Sequencing and Annotation of the Chloroplast Dnas and Identification

737 ARTICLE

Sequencing and annotation of the chloroplast DNAs and identiﬁcation of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion Christopher von Kohn, Agnieszka Kiełkowska, and Michael J. Havey

Abstract: Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classiﬁed as N or S cytoplasmic using restriction fragment length polymorphisms. Al- though cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

Key words: Allium cepa, cytoplasmic male sterility, molecular markers.

Résumé : Le cytoplasme mâle stérile (S) de l’oignon provient de l’introgression d’un cytoplasme étranger dans l’antiquité et il est largement employé pour la production de semence hybride. En raison de la nature bisannuelle de l’oignon, un schéma classique de croisement nécessitera au moins 4 ans pour classiﬁer un cytoplasme comme étant du type S ou N (pour normal et mâle fertile). Des marqueurs moléculaires provenant des organites et permettant de distinguer les cytoplasmes N et S permettent de réduire

For personal use only. le temps requis pour classifier les cytoplasmes. Dans ce travail, les auteurs ont réalisé un séquençage complet des ADN chloroplastiques chez les oignons a` cytoplasme N et S; ils ont assemblé et annoté les génomes en plus d’identifier des polymorphismes qui distinguent ces cytoplasmes. La taille du génome chloroplastique (153 536 vs. 153 355 pb) et son contenu en GC (36,8 %) étaient très semblables chez les deux types de cytoplasmes, tel qu’attendu en raison de leur proximité phylogénétique. La différence de taille provenait principalement de petits indels dans les régions intergéniques et d’une délétion au sein du gène accD chez l’oignon a` cytoplasme N. La structure du génome chloroplastique était semblable a` celle retrouvée chez la plupart des plantes terrestres avec une grande et une petite région a` simple copie, lesquelles sont séparées d’une région répétée inversée. Les auteurs ont choisi vingt-huit polymorphismes mononucléotidiques, deux polymorphismes de site de restriction, et un indel répartis parmi les 20 gènes chloroplastiques dans une grande et une petite région a` simple copie. Ces marqueurs ont été validés au sein de diverses populations d’oignons déja` classifiées comme étant N ou S a` l’aide de polymorphismes de fragments de restriction. Bien que la stérilité mâle cytoplasmique soit vraisemblablement associée a` l’ADN mitochondrial, la transmission maternelle des ADN mitochondriaux et chloroplastiques rendent utiles des polymorphismes observés dans l’un ou l’autre génome pour la classification des cytoplasmes chez l’oignon et pour aider au développement de cultivars hybrides. [Traduit par la Rédaction]

Mots-clés : Allium cepa, stérilité mâle cytoplasmique, marqueurs moléculaires.

Genome Downloaded from www.nrcresearchpress.com by UNIV OF WISC MADISON on 02/04/14 Introduction vaceae (yucca and agave), among others (Pires et al. 2006). Basal to the core Asparagales are families such as the Iridaceae Onion (Allium cepa L.) is a member of the family Alliaceae in the (irises) and Orchidaceae (orchids). These phylogenetic relation- order Asparagales, a large monophyletic group within the mono- ships have been supported by sequencing of speciﬁc chlo- cots. Within the Asparagales, the Alliaceae has been assigned to roplast genes and intergenic spacers (Pires et al. 2006; APGIII the “core” Asparagales, a monophyletic group that also includes 2009), as well as next-generation sequencing of genomic DNAs the Asparagaceae (asparagus), Amaryllidaceae (amaryllids), Aga- (Steele et al. 2012).

Received 1 October 2013. Accepted 27 November 2013. Corresponding Editor: L. Bonen. C. von Kohn. Department of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706, USA. A. Kiełkowska. Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Kraków, 31-425 Kraków, Poland. M.J. Havey. USDA–ARS and Department of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706, USA. Corresponding author: Michael J. Havey (e-mail: [email protected]).

Genome 56: 737–742 (2013) dx.doi.org/10.1139/gen-2013-0182 Published at www.nrcresearchpress.com/gen on 28 November 2013. 738 Genome Vol. 56, 2013

Restriction enzyme analyses of the chloroplast DNA of onion Table 1. Origins of onion populations and their cytoplasms as de- revealed polymorphisms distinguishing normal (N) male-fertile termined by restriction fragment length polymorphisms (Havey cytoplasm from the primary source of cytoplasmic male sterility 1993) and single nucleotide polymorphisms (SNPs) in the chlo- (CMS) used to produce hybrid seed (Havey 1993, 2000). In the roplast DNAs of normal (N) male-fertile and male-sterile (S) cyto- 1920s, Jones and colleagues identified a single male-sterile onion plasms of onion. plant (13-53) in the open-pollinated population ‘Italian Red’ (Jones Accessiona Originb Cytoplasm and Emsweller 1937). This source of male-sterile (S) cytoplasm likely originates from an alien cytoplasm introgressed into onion 164807 India N 168960 Turkey N populations in antiquity (Havey 1993; Bark and Havey 1995). In a 168967 Turkey N classic publication, Jones and Clarke (1943) demonstrated that 174021 Turkey N male sterility results from the interaction of S cytoplasm and the 175571 Turkey N homozygous recessive genotype at one nuclear male-fertility res- 177247 Syria N toration (Ms) locus. A dominant allele at Ms in an S-cytoplasmic 179167 Iraq N plant results in male fertility. Plants possessing N cytoplasm are 181929 Syria N always male-fertile regardless of the genotype at Ms. 207456 Afganistan N Owing to the biennial generation time of onion, classical cross- 210994 Afganistan N ing takes from 4 to 8 years to establish the cytoplasm of individual 218059 Pakistan N onion plants (Havey 1993). Cytoplasmic determinations in onion 222228 Iran N have been greatly simplified by molecular markers in the mito- 222698 Iran N chondrial and chloroplast DNAs distinguishing N and S cyto- 222764 Iran N plasms (Havey 1993, 1995; Sato 1998; Engelke et al. 2003; Cho et al. 233187 Former Soviet Union N 2006; Kim et al. 2009). Although these molecular markers do not 239633 Iran N shorten the generation time of onion, they represent a more ju- 243008 India N dicious use of resources because only plants with known cyto- 247067 Italy N plasms are used for crossing (Havey 1995). In this research, we 251021 Afganistan N sequenced, assembled, and annotated the chloroplast DNAs from 251509 Iran N 262915 Former Soviet Union N N and S cytoplasms of onion; compared their chloroplast DNA 262920 Former Soviet Union N structure with those of eudicot tobacco and the monocots rice and 271309 India N orchid; and identified polymorphisms in chloroplast genes that 273210 Poland N distinguish these two cytoplasms. These cytoplasmic markers, 273626 Ethiopia N coupled with single nucleotide polymorphisms (SNPs) tightly 274781 India N linked to the nuclear Ms locus (Havey 2013), can be used to select 280554 Former Soviet Union N individual plants using high-throughput genotyping platforms to 288274 India N aid in the development of male-fertile maintainer (N msms) and 288692 Former Yugoslavia N male-sterile (S msms) lines for the production of hybrid onion seed. B1750A USDA S B1750B USDA N Materials and methods B1789B USDA N For personal use only. Chloroplast DNAs were purified using percoll gradients (Eubel B2117B USDA N et al. 2007) from inbred lines of N-cytoplasmic (MSU611B, source B2133B USDA N 4414) and S-cytoplasmic (MSU611-1A, source 3405) onions (both the W101B UW N gift of Seminis Seed Company, Woodland, Calif., USA), and pre- W404B UW N pared for sequencing using the 454 FLX platform and protocols W52B UW N MSU826B USDA N as recommended by the manufacturer (Roche, Branford, Conn., MSU8155B USDA N USA). The reads from one-quarter 454 plate each for N- and S- MSU611-1B USDA N chloroplast DNAs were assembled separately using the Newbler B1828B USDA N Assembler (Roche) version 2.5.3 and aligned to the plastid DNA B1794B USDA N of the orchid Oncidium ‘Gower Ramsey’ (GenBank accession MSU2399B USDA N NC_014056). Aligned reads were visualized using Tablet (Milne MSU611B Seminis N et al. 2013) and annotations completed using Dogma (Wyman MSU611-1A Seminis S et al. 2004). Regions with poor annotations or alignments, often aSix-digit numbers indicate plant introductions from the USDA germplasm due to homopolymer runs, were identified in Tablet, and oligonu- collections. cleotide primers were designed to amplify across the problematic bUSDA and UW are the United States Department of Agriculture and Univer-

Genome Downloaded from www.nrcresearchpress.com by UNIV OF WISC MADISON on 02/04/14 regions. Amplicons were TA cloned into the pGEM-T Easy vector sity of Wisconsin, respectively, and are publicly released inbreds of onion. Semi- using the manufacturer’s protocol (Promega, Madison, Wis., USA), nis (Woodland, Calif., USA) is a vegetable seed company. Sanger sequenced (Sambrook and Russell 1989), and aligned using Sequencer version 5.1 (Gene Codes, Ann Arbor, Mich., USA) to confirm the correct sequences. Differences between the onion Table 2. Sequencing and assembly results from 454 sequencing of chloroplast sequences and tobacco (NC_001879.2), rice (JN861110.1), chloroplast DNAs from normal (N) male-fertile and male-sterile (S) and the orchid Phalaenopsis (NC_007499.1) were visualized using cytoplasms of onion. mVISTA (Frazer et al. 2004). Cytoplasm SNPs were identified in the chloroplast DNAs of N and S cytoplasm by comparing aligned sequences in Tablet. Well-supported Characteristic NS SNPs were chosen by requiring at least eight reads across the Total number of reads 87 602 41 893 region carrying the SNP (mean number of reads were 40 and 67 for Total number of bases 34 959 395 16 706 873 N and S cytoplasm, respectively) and at least 80% of the reads Number of aligned reads 56 134 27 460 supporting a specific nucleotide in each cytoplasm. SNPs at the Number of aligned bases 22 692 594 11 059 609 ends of sequence reads or near homopolymers were avoided.

Published by NRC Research Press von Kohn et al. 739

Fig. 1. Annotation of the chloroplast DNAs of normal (N) male-fertile and male-sterile (S) cytoplasms of onion. LSC and SSC refer to the large and small single copy regions, respectively, and IRA and IRB are the two inverted repeats. Genes located on the inner side of the circle are transcribed in the opposite direction of those on the outside. Grey bars on the inside of circle indicate GC contents with the line representing 50%. Gene classes are indicated by boxes. For personal use only.

Sequences flanking SNPs in chloroplast genes in the large (LSC) Results and discussion and small single copy (SSC) regions were analyzed by the Prim- Genome Downloaded from www.nrcresearchpress.com by UNIV OF WISC MADISON on 02/04/14 erpicker software for the KASPar platform (LGC Genomics, Bev- Sequencing, assembly, and annotation of the chloroplast erly, Mass., USA) to identify those conducive for genotyping. DNAs of N- and S-cytoplasmic onions Oligonucleotide primers were synthesized and the onion popula- Statistics from 454 sequencing of chloroplast DNAs from N and tions listed in Table 1 were evaluated for these SNPs using KASPar; S cytoplasms of onion are listed in Table 2. The average sizes of the cytoplasms of these onion populations were previously deter- reads were 399 base pairs (bp) for both cytoplasms, generating mined using restriction fragment length polymorphisms (RFLPs) over 34 and 16 megabases of sequence for N and S cytoplasms, (Havey 1997). An indel and SNPs resulting in polymorphic restric- respectively. Sequences and annotations of these chloroplast tion enzyme sites that distinguish N and S cytoplasms were DNAs are available from GenBank as accessions KF728080 and identified in chloroplast genes, and primers flanking these poly- KF728079 for N and S cytoplasms, respectively. The onion chlo- morphisms were designed to amplify the regions. For polymor- roplast DNA is typical of most land plants with LSC and SSC re- phic restriction enzyme sites, the amplicons were subsequently gions separated by two inverted repeats (Havey 1991; Katayama digested with the appropriate restriction enzyme according to et al. 1991). The chloroplast DNAs of N and S cytoplasms are very the manufacturer’s (Promega) recommendations. Fragment size similar in size (153 538 versus 153 355 bp, respectively) and GC differences were resolved using 9% polyacrylamide gels, silver contents (both at 36.8%), as well as sharing identical gene orders stained, and photographed. (Fig. 1) as expected given their close phylogenetic relationship.

Published by NRC Research Press 740 Genome Vol. 56, 2013

Fig. 2. Similarities between the chloroplast DNAs from normal (N) male-fertile as the reference and male-sterile (S) cytoplasms of onion across the large single copy region as visualized by mVISTA (Frazer et al. 2004). Annotated genes are displayed along the top. Right side shows percent similarities between chloroplast sequences from 50% to 100%. For personal use only.

Size difference was primarily due to small indels in intergenic in psbA (Table 3) was previously reported by Cho et al. (2006)). Two regions and a deletion of 45 bp in the accD gene of N-cytoplasmic of the chloroplast SNPs distinguishing N and S cytoplasms re- onion relative to S cytoplasm (Fig. 2), tobacco, orchid, and rice. In sulted in polymorphic sites for relatively inexpensive restriction the intergenic regions, differences were observed of 99, 59, and enzymes (Table 4). The indel and polymorphic restriction-enzyme 50 bp between trnL-UAA and trnT-UGU, psbM and petN, and rps16 sites are useful for classification of onion cytoplasms using gels as and trnQ-UUG, respectively. For each of these regions, S cytoplasm opposed to SNP-genotyping platforms. We evaluated these chlo- had the shorter sequence. The indels between trnL-UAA and trnT- roplast polymorphisms across a sample of onion germplasms UGU were previously reported as totaling 111 bp for a different (Table 1), previously classified as N or S cytoplasmic using RFLPs source of N cytoplasm (Havey 1995; Lilly and Havey 2001); how- (Havey 1993, 1997), and observed complete agreement between ever, Alcalá et al. (1999) and Terefe et al. (2002) reported that the the SNP, indel, polymorphic restriction site, and RFLP-based clas- chloroplast DNAs of N-cytoplasmic plants possesses size polymor- sifications. phisms in this intergenic region. In agreement with Steele et al. The majority of onion populations possess N cytoplasm; S cyto- (2012) for other members of the core Asparagales, trnH and rps19 plasm is likely an alien cytoplasm introgressed into onion in an- were assigned to the inverted repeats in onion as opposed to tiquity (Havey 1993, 2000). This alloplasmic origin of S cytoplasm Genome Downloaded from www.nrcresearchpress.com by UNIV OF WISC MADISON on 02/04/14 the LSC region in tobacco. We observed significant annotation is supported by numerous polymorphisms in the organellar DNAs differences between onion and the orchids across the SSC region. distinguishing it from N cytoplasm (Havey 1995, 2000; Sato 1998; Chang et al. (2006), Wu et al. (2010), and Pan et al. (2012) reported Engelke et al. 2003; Kim et al. 2009). Although the genetic basis of that the orchids Phalaenopsis, Oncidium, and Erycina do not possess CMS in onion is most likely associated with the mitochondrial functional subunits A, D, E, F, G, H, and (or) I of NADH dehy- DNA, maternal transmission of both organellar genomes means drogenase (nadh) in the SSC; however, our annotations revealed that polymorphisms in either the mitochondrial or chloroplast that these nadh subunits are full length and appear functional DNAs are useful to classify cytoplasms in onion (Havey 1993). Poly- in onion (Fig. 1), in agreement with annotations of these chlo- morphisms in the chloroplast DNAs can be easier to detect be- roplast genes in other members of the core Asparagales (Steele cause of the greater amounts of chloroplast DNA relative to et al. 2012). mitochondrial DNA in leaf tissue. Chloroplast polymorphisms can be used to confidently identify S cytoplasm; however, they do not Polymorphisms in the chloroplast DNA distinguishing N distinguish between N cytoplasm and the T-like male-sterile cyto- and S cytoplasms plasms of onion (Havey 2000). To confidently distinguish these We identified 26 SNPs (Table 3) and one indel (Table 4) in 20 chlo- more closely related cytoplasms, mitochondrial polymorphisms roplast genes that distinguish N and S cytoplasms (one of the SNPs must be evaluated (Sato 1998; Havey 2000; Engelke et al. 2003; Kim

Published by NRC Research Press o one al. et Kohn von Table 3. Sequences flanking single nucleotide polymorphisms (SNPs) in chloroplast genes that distinguish normal (N) male-fertile and male-sterile (S) cytoplasms of onion. No. Regiona Gene SNP [N/S] with flanking sequenceb 1 LSC accD GTCTAAGAGTAACAATCACGACTATTATCATTACATGTATGATACTAAATCTGGTTGGA[G/A]TAATCACATTAATAGTTGTATTGATAGTTATCTTCGT TTTGAAATCAGTATTCATAGTTA 2 LSC atpA AGAAGCAGAAATTTCACCTCTTCCATCAATAGGTTTAGCCAGAGCATTTATAACACGACC[T/C]AAATAAGCCTCGCTTACAGGTATTTGAGCAATCCTT CCTGTTGCTTTTACAGAACTTCCC 3 LSC atpF GATTCCAATATTAGCACTGATGGTATGGAAATGTAACTCGCTATTCAAACAACTATTCAG[G/A]GCTCCTATAGCCCCCTGTAAGGCTTGTTGGAAAACT TGTTGTCGGACCTGATTAATTGCT 4 LSC cemA (2 SNPs) CATTTCAAAAAAGTCTGGAACCTTGGATTATTCATTGGTGGAATACTAGGCAATCCGAAA[T/A]TTTTTTGAATGATATTCAAGAGAAAAATGTTTTAGA[A/G] AAATTCCTAGAATTAGAAGAACTATTCTTGTTGGATGAAATGATAAAAGAATACCCAGAG 5 LSC petB TGGTTATTGGGCAGTAAAAATTGTGACAGGCGTGCCTGACGCTATTCCTATAATAGGATC[T/C]CCTTTGGTAGAGTTATTACGTGGAAGTGCTAGTGT GGGTCAATCTACCTTGACTCGTTTT 6 LSC psaA TAGGTTCCAGATCCAAGTGGTAGTATCGGGGCCTTTAGCTATTGTTCTTGAGAAATGACC[A/G]GGTCTGGCCCATTCCTCGAAAGACGTTTTTACAGGAT CCCTATCTACAACAATTTTCACT 7 LSC psaB GGAGGTACCAAATAAATGACTACTGGAATCAGGGTTTTGGGCATAAAGATTCCACTGACC[C/T]GTAAGAAGTGGTTCCAACCCTTGGGGATAGGGTAAT ACGTCTAAGAAATTATTCCATCTT 8 LSC psbAc (2 SNPs) AAATGGATGCATAAGGATGTTGTGCTCTGCCTGGAATACAATCATAAAATTGAAAGTACC[G/A]GAAATTCCTAAAGGCATACCATCAGAGAAACTTCCTT GACCAATAGGATAGATCAAGAAAACAGCAGTAGCAGCTGCAACAGGAGCTGAATATGCAACAGCAATCCAAGGACGCATACCCAGACGAAAACT[A/C] AGTTCCCATTCACGACCCATGTAACAAGCTACACCAAGTAAGAAGTGTAAAACAATTAGC 9 LSC psbE ACTTCCAAATACATCGTAAGCTAAACCTGTACTGACGAATAACCAACCCGCAATGAATAG[G/C]GAAGGTATAGTAATGCTATGAATGACCCAGTATCGA ATACTGGTAATAATATCAACAAAA 10 LSC rbcL TTTACCAAGGATGATGAAAATGTGAACTCCCAGCCTTTTATGCGTTGGAGAGACCGTTTC[T/A]TATTTTGTGCTGAAGCTATTTATAAAGCGCAAGCTGA AACAGGTGAAATCAAAGGGCATT 11 LSC rpoA (3 SNPs) TATTTCCACGGAAGGCGGTGAGATGATATCTTGAGCGGTTACGTATCTAGGACCTCTAA[C/T]GCAAATGGATGCGTCTCTAACTCCATACAGATTACTTCTC AATACAATTTCTTTCAAATTTATTCAAATTTCATGTACCGATTCCTCAATACCTACTATCGTAGAATA[T/G]TCATGTGGCACTTTCTCAGATTTAGCACGT GTGATACATGTTCCTTCTATTTCTCCAAGTAAAGCCCTTCGCATGGCAATACCTATGGTATCGGCTTGCCCTTTCATGAG[T/C]GGGGACAGAATGAAACG ACCATAATAAAGACGCGTACTGTCTACTCTTGATTCAACACAT 12 LSC rpoB AAGTATGGCTCGTAATAATCTATCCTCGGGGGCATACGAGGATTCGTTCGCTGTCTGAGG[G/T]GTTAATTTACCTATTAAAATATCGCCGGTTTCTATCC AAGATCCCAGCATTACAATTCCA 13 LSC rpoB TGATAAAATGATCTTGTGAGTATCAGTATAAATGATCTTTCCTTCGCGTTCAGCTATAAC[A/G]GACACCCCCGAATCTAGAGCCGTTTGGCGTTCCAGC CCAGTTCCAACAATGCACTTCTCG For personal use only. use only. For personal 14 LSC rpoB TCTATTTGGTGATAAATAAATTATCTGTGCCTCTTTTGATCTCTCAGACATTTCATAAAG[T/C]GGACTCTCTATAGATCCCCAAGGACCAATCCTCACA TGAATAGCTAAAGATCCAATAAGT 15 LSC ycf4 TCTTTATATGGAAATCAGAGGCCAAGGAACCATCCCCTTGACTCGTACTGATGAGAATTT[G/A]ACTCCACGAGAAATTGAACAAAAAGCTGCCGAATTG GCCTATTTCCTGCGCGTACCAATT 16 SSC ccsA ACCTATGGTTAAAAAGATAAACCCTAAACTAATAACACGATAACTCCAATAATCCAAACG[C/A]TGAATTAATTGATATTTATAATAATTTGGAAATGAA AGAAAAGAAGTGCTTTTAACAACA 17 SSC ndhA AATGTGAGAGATTACCTTTTGATTTACCAGAAGCAGAGGAGGAATTAGTAGCAGGTTATC[A/G]AACAGAATATTCGGGTATTAAATATGCTCTATTTTA CCTTGCTTCTTACCTAAATTTATT 18 SSC ndhD ATATGGTTCCAATGAACCGACATTGGATTTTGACAGATTAGCTAATCAGTCATATCCTGT[T/G]ACATTAGAAATAATATTCTATTTGGGCTTCCTTATTG CTTATGCTGTCAAATCACCGATT 19 SSC ndhD ACCTATGTCTTCTGTTATCATTTCCAATTGGACGATCCATTAATTCAATTAGAAGAGGAT[G/A]CTAAATGGATAAATGTTTTCAATTTTCACTGGAGAC TGGGAATCGACGGACTTTCCATAG ulse yNCRsac Press Research NRC by Published 20 SSC ndhG (2 SNPs) GGATCGGTTCTTGTTTGTATATCCTTATTCTATATTTCATCAAACTCCTTTTTTGTAGCTG[C/T]CGCCCAGCTTCTTATTTATGTGGGAGCCATAAATGTCTTA ATCATATTTGCTGTTATGTTCGTGAATGGTTCAGAATAT[G/T]CTAACGACTCCTATCTTTGGACTATTGGAGATGGAGTCACTTCACTAGTTTGTATAAGTA 21 SSC ndhH GACCAATGTTACGGGCTTCTGGAATCCAATGGGATCTTCGTAAAGTTGATAGTTATGAGT[A/G]TTACAATAAATTTGATTGGGAAGTCCAATGGCAAAA AGAAGGAGATTCACTAGCTCGTTA 22 SSC ndhI AATCAAATTGCATTAGGTCGGTTACCAATGTCAGTAATTGGAGATTACACAAT[C/T]CAAACAATTATGAATTCCAGTCAAATCAAAATGGATAAAGA Genome Downloaded from www.nrcresearchpress.com by UNIV OF WISC MADISON on 02/04/14 OF WISC MADISON by UNIV from www.nrcresearchpress.com Downloaded Genome TAAACCCCTTGATTCAAGA 23 SSC psaC TAAGCAAATTGCTTCCGCGCCAAGAACGGAAGACTGTGTAGGTTGTAAAAGATGTGAATC[C/T]GCCTGTCCAACAGATTTTTTGAGTGTCCGTGTTTAT TTATGGCATGAAACAACTCGCAGC aLSC and SSC refer to the large and small single copy regions, respectively, in the chloroplast DNA. b[N/S] indicates specific nucleotides for N and S cytoplasms, respectively. 741 cSNP in bold was previously reported by Cho et al. (2006). 742 Genome Vol. 56, 2013

Table 4. Primer sequences amplifying regions carrying an indel or polymorphic restriction enzyme sites that distinguish normal (N) male-fertile and male-sterile (S) cytoplasms of onion. Fragment sized Markera Geneb Enzyme Primerc NS Indel accD F: AGAATGAGGAGCAGGAAAACTCT 375 420 R: AGTCGTGATTGTTACTCTTAGACCT RE atpF (3) SacI F: TTCGGAAACAAAGGGAAAAA 198 155 + 43 R: TCCGACAACAAGTTTTCCAAC RE petB (5) BamHI F: CAGGTGTGGTTCTGGCTGTA 231 126 + 105 R: CGGCAGTAAGAAGAGGCAAT aIndel refers to an insertion–deletion polymorphism; RE refers to restriction-site polymorphism. bNumber in parenthesis refers to character number in Table 3. cF, forward; R, reverse. dSizes in base pairs.

et al. 2009). The chloroplast SNPs distinguishing N and S cyto- lations of onion. Theor. Appl. Genet. 90(2): 263–268. doi:10.1007/BF00222212. plasms can be genotyped together with nuclear SNPs closely asso- PMID:24173901. Havey, M.J. 1997. On the origin and distribution of normal cytoplasm of onion. ciated with the nuclear Ms locus (Havey 2013) to efficiently identify Genet. Res. Crop Evol. 44: 307–313. doi:10.1023/A:1008680713032. maintainer (N msms) plants to seed propagate male-sterile lines for Havey, M.J. 2000. Diversity among male-sterility-inducing and male-fertile hybrid production. cytoplasms of onion. Theor. Appl. Genet. 101(5–6): 778–782. doi:10.1007/ s001220051543. Acknowledgements Havey, M.J. 2013. Single nucleotide polymorphisms in linkage disequilibrium with the male-fertility restoration (Ms) locus of onion. J. Am. Soc. Hortic. Sci. Names are necessary to report factually on available data; how- 138(4): 306–309. ever, the U.S. Department of Agriculture (USDA) neither guaran- Jones, H.A., and Clarke, A. 1943. Inheritance of male sterility in the onion and tees nor warrants the standard of the product, and the use of the the production of hybrid seed. Proc. Am. Soc. Hortic. Sci. 43: 189–194. name by USDA implies no approval of the product to the exclusion of Jones, H.A., and Emsweller, S.L. 1937. A male-sterile onion. Proc. Am. Soc. Hortic. Sci. 34: 582–585. others that may also be suitable. We gratefully acknowledge the Katayama, H., Sasakuma, T., and Ogihara, Y. 1991. Physical map of chloroplast support of grant 2008-51180-04875 from the USDA Specialty Crops DNA of the onion, Allium cepa L., showing the location of photosynthesis- Research Initiative and SNP genotyping by Ag-Biotech, San Juan Bau- related genes. Jpn. J. Genet. 66: 421–431. doi:10.1266/jjg.66.421. tista, California. Kim, S., Lee, E.-T., Cho, D.Y., Han, T., Bang, H., Patil, B.S., et al. 2009. Identifica- tion of a novel chimeric gene, orf725, and its use in development of a molecular marker for distinguishing among three cytoplasm types in onion (Allium References cepa L.). Theor. Appl. Genet. 118(3): 433–441. doi:10.1007/s00122-008-0909-x. Alcalá, J., Pike, L.M., and Giovannoni, J.J. 1999. Identification of plastome vari- PMID:18936906. ants useful for cytoplasmic selection and cultivar identification in onion. Lilly, J.W., and Havey, M.J. 2001. Sequence analysis of a chloroplast intergenic J. Am. Soc. Hort. Sci. 124: 122–127. spacer for phylogenetic estimates in Allium section Cepa and a PCR-based APGIII. 2009. An update of the Angiosperm Phylogeny Group classification for polymorphism detecting mixtures of male-fertile and male-sterile cytoplas- the orders and families of flowering plants: APG III. Bot. J. Linn. Soc. 161: mic onion. Theor. Appl. Genet. 102(1): 78–82. doi:10.1007/s001220051620.

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