The first doubled haploid linkage map for cultivated oat.
ABSTRACT To date, all linkage maps of hexaploid oat (Avena sativa L.) have been constructed using recombinant inbred lines (RILs). Doubled haploids (DHs), however, have the advantage over RILs of their comprehensive homozygosity. DHs have been used for mapping in several cereal species, but in oats the production of large DH populations has only recently become an option. A linkage map of hexaploid oat was constructed using an anther culture-derived DH population (137 individuals) from the F1 individuals of a cross between the Finnish cultivar 'Aslak' and the Swedish cultivar 'Matilda'. The map is composed of 28 linkage groups containing 625 DNA markers: 375 AFLPs (amplified fragment length polymorphisms), 3 IRAPs (inter-retrotransposon amplified polymorphisms), 12 ISSRs (inter simple sequence repeats), 12 microsatellites, 57 RAPDs (random amplified polymorphic DNAs), 59 REMAPs (retrotransposon-microsatellite amplified polymorphisms), 105 SRAPs (sequence-related amplified polymorphisms), and 2 SNPs (single-nucleotide polymorphisms). The total map size is 1526 cM. Over half of the markers in the map showed distorted segregation, with alleles from 'Aslak' usually prevailing. This is explained by the better performance of 'Aslak' in anther culture. Quantitative trait loci affecting some important quality and agronomic traits are being localized on the map.
- SourceAvailable from: James B Holland[show abstract] [hide abstract]
ABSTRACT: Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.Genome 01/2002; 44(6):1065-76. · 1.67 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Microsatellites have developed into a powerful tool for mapping mammalian genomes and first reports about their use in plants have been published. A database search of 228 barley sequences from GenBank and EMBL was made to determine which simple sequence repeat (SSR) motif prevails in barley. Nearly all types of SSRs were found. The (A)n and (T)n SSRs occurred more often than (C)n and (G)n for n > or = 10. Among the dinucleotide repeats, the (CG)n SSRs occurred least often. Trinucleotide repeats did not occur with n > 7 and there is no correlation between the GC content in the trinucleotide motifs and the number of observed SSRs. Analysing 15 different microsatellites with 11 barleys yielded 2.1 alleles per microsatellite. Sequencing 25 putative microsatellites showed that the resolution capacity of high-quality agarose gels was sufficient to determine differences of only three base pairs. Five microsatellites were mapped on three different chromosomes of a barley RFLP map.Plant Molecular Biology 03/1995; 27(4):835-45. · 3.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The colinearity of markers linked with resistance loci on linkage group A of diploid oat, on the homoeologous groups in hexaploid oat, on barley chromosome 1H, and on homoeologous maize chromosomes was determined. Thirty-two DNA probes from homoeologous group 1 chromosomes of the Gramineae were tested. Most of the heterologous probes detected polymorphisms that mapped to linkage group A of diploid oat, two linkage groups of hexaploid oat, barley chromosome 1H, and maize chromosomes 3, 6, and 8. Many of these DNA markers appeared to have conserved linkage relationships with resistance and prolamin loci in Avena, Hordeum, and Zea mays. These resistance loci included the Pca crown rust resistance cluster in diploid oat, the R203 crown rust resistance locus in hexaploid oat, the Mla powdery mildew resistance cluster in barley, and the rp3, wsm1, wsm2, mdm1, ht2, and htn1 resistance loci in maize. Prolamin encoding loci included Avn in diploid oat and Hor1 and Hor2 in barley. A high degree of colinearity was revealed among the common RFLP markers on the small chromosome fragments among these homoeologous groups. Key words : disease resistance, colinearity, Gramineae, cereals.Genome 03/1996; 39(1):155-64. · 1.67 Impact Factor
The first doubled haploid linkage map for
Pirjo Tanhuanpa ¨a ¨, Ruslan Kalendar, Alan H. Schulman, and Elina Kiviharju
Abstract: To date, all linkage maps of hexaploid oat (Avena sativa L.) have been constructed using recombinant inbred
lines (RILs). Doubled haploids (DHs), however, have the advantage over RILs of their comprehensive homozygosity. DHs
have been used for mapping in several cereal species, but in oats the production of large DH populations has only recently
become an option. A linkage map of hexaploid oat was constructed using an anther culture–derived DH population (137
individuals) from the F1 individuals of a cross between the Finnish cultivar ‘Aslak’ and the Swedish cultivar ‘Matilda’.
The map is composed of 28 linkage groups containing 625 DNA markers: 375 AFLPs (amplified fragment length poly-
morphisms), 3 IRAPs (inter-retrotransposon amplified polymorphisms), 12 ISSRs (inter simple sequence repeats), 12 mi-
crosatellites, 57 RAPDs (random amplified polymorphic DNAs), 59 REMAPs (retrotransposon-microsatellite amplified
polymorphisms), 105 SRAPs (sequence-related amplified polymorphisms), and 2 SNPs (single-nucleotide polymorphisms).
The total map size is 1526 cM. Over half of the markers in the map showed distorted segregation, with alleles from
‘Aslak’ usually prevailing. This is explained by the better performance of ‘Aslak’ in anther culture. Quantitative trait loci
affecting some important quality and agronomic traits are being localized on the map.
Key words: anther culture, Avena sativa, DNA markers, doubled haploid, linkage map, segregation distortion.
Re ´sume ´ : A`ce jour, toutes les cartes ge ´ne ´tiques de l’avoine hexaploı ¨de (Avena sativa L.) ont e ´te ´ produites avec des
populations de ligne ´es recombinantes fixe ´es (« RIL »). Par rapport aux RIL, les haploı ¨des double ´s (HD) ont cependant
l’avantage d’une homozygotie exhaustive. Les HD ont e ´te ´ employe ´s en cartographie chez plusieurs espe `ces de ce ´re ´ales,
mais jusqu’a ` re ´cemment il n’e ´tait pas possible de produire de grandes populations de ligne ´es HD chez l’avoine. Une carte
ge ´ne ´tique de l’avoine hexaploı ¨de a e ´te ´ produite gra ˆce a ` une population de 137 ligne ´es HD de ´rive ´es de la culture d’anthe `res
a ` partir d’individus F1issus du croisement entre le cultivar finlandais ‘Aslak’ et le cultivar sue ´dois ‘Matilda’. La carte
compte 28 groupes de liaison et totalise 625 marqueurs : 375 AFLP (polymorphismes de longueur de fragments amplifie ´s),
3 IRAP (polymorphismes d’amplification inter-re ´trotransposons), 12 ISSR (polymorphismes d’amplification inter-
microsatellites), 12 microsatellites, 57 RAPD (polymorphismes d’ADN amplifie ´ au hasard), 59 REMAP (polymor-
phisme d’amplification re ´trotransposon-microsatellite), 105 SRAP (polymorphismes d’amplification de se ´quences
apparente ´es) et 2 SNP (polymorphismes mononucle ´otidiques). La taille totale de la carte est de 1526 cM. Plus de la
moitie ´ des marqueurs de la carte pre ´sentaient une se ´gre ´gation biaise ´e ou ` les alle `les du parent ‘Aslak’ e ´taient ge ´ne ´rale-
ment favorise ´s. Ceci s’explique par la meilleure performance de ce cultivar en culture d’anthe `res. Des travaux de
cartographie sont en cours en vue de localiser des QTL contro ˆlant certains caracte `res agronomiques et de qualite ´.
Mots-cle ´s : culture d’anthe `res, Avena sativa, marqueurs mole ´culaires, haploı ¨des double ´s, carte ge ´ne ´tique, se ´gre ´gation
[Traduit par la Re ´daction]
Cultivated oat (Avena sativa L.) is a self-pollinating
allohexaploid (n = 3x = 21) consisting of 3 basic genomes
(A, C, and D; Rajhathy and Thomas 1974) and having a
large genome size (1C DNA content 13.7 pg; Bennett and
Smith 1976). As a consequence, molecular mapping of the
cultivated oat has been rather complicated. To simplify
mapping efforts, the earliest oat linkage maps were
constructed in diploid oat species (A genome) corresponding
to ancestors of the cultivated hexaploid oat. Populations
from two different crosses have been used: A. atlantica
Received 5 March 2008. Accepted 11 May 2008. Published on the NRC Research Press Web site at genome.nrc.ca on 2 July 2008.
Corresponding Editor: D. Somers.
P. Tanhuanpa ¨a ¨1and E. Kiviharju. Plant Genomics, Biotechnology and Food Research, MTT Agrifood Research Finland, FI-31600
R. Kalendar. MTT/BI Plant Genomics Laboratory, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, P.O. Box 56,
Viikinkaari 1, FI-00014 Helsinki, Finland.
A.H. Schulman. Plant Genomics, Biotechnology and Food Research, MTT Agrifood Research Finland, FI-31600 Jokioinen, Finland;
MTT/BI Plant Genomics Laboratory, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, P.O. Box 56, Viikinkaari 1, FI-
00014 Helsinki, Finland.
1Corresponding author (e-mail: email@example.com).
Genome 51: 560–569 (2008) doi:10.1139/G08-040
##2008 NRC Canada
Baum et Fedak ? A. hirtula Lag. (O’Donoughue et al. 1992;
Van Deynze et al. 1995) and A. strigosa Schreb. ? A. wies-
tii Steud. (Rayapati et al. 1994; Yu et al. 1996; Yu and Wise
2000; Kremer et al. 2001).
The first linkage map in hexaploid oat was built using
recombinant inbred lines (RILs) from the cross A. byzantina
C. Koch ‘Kanota’ ? A. sativa L. ‘Ogle’ (O’Donoughue et
al. 1995). This KO map has since been extended and used
in QTL (quantitative trait locus) studies (Bush and Wise
1996; Holland et al. 1997; Kianian et al. 1999, 2000; Jin et
al. 2000; Groh et al. 2001a, 2001b; Wight et al. 2003; Wooten
et al. 2008). Mapping populations of hexaploid oat have
also been produced from the crosses between genotypes
‘Kanota’ and ‘Marion’ (Kianian et al. 1999, 2000; Groh et
al. 2001a, 2001b), ‘Terra’ and ‘Marion’ (De Koeyer et al.
2004), ‘Ogle’ and ‘TAM O-301’ (A. sativa subsp. byzantina
C. Koch) (the so-called OT map; Portyanko et al. 2001;
Jackson et al. 2008), ‘Clintland64’ and ‘IL86-5698’ (Jin et
al. 2000), ‘Ogle’ and ‘MAM17-5’ (the OM map; Zhu and
Kaeppler 2003; Zhu et al. 2003), and ‘MN841801’ and
‘Noble’ (Portyanko et al. 2005).
All the maps of hexaploid oat have been constructed
using RILs. Compared with F2 and backcross mapping
populations, RILs represent a permanent population. This
allows character measurements in several locations and over
several years. In addition, because heterozygotes are for the
most part lacking, dominant markers are as informative as
codominant ones when RILs are used in mapping. In an F2
population, the information content of dominant markers is
low, especially when the markers are in repulsion (Ott
1985). Another permanent population type is a doubled
haploid (DH) population. In contrast to RILs, DHs contain
no residual heterozygosity. However, because DHs have
undergone only one cycle of meiosis compared with the
several cycles that are used for development of RILs, RILs
have higher recombination. Thus, more accurate maps may
be obtained with RILs than with DHs (Ferreira et al. 2006).
DHs have been used for mapping in several cereal species
(Forster and Thomas 2003). However, in oats the production
of DH mapping populations has only recently become an
option (Kiviharju et al. 2005).
In this paper, we present the first linkage map of
hexaploid oat (Avena sativa L.) constructed using a DH
population. The map is composed of various types of
PCR-based DNA markers; both SRAPs (sequence-related
amplified polymorphisms) and the retrotransposon-based
marker methodsIRAP (inter-retrotransposon
polymorphism) and REMAP (retrotransposon-microsatellite
amplified polymorphism) were used here for the first time
in oat. QTLs for some important quality and agronomical
traits are now being placed onto the map, and the results
will be presented in a subsequent paper.
Materials and methods
The Nordic hexaploid oat cultivars ‘Aslak’ and ‘Mat-
ilda’ were used as parental lines. ‘Aslak’ was bred by
Boreal Plant Breeding Ltd. (Finland) and ‘Matilda’ by
crossed, and 200 ‘Aslak’ ? ‘Matilda’ F1seeds were pro-
duced by Boreal. The F1plants were sown, and 148 DH
plants were derived from anther culture by the method of
Kiviharju et al. (2005) in 3 separate experiments. Cold
treatment was used for cut tillers and heat treatment for
isolated anthers. The induction medium contained W14
salts and vitamins (Ouyang et al. 1989) supplemented
with 5 mg/L 2,4-dichlorophenoxyacetic acid (Sigma, Oslo,
Norway), 0.5 mg/L kinetin (Sigma), 20 mg/L ethylene re-
leasing compound Ethephon (Dr. Ehrenstorfer GmbH,
Augsburg, Germany), 50 mg/L L-cysteine (Merck, Espoo,
Finland), 500 mg/L myo-inositol (Merck), and 10% mal-
tose, and the pH was adjusted to 6.0. Note that the com-
position of W14macro salts was incorrectly presented in
Kiviharju et al. (2005), the correct one (in mg/L) being
CaCl2?2H2O, 140; K2SO4, 700. A solid medium (solidified
with 0.3% Phytagel, Sigma, USA) covered with a liquid
layer (with 10% Ficoll 400, Pharmacia Biotech, Sweden)
was used for induction culture. Regeneration and rooting
media were the same as in Kiviharju et al. (2005). The
induction was carried out at 28 8C in the dark or under
dim light (approx. 40 mmol photons?m–2?s–1), and regener-
ation and rooting were carried out at 25 8C under dim
light with a 16 h photoperiod. The ploidy level of the re-
generants was determined by flow cytometry (Becton
Dickinson FACSort, USA), and the haploid genome was
doubled by colchicine as described in Kiviharju et al.
DNA was extracted from oat leaves using a CTAB
Poulsen et al. (1993), except that the DNA was treated
additionally with 10 mg/mL RNase (Sigma) for 30 min at
37 8C and was not centrifuged through a CsCl density
gradient. DNA concentrations were measured using the
GeneQuant II RNA/DNA Calculator (Pharmacia Biotech
Ltd., Cambridge, UK).
Unless mentioned otherwise, PCR amplifications were
carried out with Biotools DNA polymerase (Biotools
B&M Labs, S.A., Madrid, Spain), with the buffer (con-
taining 2 mmol/L MgCl2) supplied by the enzyme manu-
facturer, in a PTC-220 DNA Engine Dyad Peltier Thermal
Cycler (MJ Research, Waltham, Massachusetts, USA). Oat
microsatellites from various sources were used (Li et al.
2000; Holland et al. 2001; Pal et al. 2002; Jannink and
Gardner 2005). Two different PCR programs were carried
out in a Mastercycler gradient thermal cycler (Eppendorf,
Hamburg, Germany) as described by Li et al. (2000), ex-
cept that the number of cycles was reduced from 48 to 43
(program 1) and from 38 to 33 (program 2) when the
whole population was analyzed. The amplification reac-
tions (25 mL) contained 0.75 U of Taq polymerase (MBI
Fermentas, St. Leon-Rot, Germany) or 0.4 U of Biotools
DNA polymerase (Biotools B&M Labs), the buffer sup-
plied by the respective enzyme manufacturer, 1.5–2 mmol/
L MgCl2, 100 mmol/L each dNTP, 400 nmol/L each pri-
mer, and 20–40 ng of DNA. One primer of each primer
pair was labeled with a fluorescent dye, FAM (5-carboxy-
fluorescein) or TET (6-carboxytetrachlorofluorescein), and
the amplification products were resolved and visualized on
Tanhuanpa ¨a ¨ et al.561
##2008 NRC Canada
a MegaBACE 500 Sequencer (GE Healthcare, Bucking-
In addition to oat microsatellites, barley (Becker and Heun
1995; Liu et al. 1996; Ramsay et al. 2000) and rye (Hackauf
and Wehling 2002; Lochow-Petkus GmbH) microsatellites
were tested in the oat parents. Various PCR conditions were
used for the amplifications. The only polymorphic barley
microsatellite in the DH population (HVM20) was amplified,
using the MSA program according to Liu et al. (1996), in a
reaction volume of 25 mL containing 0.4 U of Red Hot DNA
polymerase (Advanced Biotechnologies, Epsom, Surrey, UK),
the buffer supplied by the enzyme manufacturer, 2.5 mmol/L
MgCl2, 100 mmol/L each dNTP, 160 nmol/L each primer, and
20 ng of DNA.
RAPD (random amplified polymorphic DNA) and ISSR
(inter simple sequence repeat) analyses were carried out
basically as described in Tanhuanpa ¨a ¨ et al. (2006), using
Biotools DNA polymerase. The ISSR technique amplifies
DNA segments lying between two identical microsatellite
repeat regions (Zietkiewicz et al. 1994). The primers con-
tained a microsatellite repeat sequence anchored at the 3’ end.
The IRAP and REMAP marker systems (Kalendar et al.
1999; Kalendar and Schulman 2006) are based on retro-
transposons. In the IRAP method, polymorphisms are found
among amplification products between the long terminal
repeats (LTRs) of retrotransposons, but in the REMAP
method they are found between retrotransposons and
thereby reflect retrotransposon insertions that took place
following divergence from the last common ancestor. Pri-
mers were designed by cloning retrotransposon regions
from different plant species (mostly from oat but also
from barley, rye, and timothy), identifying the LTRs, and
choosing conserved motifs at or near their termini. The mi-
crosatellite-based primers contained repeat units (composed
of 2 or 3 bases) anchored at their 3’ ends by a single nu-
cleotide. The PCR programs for IRAP and REMAP were
as described in Tanhuanpa ¨a ¨ et al. (2006). IRAP markers
were amplified in a reaction volume of 20 mL, using 1 U
of polymerase, Failsafe 2? PCR PreMix D (Epicentre,
Madison, Wisconsin) containing
250 nmol/L primers, and 25 ng of DNA. The amplification
conditions for REMAP were the same except that 1.5 U of
polymerase, 500 nmol/L each primer, and 50 ng of DNA
AFLP (amplified fragment length polymorphism) analysis
was based on Vos et al. (1995) and used the restriction
enzymes EcoRI and MseI. Restriction and ligation were per-
formed simultaneously in a 30 mL volume containing 0.5 mg
of DNA, 5 U of each restriction enzyme (New England Bio-
labs, Ipswich, Massachusetts), 1? NEBuffer2, 100 mg/mL
bovine serum albumin, 0.4 mmol/L ATP (MBI Fermentas),
33 U (cohesive-end ligation units) of T4 DNA ligase
(New England Biolabs), 2.5 mmol/L MseI adapters, and
0.25 mmol/L EcoRI adapters. Adapters were prepared by
mixing equimolar amounts of both strands, heating the
mixture to 95 8C for 5 min, and then cooling it down to
room temperature. The restriction–ligation reaction mixture
was incubated for 3 h at 37 8C, 4 h at 21 8C, and 15 min
were performed in 20 mL reactions containing 0.25 U of
polymerase, 200 mmol/L each dNTP, primers, and DNA.
The pre-selective amplifications contained 375 nmol/L pri-
mers (EcoRI + A, C, or G and MseI + A or C) and 50 ng
(3 mL from the restriction–ligation reaction) of DNA. The
program for pre-selective amplification consisted of an ini-
tial incubation at 72 8C for 2 min, followed by 28 cycles of
30 s at 94 8C, 1 min at 56 8C, and 1 min at 72 8C. The selec-
tive amplifications contained 250 nmol/L EcoRI primers,
500 nmol/L MseI primers, and 5 mL from a 10-fold dilution
of the pre-selective amplification reaction as template DNA.
The selective amplification reactions were performed for 37
cycles with the following ‘‘touchdown’’ profile: 30 s at
94 8C, 30 s at the annealing temperature, and 1 min at
72 8C. The annealing temperature was 65 8C in the first
cycle, was reduced by 1 8C per cycle for the next 8 cycles,
and then remained at 56 8C for the following 28 cycles. The
selective EcoRI primers were labeled with a fluorescent
dye — FAM, HEX (hexachloro-6-carboxyfluorescein), or
TET — and the amplified fragments were analyzed on a
MegaBACE 500 Sequencer (GE Healthcare). AFLP reac-
tions made with different dyes were multiplexed for analysis.
The SRAP marker technique is designed to amplify open
reading frames (Li and Quiros 2001). The forward primers
(‘‘me’’) preferentially anneal to exonic regions and the re-
verse primers (‘‘em’’) to intronic regions and promoters.
SRAP analyses were carried out as described in Tanhuanpa ¨a ¨
et al. (2007). The primer sequences are presented in Budak
et al. (2004). Three SNPs (single-nucleotide polymor-
phisms) were also analyzed in the progeny. The SNPI11
marker (previously named SNP-RAPD) associated with
short straw in oat was described by Tanhuanpa ¨a ¨ et al.
(2006), who also presented its amplification conditions. The
development of SNPs for ACCase (acetyl-CoA carboxylase)
1 and 2 will be described in a forthcoming article concern-
ing the localization of an associated QTL on the doubled
haploid oat map.
The designation of markers is explained in Table 1. Ad-
ditional information concerning the markers on the final
map, as well as the sequences of primers containing a
microsatelliterepeat, ispresented inthe electronic
Table 1. Marker names in the ‘Aslak’ ? ‘Matilda’ doubled
haploid oat map.
Selective bases with Eco primer –
selective bases with Mse primer
Retroprimer (A, D, or F + number)
Prefix AM, and Astavea
Retroprimer + ISSR
me1em1 through me5em16
ACCase1 and 2
aLetters from ‘‘a’’ to ‘‘r’’ at the end of marker names indicate
different-sized markers (in order of increasing molecular weight)
amplified with the same primer(s).
562Genome Vol. 51, 2008
##2008 NRC Canada
supplementary material (Tables S1 and S22). The primers
designed for the oat retrotransposons are owned by Boreal
Plant Breeding Ltd. and their sequences can be acquired
from them. The primer sequences for a barley retro-
transposon (F0004) and a timothy retrotransposon (F0778)
that were used in the construction of the map are CGAGT-
GAGGACAAAGTGCGCA and ACCAGCCCGGGCCGTC-
JoinMap 3.0 (Van Ooijen and Voorrips 2001) was used
for map construction, using primarily a LOD (logarithm of
odds) score of 9.0 but in some cases 8.0 (groups 8, 23, and
25) or 10.0 (groups 5 and 6). Map distances in centimorgans
(cM) were calculated by Kosambi’s mapping function (Ko-
sambi 1944). Maps for each linkage group were constructed
using first- or second-round maps. The jump threshold was
Table 2. ‘Aslak’ ? ‘Matilda’ F1anther culture.
No. of ELSNo. of green regenerants
No. of plants in the greenhouse
Note: ELS, embryo-like structures.
aInduction in this set was made in dim light, in other sets in the dark.
Table 3. Summary data for the doubled haploid oat map.
No. of lociSkewed markers (P < 0.05) in the map
2Supplementary data for this article are available on the journal Web site (http://genome.nrc.ca) or may be purchased from the Depository
of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ON
K1A 0R6, Canada. DUD 3765. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/cms/unpub_e.html.
Tanhuanpa ¨a ¨ et al.563
##2008 NRC Canada
5.0. The segregation of markers was tested against an ex-
pected 1:1 ratio using the ?2test in the JoinMap program.
Results and discussion
In total, 148 ‘Aslak’ ? ‘Matilda’ regenerants were pro-
duced in 3 separate anther culture experiments (Table 2).
Eleven regenerants were rejected because they did not pro-
duce seeds (5 individuals) or were potential heterozygotes
(5), or for technical reasons (1). As a consequence, the map-
ping population included 137 DH individuals; the genome of
82 individuals (60%) was doubled by colchicine and 55
individuals were spontaneously doubled haploids. Although
the use of dim light in the induction phase of ‘Aslak’ anther
culture previously showed positive effects on the induction
of embryo-like structures (Kiviharju et al. 2005), it de-
creased the response in this experiment. Without that trial,
the average green plant regeneration rate would have been
1.1/100 anthers for the ‘Aslak’ ? ‘Matilda’ progeny. Only a
few albino plants were regenerated.
The mapping population was screened with 717 DNA
markers polymorphic between the parents; 668 formed 28
linkage groups longer than 10 cM and containing more than
4 markers (Table 3). When only first- or second-round maps
were used for determining the order of markers in the
groups, the number of markers in the final map was reduced
to 625 (Table 3, Fig. 1). The linkage groups contained from
6 to 70 markers, of which 13% were codominant. The
SRAPs demonstrated the highest frequency of codominance
(18%), whereas among the other marker types the frequency
was around 10%. Cultivated hexaploid oat has 21 chromo-
some pairs, but we found 28 linkage groups. Therefore,
some of the linkage groups in our map belong to the same
chromosome but probably reside at the extreme ends,
thereby becoming impossible to join without additional
markers. The total map size was 1526 cM, whereas the esti-
mated complete map size of hexaploid oat is 2932 cM
(O’Donoughue et al. 1995).
In the map construction, a LOD score of 9.0 was suitable
for creating linkage groups. However, a lower LOD score
(8.0) was used for creating linkage groups 8, 23, and 25. In
linkage group 8, use of a LOD score of 9 would have
Fig. 1. A doubled haploid linkage map of oat from the cross ‘Aslak’ ? ‘Matilda’. The designations of markers are explained in Table 1.
The linkage group identifications are based on microsatellites (marked in bold) that have been located in previous studies:1Jannink and
Gardner 2005;2Pal et al. 2002;3Wight et al. 2003;4Zhu and Kaeppler 2003. Relationships between KO and OT maps are from Portyanko et
al. 2001, and those between KO and OM maps are from Zhu and Kaeppler 2003.
564Genome Vol. 51, 2008
##2008 NRC Canada
dropped marker ACC-CGTe from the end of the group.
However, including this marker lengthens the group from
25 to 42 cM, and the marker seems to be reliable and well
linked to other markers. The linkage groups 23 and 25
would split into two parts with a LOD score of 9.0, although
all the markers in both groups are well linked to each other,
and therefore were kept together with a lower LOD score.
On the other hand, linkage groups 5 and 6 were kept apart
using a LOD score of 10.0 because with a lower LOD score,
half of the markers could not be ordered until the last (third)
mapping round. It seems that only one marker connects
Fifty-four percent of the mapped markers showed dis-
torted segregation (P < 0.05, Table 3). About the same level
of distortion was observed in every marker type (results not
shown). Distorted markers were often confined to certain re-
gions, and sometimes all the markers in a linkage group
were skewed. The distortion in these regions was towards
only one of the parental alleles, usually ‘Aslak’ (19 groups),
but in 3 linkage groups (7, 16, 21) it was towards ‘Matilda’.
In linkage group 7, there were distorted regions towards
‘Aslak’ and ‘Matilda’. Only 2 linkage groups (23, 26) con-
tained normally segregating markers exclusively.
Distorted segregation in anther culture–derived progenies
is a generally acknowledged feature in many species, as is
the clustering of distorted markers (Foisset and Delourme
1996). In the distorted areas, alleles from the parent that
responds better in anther culture often prevail among the
progeny, implying that there are genes that affect anther cul-
ture response (Foisset and Delourme 1996). In our earlier
experiments, ‘Aslak’ produced as many as 8 green plants
per 100 anthers, whereas no regenerants have been obtained
from ‘Matilda’. On the other hand, alleles that favour anther
culture traits can also be derived from the less responsive
parent, which probably explains the skewing of markers
towards ‘Matilda’ alleles in 3 linkage groups. This has pre-
viously been shown in oat (Kiviharju et al. 2004) as well as
in other cereals (Martinez et al. 1994; Beckert 1998; Torp et
al. 2001). Distorted segregation should not significantly
affect linkage analysis in large populations (Devaux et al.
1995; Van Ooijen and Voorrips 2001; Hackett and Broad-
In addition to anther culture, parental heterogeneity may
partly explain segregation distortion in the present study.
We were forced to use many parents in several crosses to
get enough F1seed. Although oat is a self-pollinator, culti-
Fig. 1 (continued).
Tanhuanpa ¨a ¨ et al.565
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vars are not completely homogeneous. According to our
‘Matilda’ is more variable than ‘Aslak’, which seems to be
Dense clusters of normally segregating markers were also
observed in several linkage groups and involved all marker
types. Clustering is a general phenomenon in linkage maps
and is due to non-equal frequency of recombination along
chromosomes, recombination being low near centromeres
and in heterochromatic and introgressed regions (Young
Different linkage maps can be compared with each other
using anchor markers, usually RFLPs or microsatellites. Be-
cause analyzing RFLPs is very laborious, microsatellites
were chosen as anchors in the current study even though
only a few microsatellites have been located to oat chromo-
somes (after the current study was completed, some addi-
tional microsatellites were reported by Becher 2007).
Consequently, our linkage groups could be compared only
partly with published maps. Of the 51 tested oat micro-
satellites, 6 could not be amplified. Twelve were polymor-
phic in the parents (27% of the functioning microsatellites)
and were analyzed in the progeny, producing 14 loci (AM1
amplified 3 dominant loci as in Zhu and Kaeppler 2003).
Eleven loci were located on the map and 4 linkage groups
could be identified with the aid of microsatellites (Fig. 1).
Linkage groups 17, 24, and 25 (AM1c belongs to this group
but could not be located until after the third mapping round)
all belong to linkage group KO22. Groups 24 and 25 would
have been united with a LOD score of 6.
In addition to microsatellites developed from oat, 28 bar-
ley and 22 rye microsatellites were tested in the parents of
the mapping population. Nineteen (68%) barley and 18 (82%)
rye microsatellites were also amplified in oat, although
sometimes weakly, probably because of sequence diver-
gence. In previous studies, 42% of wheat (Hu et al. 2007),
38% of barley (Hu et al. 2007), and 12% of ryegrass
(Jones et al. 2001) microsatellites were successfully ampli-
fied in oat. Li et al. (2000) showed that the percentage of
functioning barley microsatellites in oat could be increased
from 5% to 26% by lowering annealing temperatures in
PCR. In addition to low reproducibility, the polymorphism
in microsatellites borrowed from other species is usually
low: in our study, only one (5%) of the barley micro-
satellites and two (11%) of the rye microsatellites were
polymorphic in the parents. The barley microsatellite HVM20
Fig. 1 (continued).
566 Genome Vol. 51, 2008
##2008 NRC Canada
mapped to linkage group 28, which thus corresponds to
barley chromosome 5 (1H) (Liu et al. 1996). The polymor-
phic rye microsatellites were not analyzed in the whole
progeny because there were problems in their amplification.
Acetyl-CoA carboxylase plays a major regulatory role in
fatty acid synthesis (reviewed by Ohlrogge and Jaworski
1997) and is therefore a good candidate gene for oil content.
Because our purpose is to locate QTLs for oil content on our
map, SNP analyses were done for two different ACCase loci
and the SNPs were located on groups 11 and 12. Kianian et
al. (1999) identified two ACCase loci, AccaseA and AccaseB,
of which AccaseA mapped to linkage group 11 in the KO
population. As a consequence, either of our groups, 11 or
12, corresponds to KO11.
Previously, we identified markers associated with the
dwarfing gene Dw6 (Tanhuanpa ¨a ¨ et al. 2006) and Cd accu-
mulation in oat (Tanhuanpa ¨a ¨ et al. 2007). One of the
markers linked to Dw6, SNPI11, belongs to linkage group
14 but could be ordered only after the third mapping round
(to the end of the group, between markers CAC-CCGb and
me4em12a). The SRAP marker me1em6e, which is linked to
Cd accumulation, resides on linkage group 8.
As far as we know, our linkage map of oat is the first one
constructed using a DH population and also the first one
with Nordic cultivars as parents. In addition, SRAP and
retrotransposon-based IRAP and REMAP markers have
been located on an oat map for the first time. Although Yu
and Wise (2000) used a primer for the barley BARE1 retro-
transposon for SSAP (sequence-specific amplified polymor-
phism) in oat, which is a hybrid retrotransposon and AFLP
technique, here we used a set of native oat retrotransposons
for the first time. The 62 retrotransposon-based markers
seemed to be uniformly distributed on the map, as could be
expected because of the dispersion of retrotransposons and
microsatellites in genomes and from previous mapping stud-
ies in other species (Schulman et al. 2004). SRAPs were
also evenly distributed on the map, as previously reported
for Brassica oleracea (Li and Quiros 2001). Currently, we
are placing QTLs affecting oil content, ?-glucan content,
leaf blotch disease resistance, and some agronomic charac-
ters onto the map.
We wish to thank Boreal Plant Breeding Ltd., especially
Leena Pietila ¨, for cooperation and Anu Kostamo, Ulla-Maija
Kuronen, Leena Lamminpa ¨a ¨, and Aija Viljanen for oat
Fig. 1 (concluded).
Tanhuanpa ¨a ¨ et al. 567
##2008 NRC Canada
hybridizations. We also thank Anneli Virta for MegaBACE
runs, and Marja-Riitta Araja ¨rvi, Sirpa Moisander, Kirsti
Salmi, Tarja Hovivuori, Ritva Koskenoja, and Yrjo ¨ Karppinen
for their excellent technical assistance. The Ministry of
Agriculture and Forestry in Finland is acknowledged for
their financial support.
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