated the plastid genomes, M.B. performed molecular experiments, M.B, C.G. and P.M. 59
analyzed data, M.B., C.G. and P.M. wrote the article. 60
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ABSTRACT 63
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Cytoplasmic male sterile (CMS) lines in maize have been classified by their response to specific 65
restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome 66
representing each of the CMS cytotypes has been sequenced and male sterility in the cms-S and 67
cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid 68
genotyping, we sequenced the plastid genomes (ptDNA) of three fertile maize lines (B37, B73, 69
A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the 70
plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid 71
genomes are conserved, differing only by 0-3 single nucleotide polymorphisms (SNPs) in coding 72
regions and 8-22 SNPs and 10-21 short insertions/deletions in noncoding regions. To gain insight 73
into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T 74
plastids, and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS 75
and T male sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have 76
the same three diagnostic plastid SNPs indicating a single origin and maternal co-transmission of 77
the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen 78
transmission of organelles or multiple horizontal gene transfer events as the source of the urf13-T 79
gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of evolutionary 80
relationships of cytoplasms in cultivated maize. 81
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Cytoplasmic Male Sterility (CMS) has been described in many flowering plant species, 85
and is linked to mitochondrial genes encoding toxic proteins. CMS proteins are typically 86
encoded by a chimeric mitochondrial gene assembled from rearranged mitochondrial DNA 87
sequences, and contain a hydrophobic, membrane-spanning domain (Hanson and Bentolila, 88
2004; Chase, 2007; Carlsson et al., 2008; Kubo and Newton, 2008; Chen and Liu, 2013). CMS 89
cytoplasms in maize are well characterized. 38 sources of cytoplasmic male sterility have been 90
examined for fertility restoration in 28 inbred backgrounds and classified by their response to 91
specific restorer genes into the cms-C, cms-S, and cms-T group. The cms-T group is comprised 92
of the earlier identified HA, I, Q, RS and T cytoplasms, which all respond to the same fertility 93
restorers (Rf1, Rf2). Plants with the cms-T cytotype are susceptible to Bipolaris 94
(Helminthosporium) maydis race T, a fungal pathogen that causes Southern Corn Leaf Blight 95
(Beckett, 1971; Gracen and Grogan, 1974). A distinct mitochondrial genome representing each 96
of the CMS cytotypes has been sequenced (Allen et al., 2007) with male sterility linked to the 97
urf13-T gene in cms-T (Dewey et al., 1987) and the cotranscribed orf355/orf77 in cms-S (Zabala 98
et al., 1997). The 13-kd maize mitochondrial protein encoded by the urf13-T gene was shown to 99
confer sensitivity to the T-toxin produced by Bipolaris maydis in E. coli, firmly establishing the 100
linkage between the T-type cytoplasmic male sterility and T-toxin sensitivity (Dewey et al., 101
1988). A collection of CMS lines is searchable at the Maize Genetics and Genomics Database 102
(MaizeGDB) website (Sen et al., 2009) and seed may be obtained upon request. We used this 103
resource to learn whether or not the independently isolated cms-T cytoplasms in cultivated maize 104
are related. In maize, plastids and mitochondria are transmitted to the seed progeny from the 105
maternal parent (Conde et al., 1979). However, maize yields hybrids with relatively distant wild 106
relatives such as Z. luxurians, Z. diploperennis and Z. perennis (Allen, 2005), and when doing 107
so, it is possible that the mode of organelle inheritance may change to biparental with an 108
increased frequency of organelle “leakage” via pollen. In an extreme case of an inter-species 109
hybrid, a shift from maternal to paternal inheritance was documented (Hansen et al., 2007). 110
Lineages arising outside a strict maternal mode of organelle inheritance in the cms-T cytoplasm 111
can be reconstructed by analyzing plastid types in the cms-T collection. This necessitated a 112
search for plastid markers. 113
Earlier work yielded very few markers that would be useful for plastid genotyping in 114
cultivated maize (Pring and Levings, 1978). Complete plastid genome sequences are convenient 115
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sources of ptDNA markers. Prior to our study, the only annotated maize ptDNA sequence in 116
GenBank (NC_001666) (Maier et al., 1995) was assembled from sequencing clones of two 117
maize hybrids (see Discussion). To provide markers specific to the lines used in this study, we 118
sequenced the plastid genomes of three fertile lines: A188 representing cytotype NA and B37 119
representing cytotype NB (Clifton et al., 2004; Allen et al., 2007). The third fertile line, B73, was 120
chosen because its nuclear genome has been sequenced (Schnable et al., 2009). We also 121
sequenced the plastid genome of cms-C, cms-S and cms-T lines in the B37 nuclear background 122
(B37C, B37S, B37T lines), in which the mitochondrial genome sequence has been determined 123
(Clifton et al., 2004; Allen et al., 2007). The maize lines with sequenced plastid and 124
mitochondrial genomes are listed in Table I. 125
An alignment of the completed plastid genome sequences facilitated the identification of 126
three SNPs that are unique to the cms-T plastid haplotype. We report here that each of the tested 127
27 cms-T accessions has the same three diagnostic plastid SNPs indicating a single origin. Our 128
data exclude exceptional transmission of organelles by pollen or independent horizontal transfer 129
of the urf13-T gene to fertile mitochondrial genomes during domestication as the source of 130
urf13-T gene in the cms-T cytoplasm. 131
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RESULTS 135
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Plastid Genomes of Fertile Maize Lines 137
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Biotechnological applications require a maize line amenable to plant regeneration. A188 is such 139
a line because it maintains its potential for plant regeneration from cultured cells over an 140
extended period of time. Sustained regeneration potential is linked to a morphology known as 141
Type II callus, which is friable and embryogenic (Armstrong and Green, 1985). The Hi-II maize 142
line was developed to combine the sustained regeneration potential of the A188 line with the 143
superior agronomic performance of B73. B73 is an important breeding line with poor tissue 144
culture regeneration potential. The two lines were crossed using A188 as the maternal parent and 145
the segregating progeny selected over several seed generations for tissue culture response. The 146
resulting A and B maize lines are crossed to provide highly regenerable Hi-II immature embryos 147
for transformation (Armstrong et al., 1991). We sequenced total cellular DNA isolated from the 148
A188 line, the Hi-II A and B lines and B73. The plastid genomes of the Hi-II A and B lines are 149
identical to the A188 line (KF241980), as expected. The B73 plastid genome (KF241981) is 150
somewhat larger (140,447 nt as compared to 140,437 nt) (Fig. 1), and differs from A188 by 17 151
SNPs and 11 indels, which are one or two nucleotide differences in length (Fig. 1, Table II). Our 152
B73 ptDNA sequence (KF241981) is 8 nt longer than the non-annotated B73 ptDNA sequence in 153
GenBank (AY928077). This discrepancy is due to differences in the length of mononucleotide 154
repeats that were verified in our genomes by direct sequencing of PCR amplicons. 155
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Diversity of Plastid Genomes of CMS Maize Lines 157
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The mitochondrial genomes of the fertile B37 maize (referred to as wild-type or normal 159
NB)(Clifton et al., 2004) and the three main CMS types have been sequenced in the B37 nuclear 160
background (Allen et al., 2007). Therefore, we decided to sequence the ptDNA of the fertile B37 161
(B37N) line and the three CMS cytoplasmic substitution lines. The ptDNA sequences of the 162
B37N (KP966114) and the B73 (KF241981) lines are identical. This is not surprising, since these 163
particular B lines may share a common ancestry. Both lines were developed from different 164
cycles of recurrent selection on Iowa Stiff Stalk Synthetic at Iowa State University. The plastid 165
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166 genome of the cytoplasmic male sterile B37 cms-C, B37 cms-S and B37 cms-T lines differ from 167
their fertile counterpart by 25/18, 21/20 and 17/18 nucleotide substitutions/indels, respectively. 168
Most of the SNPs and all of the indels are in non-coding, intergenic regions, Relative to B37N, 169
four genes carry nucleotide substitutions in their coding regions. Two of these substitutions 170
result in amino acid changes: in the rpoC2 gene of all lines and in the infA reading frames of 171
cms-C and cms-S. The remaining two polymorphisms (in the rpl36 gene of cms-C and the psbD 172
gene of cms-S) are silent. The map positions of all SNPs and substantial insertions/deletions, 173
which may be useful as markers, are shown in Fig. 1. A complete listing of ptDNA 174
polymorphisms is in Table II. 175
The ancestral state of the polymorphic nucleotides could be determined by comparing 176
them to sugarcane (AE009947), the closest maize relative with an available plastid sequence. 177
The ancestors of maize diverged 11.9 million years ago from the lineage leading to sugarcane 178
and sorghum (Swigonova et al., 2004). With one exception, sugarcane ptDNA carries one of two 179
maize alleles for every SNP in maize, the allele we consider the ancestral state. The exception is 180
at nucleotide position 20,313 (Table II), where sugarcane carries a C, while maize lines harbor an 181
A or G. The ancestral state was determined to be A after comparison of this region with publicly 182
available plastid sequences. 183
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We aligned the 1995 maize ptDNA (NC_001666) with the B37 NB ptDNA (KP966115). 184
The two ptDNAs differ by 235 SNPs and 144 indels. This deviation is much more extensive than 185
variation among the newly sequenced genomes, which differ from B37N only by 0-3 SNPs in 186
coding regions, and 8-22 SNPs and 11-21 short indels in noncoding regions. We also compared 187
the 1995 sequence (NC_001666) to the ptDNA sequence of sugarcane (AE009947) and sorghum 188
(NC_008602), the two closest relatives of maize with a sequenced chloroplast genome. The 189
overwhelming majority of SNPs in the 1995 sequence (NC_001666) are unique to it, while 190
sorghum, sugarcane and the six maize plastid genomes reported here carry the same nucleotide. 191
We therefore believe that most of the differences in the 1995 sequence are due to sequencing 192
errors inherent to the technology available at the time. With the exception of one locus (Tillich et 193
al., 2001), the sequence has not been updated since the original submission. 194
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Genotyping CMS-T Plastid Genomes 197
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CMS lines were collected independently from multiple sources and subsequently classified by 199
their response to restorers of male fertility. Five different groups of cms-T isolates derived from 200
different maize varieties: HA (Hasting’s Yellow variety), Q (uncertain, perhaps Mo988), T 201
(Texas sterile cytoplasm or cms-T, derived from Golden June variety), I (derived from a line 202
carrying the iojap gene) and RS (unknown) (Beckett, 1971; Gracen and Grogan, 1974). Male 203
sterility in the cms-T lines is conferred by urf13-T, a chimeric mitochondrial gene which is toxic 204
to plant cells, and whose effect is masked at the post-transcriptional level by certain fertility 205
restorer genes (Dewey et al., 1987). Cytotype-specific ptDNA markers, based on our sequence 206
information, allowed us to test whether or not all T-cytotype lines carry the same ptDNA. We 207
acquired 27 seed accessions representing the five different groups of origin, and tested them for 208
the three cms-T specific ptDNA polymorphisms (at positions 6422, 6423, 66839) and three 209
polymorphisms shared only with the A188 ptDNA (at positions 7423, 52307, 61312) (Table II). 210
All 27 seed accessions listed in Supplementary Table I carried the six ptDNA polymorphisms 211
characteristic of cms-T plastids. 212
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DISCUSSION 216
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Sequencing Maize Plastid Genomes Using Total Cellular DNA 218
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Plastid genome assembly from total cellular DNA sequence reads has become the predominant 220
method to obtain complete chloroplast genomes (Nock et al., 2011). The feasibility of this 221
approach is based on the relatively high number of plastid genome copies compared to the 222
mitochondrial and nuclear genomes per cell. Estimates of plastid genomes per leaf cell range 223
from 700 to 1,400 (Golczyk et al., 2014) and 3,000 to 4,000 copies (Ma and Li, 2015) in maize. 224
The number of maize mitochondrial DNA copies are 30 to 110 in young leaf cells (Ma and Li, 225
2015). Plastid DNA fragments are also present in the other genetic compartments: 7 to 10 226
genome equivalents in the maize nuclear genome (Roark et al., 2010; Yoshida et al., 2014) and 227
17- to 29-kb of the 140-kb ptDNA in the mitochondrial genome (Allen et al., 2007). PtDNA in 228
the heterologous compartments accumulates mutations over time. The ptDNA sequence is a 229
majority consensus of thousands of sequence reads, thus low coverage sequences derived from 230
the ptDNA copies integrated in the nuclear and mitochondrial genomes are excluded from the 231
majority consensus. The relatively modest Illumina MySeq V3 platform we use today generates 232
25 million 2x300 nucleotide long reads generating ~15 GB of useful data that is sufficient to 233
assemble multiple plastid genomes in a single run. De novo assembly of paired-end sequence 234
reads yielded complete plastid genomes with ambiguity only about the length of mononucleotide 235
runs. These ambiguities were eliminated by Sanger sequencing of PCR amplicons. Additionally, 236
we confirmed each polymorphism in the assembled plastid genomes by Sanger sequencing, as 237
we believe that error-free chloroplast sequences are necessary for phylogenetic comparisons. 238
The 1995 maize plastid genome sequence (NC_001666) was obtained using dideoxy 239
chain termination sequencing of ptDNA libraries. This sequence data was compared to the rice 240
and wheat chloroplast genomes in a 2002 evolutionary study, which identified five genes with a 241
higher gene divergence in maize than wheat or rice (Matsuoka et al., 2002). Four of the maize 242
genes (ndhK, psbD, rrn16, rrn23) harbor 35 SNPs in the 1995 maize sequence NC_001666, 243
which are absent in the six maize ptDNA sequences we report here. We conclude that these 244
polymorphisms are most likely sequencing errors; therefore the rate of evolution of the four 245
maize genes may not be as rapid as assumed (Matsuoka et al., 2002). 246
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247
248
Plastid Genotyping Markers 249
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The plastid genotyping markers reported here are useful for quick classification of plastid types. 251
Identical plastid genomes in the B37 and B73 lines reflect the fact that these inbreds, both 252
developed at Iowa State University, share a common lineage. However, not all B lines 253
necessarily have identical cytoplasms. The B designation reflects the location of the breeding 254
program and has no biological significance. The NA (A188; a line developed at the University of 255
Minnesota) and NB (B37, B73) fertile lines differ in the structure of their mitochondrial 256
genomes, and have a total of 17 distinguishing SNPs between their plastid genomes. The plastid 257
genome of cms-C, cms-S and cms-T lines differ from the B37/B73 fertile lines by 25/18, 21/20 258
and 17/18 nucleotide substitutions/indels, respectively. The number of plastid polymorphic sites 259
in the maize cultivars reported here is somewhat lower than between indica and japonica rice (72 260
SNPs, 27 indels (Tang et al., 2004), or between upland and lowland switchgrass (116 SNPs, 46 261
indels) (Young et al., 2011). 262
There are substantial differences between our maize plastid genomes sequences and the 263
only one previously available in GenBank (NC_001666). Relative to our B37/B73 sequence, 264
NC_001666 has 235 SNPs, of which 73 are in coding regions. These would cause 37 amino acid 265
changes in 15 genes. In addition, the two ptDNAs differ by 144 indels. However, we believe that 266
a significant fraction of differences are due to sequencing errors in the original study. The first 267
maize plastid genome sequence was reported relatively early, in a pioneering paper on plastid 268
RNA editing (Maier et al., 1995). The somewhat older tobacco ptDNA sequence published in 269
1986 (Shinozaki et al., 1986) has been updated twice by the original research group. In the 270
process the plastid genome of Nicotiana tabacum cv. Bright Yellow 4 has grown in size, first to 271
155,939 bp from the original 155,844 bp, and more recently to the current 155,943 bp. The 272
maize ptDNA sequence had only one minor update since originally published (Tillich et al., 273
2001). Although genotype information was not given in the original publication, we were able to 274
reconstruct which maize lines were used to obtain the original deposited sequence. We know 275
from the PhD thesis of Elisabeth Fritzsche (Fritzsche, 1988) cited in (Maier et al., 1995) that the 276
source of plastid DNA was Inrakorn or INRA 258, a four-way hybrid (F115-W33xF7-EP1; 277
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MaizeGDB) cited in ref. (Chaubet et al., 1989). The plastid rRNA region in GenBank Accession 278
NC_001666 was obtained by sequencing clone pZmc134 obtained from the Bogorad laboratory 279
(Edwards and Kossel, 1981). This fact was pointed out in a personal note to P.M. in a document 280
that accompanied the maize ptDNA library. The pZmc134 plasmid was obtained by cloning 281
ptDNA fragments from the maize hybrid WFGTMSxBS7 (Bedbrook et al., 1977). Thus, 282
GenBank Accession NC_001666 is a compilation of sequence obtained from two different maize 283
lines, neither of which is used in research or grown commercially today. The sequencing errors 284
do not affect the conclusions of the original publication, which is the first genome-wide study of 285
plastid organellar editing and is a classic in the field. 286
287
Phylogenetic Tree of Plastid Genomes in Maize 288
289
Plastid and mitochondrial DNA sequence variation in species and subspecies of the genus Zea 290
has been studied to obtain information about the progenitors of cultivated maize (Timothy et al., 291
1979; Doebley et al., 1987; Doebley et al., 1987); reviewed in (Doebley, 2004). Without the 292
benefit of full sequence information, the RFLP analysis used at that time could distinguish only 293
the cms-S plastids from normal (fertile), cms-C and cms-T ptDNAs (Pring and Levings, 1978). 294
Phylogenetic relationships based on the mitochondrial genome sequences describe NA and NB 295
as being the most closely related mitochondrial genomes, followed by cms-C, cms-S and cms-T. 296
On the basis of their nucleotide divergence, cms-S and cms-T were suggested to be the earliest 297
diverged cytotypes. Cms-S was considered to be a relatively ancient cytoplasm, most likely 298
derived from Zea mays ssp. mexicana (Allen et al., 2007; Darracq et al., 2010). 299
Our ptDNA sequence data allows us to re-evaluate the taxonomic position of the cms-T 300
cytotype. We built phylogenetic trees from the chloroplast sequences of the same CMS and 301
fertile lines to find whether chloroplast data independently validate the mitochondrial phylogeny. 302
The sugarcane plastid genome (Saccharum ssp., AE009947) was used as outgroup, because 303
chloroplast sequences from wild Zea lines are lacking. The ancestor of sorghum and sugarcane 304
split from the progenitors of maize 11.9 million years ago (Swigonova et al., 2004) and 305
sugarcane and sorghum ancestors split 8-9 million years ago (Jannoo et al., 2007). Thus, 306
sorghum and sugarcane are sister clades equidistant from maize. Phylogenetic trees were built 307
from the single copy regions of ptDNAs, as there was only one polymorphism in the inverted 308
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repeats (see Table II). Using all data (Fig. 2) 309
resulted in the same tree as the tree based on SNPs only (not shown). As the maize plastid 310
genomes harbor few intra-species differences, branch lengths are very short, and bootstrap 311
support is low for some branches. However, we can conclude that cms-T is closest to the fertile 312
NA and NB plastid genomes, while the cms-S and cms-C ptDNAs are more ancient. The cms-S 313
plastid type shares an 83-nt insertion and a 5-nt deletion with sugarcane relative to B37 (Table 314
II). Its placement as part of a basal branch on the maize phylogenetic tree is in accord with the 315
phylogenetic placement of the cms-S mtDNA, the origins of which predate the domesticated 316
maize (Allen et al., 2007; Darracq et al., 2010). 317
318
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Single Origin of CMS-T Cytoplasm in Maize 320
321
Plastids and mitochondria in flowering plants may be inherited maternally, paternally or 322
biparentally (Mogensen, 1996; Nagata, 2010; Hagemann, 2013). In maize, both organelles are 323
assumed to follow a maternal mode of inheritance. Data supporting maternal inheritance of 324
maize plastids was obtained in reciprocal crosses of Zea mays and Zea perennis inter-species 325
hybrids by testing RFLPs in organellar DNAs (Conde et al., 1979). Evidence supporting 326
maternal inheritance of mitochondria came from studies of non-chromosomal stripe mutations 327
and cytoplasmic male sterility encoded in mitochondrial genes (Kubo and Newton, 2008). Our 328
objective was to test whether exceptional transmission of organelles by pollen, or independent 329
horizontal transfer of the urf13-T gene to fertile mitochondrial genomes could have contributed 330
to the evolution of cms-T cytoplasm during domestication. 331
One mechanism that could yield new combinations of plastids and mitochondria is the 332
exceptional pollen transmission of plastids, a well-documented process in species with a 333
maternal mode of plastid inheritance. Examples include the monocot species Setaria italica 334
(Wang et al., 2004) and the dicots tobacco (Avni and Edelman, 1991; Ruf et al., 2007; Svab and 335
Maliga, 2007), Petunia (Derepas and Dulieu, 1992) and Arabidopsis (Azhagiri and Maliga, 336
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2007). Another mechanism that could yield new combination of plastids and CMS mitochondria 337
is the horizontal gene transfer of mitochondrial DNA, an evolutionary mechanism described in 338
multiple species (Richardson and Palmer, 2007; Bock, 2010). 339
We tested six cms-T plastid markers to determine whether or not the different cms-T 340
lines have the same ptDNA. Three of the markers are unique to cms-T plastids and three are 341
shared only with NA plastids. We have found that, based on the six markers, all 27 cms-T 342
accessions have the same plastid type. This indicates that the cms-T cytotype originated from a 343
single event. Furthermore, it suggests that the cms-T plastids and mitochondria have always been 344
co-transmitted, with both organelles following a strict maternal mode of inheritance. These 345
findings exclude exceptional pollen transmission of plastids, or horizontal transfer of the urf13-T 346
gene, as mechanisms contributing to the evolution cms-T cytoplasm during domestication. 347
348
349
MATERIALS AND METHODS 350
351
Plant Lines 352
353
Seeds of lines B73, A188 and Hi-II A and B lines were obtained from Hugo Dooner, Rutgers 354
University, Piscataway, NJ. Kathleen Newton, University of Missouri, Columbia, MO, provided 355
the seeds of B37N, B37C, B37S, B37T. We searched The Maize Genomics Database 356
(MaizeGDB) (Lawrence et al., 2004; Sen et al., 2009) to identify available CMS-T accessions, 357
which were subsequently ordered from the Maize Genetics Cooperation Stock Center, Urbana, 358
IL. Plants were grown in soil in the greenhouse from seed. 359
360
Sequencing and Assembly of Plastid Genomes 361
362
Total cellular DNA was isolated from leaves using the CTAB method (Murray and Thompson, 363
1980). B73, A188, “A” line and “B” line DNA was sequenced on the SOLiD 5500XL platform 364
using 75 nt reads. The reads were mapped to the B73 ptDNA sequence (AY928077), one 365
inverted repeat removed, using the Burrows–Wheeler Alignment Tool reference-guided 366
assembly program, version 0.7.1 (Li and Durbin, 2009). De novo contigs were assembled from 367
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quality-filtered (90% of bases having a quality cutoff value of 20) and unfiltered reads using the 368
Velvet de novo assembly program, version 1.1 at hash length 67, with otherwise default settings 369
(Zerbino and Birney, 2008). B37N, B37C, B37S and B37T lines were sequenced on the Illumina 370
MiSeq platform using 300 nt paired-end reads and 600 nt insert paired-end libraries made from 371
total cellular DNA. De novo contigs were assembled using the ABySS program, version 1.3.7 at 372
default settings (Simpson et al., 2009). Sanger sequencing of PCR amplicons was used to 373
eliminate ambiguities and to confirm each ptDNA polymorphic site. 374
Fully assembled plastid genome sequences were annotated using DOGMA, a software 375
tool developed specifically for organellar genomes (Wyman et al., 2004). DOGMA output 376
(features table) was manually adjusted in Microsoft Excel to resolve intron/exon borders and 377
include stop codons in protein coding genes. Gene annotation was verified by comparison with 378
the Inrakorn ptDNA (NC_001666). Each fully assembled plastid genome sequence was 379
deposited as a FASTA file, along with corrected DOGMA features table into Sequin (NCBI), 380
which compiled sequence and annotation information into the correct format for submission to 381
GenBank. The ptDNA sequences have been deposited in GenBank: A188, KF241980; B73, 382
KF241981; B37N, KP966114; B37C, KP966115; B37S, KP966116; B37T, KP966117. Maps 383
were prepared using OrganellarGenomeDRAW (Lohse et al., 2013). 384
385
PCR Genotyping of CMS-T ptDNA 386
387
Seed of 27 cms-T accessions were provided by the Maize Genetics COOP Stock Center 388
(University of Illinois, Urbana/Champaign). Total cellular DNA was isolated from leaves of 10 389
day-old seedlings using the CTAB method (Murray and Thompson, 1980). PCR primers 390
(Supplemental Table II) were designed to amplify each of the six SNPs used as markers. PCR 391
products ranged from 335 bp to 465 bp in length. The amplicons were treated with ExoSAP-IT 392
(Affymetrix, Santa Clara, CA) and Sanger sequenced. Sequence reads were aligned using 393
SeqMan Pro (DNASTAR) software. We checked for cms-T polymorphisms at positions 6422, 394
6423, 7423, 52307, 61312 and 66839. 395
396
Phylogenetic Analysis 397
398
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17
The concatenated large and small single copy regions of the five maize chloroplast 399
sequences, and the single copy region of sugarcane (Saccharum hybrid cultivar SP-80-3280, 400
NCBI locus AE009947) were aligned by MUSCLE (Edgar, 2004), then the alignment was 401
manually checked and adjusted when necessary. Maximum Likelihood trees were assembled 402
from the alignment in MEGA6 (Tamura et al., 2013), using the Kimura 2-parameter model 403
(Kimura, 1980), allowing some sites to be evolutionarily invariable, with 1000X bootstrap 404
(Felsenstein, 1985). Initial trees for the heuristic search were obtained by applying Neighbor-Join 405
and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite 406
Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. 407
Trees were assembled with or without the sugarcane outgroup: using gaps (96,131 positions used 408
with or without sugarcane), and discarding gaps (94,406 positions with sugarcane, 94,880 409
positions without). 410
411
ACKNOWLEDGEMENTS 412
413
We thank Kathleen Newton for the B37N, B37C, B37S and B37T maize seed and discussions 414
about the CMS cytoplasms in maize. We also thank Hugo K. Dooner for discussions and 415
providing B73, A188 and Hi-II A and B maize seed. Gabor L. Igloi is acknowledged for helping 416
to uncover the origins of Inrakorn as the source of most chloroplast DNA in the 1995 maize 417
plastid genome sequence, and Mary Schaeffer for facilitating our inquiry. 418
419
420
421
422
423
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18
424
425
FIGURE LEGENDS 426
427
Figure 1. Linear map of maize plastid genomes, with SNPs and select large indels. Large Single 428
Copy Region (LSC), Small Single Copy Region (LSC) and Inverted Repeats (IRA and IRB) are 429
marked on top. In linear maps, unique regions are in black; repeated regions in green; red circles 430
represent intergenic SNPs; yellow circles represent SNPs in coding regions, with gene name and 431
amino acid change. Insertions are on top, deletions on bottom. SNPs and indels are represented 432
in relation to the B73 or B37 genomes, which are identical. 433
434
Figure 2. Phylogenetic relationship of maize plastid genomes. Bootstrap consensus tree of maize 435
plastid genomes and sugarcane as outgroup is shown. Branch distances are shown above and 436
bootstrap support below branches. The tree was obtained using concatenated single copy regions. 437
Note that the distances are short because the maize plastid genomes are conserved and bootstrap 438
support is low for the branches. 439
440
441
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19
442 443
444
Table I. Plastid and mitochondrial genomes of maize 445
446
Line Cytotype ptDNA Accession No.
Nucleotides ptDNA Ref.
mtDNA Accession No.
mtDNA Ref.
A188 NA KF241980 140,437 This
paper
DQ490952 (Allen et al.,
2007)
B73 NB KF241981 140,447 This
paper
B37N NB KP966114 140,447 This
paper
AY506529.1 (Clifton et
al., 2004)
B37C cms-C KP966115 140,457 This
paper
DQ645536 (Allen et al.,
2007)
B37S cms-S KP966116 140,534 This
paper
DQ490951 (Allen et al.,
2007)
B37T cms-T KP966117 140,479 This
paper
DQ490953 (Allen et al.,
2007)
447
448
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Table II. PtDNA polymorphisms to distinguish plastid types 449
Homologs in sugarcane (AE009947) are shown for comparison. Numbering refers to location in 450
the B37 plastid genome (KP966114). SNPs that differ from B37 are shown in bold; maize 451
polymorphisms that differ from the ancestral state in sugarcane are marked by stars. Amino acids 452
encoded by the codons that contain genic polymorphisms are shown in B37 and the polymorphic 453
line (B37N AA, SNP AA). 454
455
456
457
458
459
460
461
462
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21
463
464 465
466
Substitutions Location B73/B37 B37 cms-C B37 cms-S B37 cms-T A188 Sugarcane B37N AA SNP AA Region1338 intergenic *T* G G G G G3353 intergenic *A* G G G G G6422 intergenic C C C *G* C C6423 intergenic A A A *G* A A7423 intergenic C C C *A* *A* C7527 intergenic G G G G *T* G8695 intergenic C C *A* C C C9633 psbD C C *T* C C C F F12449 intergenic C C *A* C C C14856 intergenic *A* G G G G G16048 intergenic *C* T T T *C* T16107 intergenic T *G* T T T T16293 intergenic A A A A *C* A20313 intergenic A A *G* A A C20656 intergenic T T *G* T T T29991 rpoC2 *A* G G G G G31586 intergenic T T *G* T T T31652 intergenic T T *G* T T T33790 intergenic G *A* G G G G34115 intergenic *A* C *A* *A* *A* C35732 intergenic *A* T T T T T K R38167 intergenic *A* T *A* *A* *A* T44891 intergenic A *C* A A A A45973 intergenic T T *G* T T T49356 intergenic T *A* T T T T49532 intergenic T T *G* T T T52307 intergenic T T T *C* *C* T53045 intergenic G *A* G G G G53751 intergenic *A* T T T T T56123 intergenic G *A* G G G G61312 intergenic A A A *G* *G* A64854 intergenic *T* A A A A A66701 intergenic T *C* T T T T66839 intergenic T T T *C* T T66848 intergenic *T* G G G G G69228 intergenic C *A* C C C C69229 intergenic T *A* T T T T69230 intergenic T *G* T T T T69522 intergenic G G *T* G G G77919 rpl36 G *A* G G G G Y Y78382 infA G *T* *T* G G G N K78983 intergenic *T* A A A A A108040 intergenic A A A A *C* A108639 intergenic C C C C *T* T108873 intergenic *T* A A A A A
LSC
SSC
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467
468 469 470 471 472 473 474 475 476
InDels Location B73/B37 B37 cms-C B37 cms-S B37 cms-T A188 Sugarcane Region3543-3553 intergenic (A)11 (A)10 (A)12 (A)11 (A)11 (A)93755-3765 intergenic (A)11 (A)13 (A)13 (A)14 (A)9 (A)154097-4107 intergenic (A)11 (A)11 (A)11 (A)10 (A)11 (A)107424-7434 intergenic (A)11 (A)17 (A)15 (A)16 (A)9 (A)118366-8376 intergenic (A)11 (A)12 (A)11 (A)11 (A)11 AT(A)78711-8720 intergenic (T)11 (T)11 (T)11 (T)12 (T)11 (T)1111851-11852 intergenic 83bp ins 83bp ins12962-12973 intergenic (T)12 (T)17 (T)17 (T)16 (T)11 (T)1112987-12995 intergenic (A)9 (A)10 (A)9 (A)9 (A)9 (A)5T(A)816052-16065 intergenic (T)14 (T)13 (T)14 (T)14 (T)14 (T)1416145-16153 intergenic (A)9 (A)9 (A)9 (A)10 (A)9 (A)917177-17188 intergenic (G)12 (G)13 (G)13 (G)12 (G)12 A(G)820571-20579 intergenic 9bp del 53bp del20815-20825 intergenic (A)11 (A)9 (A)9 (A)9 (A)9 (A)831819-31824 intergenic (A)6 (A)6 (A)5 (A)6 (A)6 (A)635298-35305 intergenic (T)8 (T)9 (T)9 (T)9 (T)9 (T)1036534-36551 intergenic (A)13(T)2 (A)12(T)2 (A)13(T)2 (A)13(T)3 (A)13(T)2 (A)9(T)238107-38108 intergenic AT AT AT ATT AT AT38168-38175 intergenic (T)8 (T)9 (T)8 (T)8 (T)8 (T)1043710-43721 intergenic (T)12 (T)11 (T)11 (T)11 (T)12 (T)948257-48267 intergenic (A)11 (A)11 (A)10 (A)10 (A)10 (A)951065-51069 intergenic 5bp del 5bp del52733-52747 intergenic (T)15 (T)16 (T)15 (T)15 (T)15 (T)758311-58320 intergenic (A)10 (A)9 (A)9 (A)9 (A)9 (A)6G(A)259489-59499 intergenic (T)11 (T)11 (T)11 (T)10 (T)10 (T)1165014-65029 intergenic (T)16 (T)12 (T)17 (T)19 (T)16 (T)3C(T)665809-65817 intergenic (T)9 (T)9 (T)10 (T)9 (T)9 (T)1065936-65946 intergenic (A)11 (A)12 (A)12 (A)13 (A)11 (A)967503-67513 intergenic (T)11 (T)11 (T)14 (T)11 (T)12 (T)1273336-73345 intergenic (A)10 (A)10 (A)12 (A)10 (A)10 (A)1177833-77834 intergenic 16bp ins ambigous 81829-81838 intergenic (T)10 (T)11 (T)11 (T)11 (T)10 (T)888310-88317 intergenic (A)8 (A)8 (A)8 (A)8 (A)7 (A)8 IRB134506-134513 intergenic (T)8 (T)8 (T)8 (T)8 (T)7 (T)8 IRA
LSC
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477 478
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