Chromosomal Assignment of Microsatellite
Loci in Cotton
S. Liu, S. Saha, D. Stelly, B. Burr, and R. G. Cantrell
Microsatellite markers or simple sequence repeats (SSRs) represent a new class
of genetic markers for cotton (Gossypium sp.). Sixty-five SSR primer pairs were
used to amplify 71 marker loci and genotype 13 monosomic and 27 monotelodisomic cotton cytogenetic stocks. Forty-two SSR loci were assigned to cotton chromosomes or chromosome arms. Thirty SSRs were not located to specific chromosomes in this study. Nineteen marker loci were shown to occur on the A
subgenome and 11 on the D subgenome by screening accessions of G. herbaceum
(2n ⴝ 2x ⴝ 26 ⴝ 2A1) and G. raimondii (2n ⴝ 2x ⴝ 26 ⴝ 2D5). The aneuploid stocks
proved to be very powerful tools for localizing SSR markers to individual cotton
chromosomes. Multiplex PCR bins of the SSR primers and semiautomated detection of the amplified products were optimized in this experiment. Thirteen multiplex
PCR bins were optimized to contain an average of 4 SSR primer pairs per bin. This
provides a protocol for high-throughput genotyping of cotton SSRs that improves
the efficiency of genetic mapping and marker-assisted programs utilizing SSR
markers.
From the Department of Agronomy and Horticulture,
New Mexico State University, Las Cruces, NM 880038003 ( Liu and Cantrell), Crop Science Research Laboratory, USDA-ARS, Mississippi State, Mississippi
(Saha), Department of Soil and Crop Sciences, Texas
A&M University, College Station, Texas (Stelly), and
Brookhaven National Laboratory, Upton, New York
( Burr). Address correspondence to R. G. Cantrell at the
address above or e-mail: [email protected]. We thank
J. Pederson and D. A. Raska for technical assistance on
this project.
2000 The American Genetic Association 91:326–332
326
Microsatellites or simple sequence repeats (SSRs) are polymerase chain reaction (PCR)-based genetic markers comprised of tandemly arranged sequence
repeats of one to six nucleotides. They are
abundant and widely distributed throughout the genomes of many higher plants
and animals (Schmidt and Heslop-Harrison 1998). SSR loci tend to be both multiallelic and polymorphic for repeat number, which is easily scored and used for
genotyping (Goldstein and Pollock 1997;
Yu et al. 1999). SSRs can be revealed using
multiplex PCR systems, rendering SSRs
amenable to analysis on automated DNA
sequencers ( Diwan and Cregan 1997;
Mitchell et al. 1997). SSR analysis can thus
be adapted to high-throughput genotyping. The informativeness, wide distribution in the genome, and the ability to evaluate multiple markers of known location
make SSRs extraordinarily useful in genetic map construction ( Burow and Blake
1998).
Microsatellites have great utility in
bridging the gap between genetic mapping
and genome sequencing (physical mapping) (Schuler 1998). Microsatellites were
converted to sequence-tagged sites (STSs)
to facilitate the construction of high-resolution genetic maps of the human ( Dib et
al. 1996), mouse ( Dietrich et al. 1996), and
rice (Chen et al. 1997). STS markers can
thus serve as a framework for saturation
and expansion of genetic maps with additional DNA markers to achieve genomewide coverage. This will facilitate integration of the genetic and physical maps
(Schuler 1998).
Framework DNA markers are especially
useful in a disomic tetraploid, such as cotton (Gossypium hirsutum L., 2n ⫽ 4x ⫽ 52,
AADD) where restriction fragment length
polymorphism (RFLP) maps have been
constructed from both interspecific (Reinisch et al. 1994) and intraspecific (Shappley et al. 1998) mapping populations. Of
the 705 RFLP loci mapped to 41 linkage
groups in the interspecific Gossypium population, the actual chromosome identity of
only 14 of the linkage groups was presented (Reinisch et al. 1994). Genomic map development in a complex polyploid involves a stepwise process that builds
upon previous genetic and cytogenetic information (Stelly 1993). Aneuploid stocks
are employed to locate markers to individual chromosomes and identify linkage
groups to chromosomes. In cotton, monotelodisomic stocks that are hemizygous
for one arm provide a facile means to localize genes and marker loci to one arm
or the other of a given chromosome ( Endrizzi et al. 1985; Saha and Stelly 1994).
Figure 1. Electrophoretograms showing the inheritance pattern of three polymorphic SSRs in a cross between
TM-1 (G. hirsutum) and 3-79 (G. barbadense). (a) TM-1; (b) 3-79; (c) euploid F1 from TM-1 ⫻ 3-79 cross; (d) Tel9Lo
monotelodisomic interspecific F1 substitution stock ( hemizygous for 9sh). Note in (d) the BNL686-2 185 bp fragment
from TM-1 is missing, thus the marker resides on the short arm of chromosome 9. The scale across the top denotes
the allele size in nucleotides and the scale on the side (not shown) is fluorescent intensity. SSRs fragments are
labeled for each peak with allele sizes designated in parentheses. The BNL686 primer pair amplifies two loci labeled
as BNL686-1 and BNL686-2.
SSR markers can be used in combination
with cytogenetic stocks to create framework maps and further localize markers to
chromosome regions. Röder et al. (1998b)
first localized about 200 SSR markers to
the chromosomes of wheat (Triticum aestivum L.) and then used deletion stocks to
study the physical distribution of the
markers on homologous group 2 chromosomes (Röder et al. 1998a).
Genomewide genetic maps of DNA
markers can assist in determining the correct homologous relationships of chromosomes in a disomic tetraploid like cotton. In many species, the chromosomes
are designated in sequential order based
on their relative sizes. Current genetic
maps in cotton do not cover all cotton
chromosomes. Thus identification of all
homologies is not yet achieved. Chromosome numbers in cotton are presently assigned based on pairing relationships in
diploid ⫻ tetraploid crosses ( Kimber
1961). Chromosomes are ordered 1–13 for
the A genome and 14–26 for the D genome
(Stelly 1993). Classical evidence for homology was based on the similarity of mutants phenotypes, genetic complementation, synteny, and linkage between
chromosomes 1 and 15, 7 and 16, and 12
and 26 as reported by Endrizzi et al.
(1985). Homology of chromosomes 9 and
23 was detected by conserved synteny between 5S and major 18S–28S rDNA loci.
Duplication, synteny, and linkages among
RFLP loci reinforced evidence of several of
these homologies, and provided the first
clear evidence of homology between chromosomes 5 and 20, and 6 and 25 (Reinisch
et al. 1994). Homologous assemblages
have been constructed for the allotetraploid genome based on gene synteny and
colinear regions with the A and D genome
diploid maps ( Brubaker et al. 1999).
The objective of this study was to assign newly developed SSR markers to cotton chromosomes that would provide the
basis for a framework genetic map. This
framework map of portable PCR-based codominant markers could be integrated
with existing and future genetic maps.
This will enhance the development of consensus genetic maps with genomewide
coverage of the 26 chromosomes of cotton.
Materials and Methods
SSR primer pairs were derived by screening a repeat-enriched cotton genomic library at Brookhaven National Laboratory
( BNL). Positive clones were sequenced
and further screened for SSR development. All BNL clone sequences used for
SSR primer construction are available at
CottonDB ( http://ars-genome.cornell.edu).
SSRs were assigned to cotton chromosome and chromosome arms in a manner
described by Stelly (1993) for codominant
DNA markers. Genetic stocks monosomic
for G. barbadense chromosomes 1, 2, 3, 4,
6, 7, 9, 10, 12, 16, 18, 20, and 25 were available for assignment of DNA markers to entire chromosomes. In addition, stocks
monotelodisomic for G. barbadense chromosome arms 1Lo, 2Lo, 3Lo, 4Lo, 5Lo, 6Lo,
9Lo, 10Lo, 12Lo, 14Lo, 15Lo, 18Lo, 20Lo,
22Lo, 25Lo, 2sh, 3sh, 4sh, 6sh, 7sh, 10sh,
16sh, 17sh, 18sh, 20sh, 22sh, and 26sh were
used. These stocks were obtained from
the Cotton Cytogenetics Collection at Texas A&M University and evaluated as
monosomic or monotelodisomic TM1/3-79
F1s. In each F1, the ‘‘donor genotype’’ is euploid G. barbadense accession 3-79 and the
‘‘recipient genotype’’ is hypoaneuploid G.
hirsutum, usually a backcross derivative of
accession TM-1. TM-1 is an inbred line derived from ‘‘Deltapine 14’’ and is considered the genetic standard of Upland cotton (G. hirsutum) ( Kohel et al. 1970). The
inbred 3-79 is a doubled haploid derived
from G. barbadense. A monosomic F1 substitution stock has a single chromosome
from the donor substituted for the corresponding chromosome pair of the recipient genotype. Similarly, monotelodisomic
F1 stocks lack alleles from the recurrent
parent in the hemizygous chromosome
arm from the donor, but carry alleles of
the recurrent parent in the opposing arm
(either in homozygous or heterozygous
condition, depending on the patterns of
crossing over). Reinisch et al. (1994) described an informative chromosome as
one where only the 3-79 allele is exhibited,
not the TM-1, for the respective whole
chromosome or chromosome arm. If the
SSR locus is at some site other than the
deficient segment, all F1 monosomic substitution stocks will be heterozygous for
TM-1 and 3-79 alleles. For an informative
chromosome or chromosome arm, the interspecific
chromatin-substitution
F1
stocks will be hemizygous for the 3-79 SSR
allele, that is, deficient for the TM-1 allele.
Monosomic or monoditelosomic stocks
were unavailable for cotton chromosomes
8, 11, 13, 19, 21, 23, or 24. In addition to
the aneuploid stocks, euploid TM-1, 3-79,
and TM-1/3-79 F1 hybrids were analyzed as
controls.
Genomic identity of SSR loci amplified in
TM-1 and 3-79 were determined by screening representatives of the putative donors
of the A genome (G. herbaceum, PI408780)
and the D genome (G. raimondii,
PI530947). Several accessions from each
species were tested for polymorphisms
with the SSR markers and found to be remarkably intraspecifically monomorphic;
Liu et al • Cotton Microsatellite Loci 327
thus a single representative accession per
species was chosen for this study.
DNA Isolation
DNA was isolated from plants of TM1, 379, and euploid TM1/3-79 F1, and all the
aneuploid genetic stocks. We used a modified method of DNA extraction with the
DNAeasy Plant Mini Kit (Qiagen, Santa
Clarita, CA). Ground freeze-dried tissue
from fresh young leaves was used instead
of tissue ground in liquid nitrogen. The remainder of the protocol was described in
the manufacturer’s instructions. DNA solutions were quantified using a TKO 100
fluorometer ( Hoefer Scientific Instruments, San Francisco, CA) and working
concentrations diluted in 1⫻ TE buffer (10
ng/␮l). Working stocks were stored at
⫺20⬚C until PCR amplification.
Table 1. Description of the 65 SSR primer pairs used to amplify marker loci in the cotton aneuploid
stocks
Microsatellite
name
Bin 1
BNL2572
BNL3442
BNL3084
BNL2634
BNL3556
BNL3147
BNL3563
Bin 2Ac
BNL1672
BNL1317
Amplification Protocols
SSR primers were selected based on their
ability to yield polymorphic products
when screened on TM1 and 3-79. Most
SSRs revealed two alleles, one for G. hirsutum ( TM1) and one for G. barbadense (379). In addition, based on agarose gels,
they yielded PCR fragments suitable for
further multiplex PCR. Initially, unlabeled
oligonucleotide primers were obtained
from Research Genetics Inc. ( Huntsville,
AL) and a few primers, those denoted as
CML, were provided by A. S. Reddy ( Texas
A&M University, College Station, TX). PCR
multiplex primer bins were constructed
according to the following criteria: primers in the same bin yielding PCR fragments
(alleles) of similar size were labeled with
different fluorescent dyes, and PCR fragments in the same bin labeled with the
same dye had to differ by at least 50 bp.
The arrangement of SSR primer pairs in
the different multiplex bins is shown in Table 1. Some primer bins were combined
after PCR to make a multiloading bin. Ten
SSR primer pairs were run individually because they were incompatible with multiplex PCR due to the amplified product
size, primer composition, or both.
Multiplex PCR conditions were used to
develop high-throughput assays for SSR
loci detection and scoring. Fifty-five fluorescent oligonucleotides ( Table 1) were
obtained from Perkin-Elmer/Applied Biosystems, Norwalk, CT. The 5⬘ end of the
‘‘forward’’ primer was labeled with either
6-FAM (6-carboxyfluorescein), HEX (4,7,2⬘,
4⬘, 5⬘, 7⬘-hexachloro-6-carboxyfluorescein),
or NED (7⬘, 8⬘-benzo-5⬘-fluoro-2⬘, 4,7-trichloro-5-carboxyfluorescein).
Both multiplex and single PCR reactions
328 The Journal of Heredity 2000:91(4)
BNL1414
BNL1053
BNL1064
BNL3065
Bin 2Bc
BNL2960
BNL1679
BNL169
Bin 3
BNL3279
BNL3482
BNL3599
BNL840
BNL1350
Bin 4Ad
BNL786
BNL3816
BNL3971
Bin 4Bd
BNL1721
BNL3255
Bin 5
BNL3449
BNL3627
CML60
CML63
Flanking primer sequence (5⬘→3⬘)a
Microsatellite compositionb
MgCl2 (mM)
3.00
6FAM-GTCCTATTACTAAAATTGTTAATTTAGCC
CGATGTTAAATCAATCAGGTCA
HEX-CATTAGCGGATTTGTCGTGA
AACGAACAAAGCAAAGCGAT
HEX-TGTTCATAAAATGAAATCCAAGC
AGTGCGCGACGTAAGTAACC
HEX-AACAACATTGAAAGTCGGGG
CCCAGCTGCTTATTGGTTTC
NED-CCTTTCATGACCCTGCAAAT
AGATGGGGAATGGATCTGTG
NED-ATGGCTCTCTCTGAGCGTGT
CGGTTCAGAGGCTTTGTTGT
NED-AAGCATAAACTTGACACAAGCC
AATGGGCAAGAAAAGGGAAC
(GA)23
(CA)14( TA)5
(GA)12
(AG)11
(AC)12(AT )4
(AG)11
(CA)13( TA)4
3.00
6FAM-TGGATTTGTCCCTCTGTGTG
AACCAACTTTTCCAACACCG
6FAM-AAAAATCAGCCAAATTGGGA
CGTCAACAATTGTCCCAAGA
HEX-AAAAACCCCTTTCCATCCAT
GGGTGTCCTTCCCAAAAATT
HEX-AGGGTCTGTCATGGTTGGAG
CATGCATGCGTACGTGTGTA
NED-TTTGCGGGTAATCCTATTGC
TGTCTATGGGACATTTCGCA
NED-CAAACGGGAGACCAAAAAAA
CGAACTGGCGAGTTAGTGCT
(AG)14
(AG)14
(AG)16
(AC)16
(CA)13
(AG)21
3.00
6FAM-TAAGCTCTGGAGGCCAAAAA
CCATTTCAATTTCAAGCATACG
HEX-AATTGAGTGATACTAGCATTTCAGC
AAAGGGATTTGCTGGCAGTA
NED-TCACAAATAAAAGTGAAATTGCG
GGCTGGTGACCATAAAAGGA
(GA)10
6FAM-CATGTCCAATGGATGTGTCA
GGGCCACTTAAAGGCATTCT
HEX-ATTTGCCCCAGGTTTTTTTT
GCAACACCTTTTCCTCCCTA
HEX-TTTAGCCCCAGTAACATGCC
ACTGCAAGCTCTGCCCTAAA
NED-CTCGTGGAAACACCAGGAAT
TCTCGCCATTAAACTGCCTT
NEE-TAGGAGGAGAAGTTGGCGAA
CAAGATGTGACCTTACCGCC
(AG)15
(AG)17
(GA)15
2.50
(AC)12
( TC)15
(CA)19
(CA)8(GA)16
2.50
6FAM-CTTTCCACGTGTAATTTGTTGATA
GATCTTAACTCTTGCTCTCTCTCTCTC
NED-GTTAGCCACGTGTTAGTTCTATG
ATCGATCACTTGCTGGTTCC
NED-CACATATTTTTGCCTCACGC
TGTGGACCCAAAAAGGAAGA
(AG)14
6FAM-TGTCGGAATCTTAAGACCGG
GCGCAGATCCTCTTACCAAA
HEX-GACAGTCAAACAGAACAGATATGC
TTACACGACTTGTTCCCACG
(AG)17
6FAM-AAGCTGTGGCTATGATGCCT
AGAGCAAAAAACAATTACAAAAGC
6FAM-TATGGGCCTGTCCACCTAAG
CAAAGCAACATGCACACACA
HEX-GAAGATTCCATCTGCAGACCCAG
CCAACAAAACCATAAACATGAACTC
NED-GTCTGCACTGCTCGGTTATGTGAG
GCAGAAAAGTGTTTAACTTGCGACAG
(CT )6TA(CA)12
( TG)5TA( TG)15
( TC)15
2.50
(GC)6AT(AC)14
2.50
( TC)17
—
—
Table 1. Continued.
Microsatellite
name
BNL1665
BNL3441
Bin 6
BNL3090
BNL256
BNL3792
BNL686
BNL2895
BNL252
Bin 7
BNL3034
BNL3408
BNL3383
Bin 8
BNL3902
BNL3895
Bin 9
BNL3955
BNL3649
Bin 10
BNL3103
BNL3995
Bin 11
CML66
BNL1440
BNL2590
CML43
BNL3452
BNL1059
BNL2544
BNL3479
Flanking primer sequence (5⬘→3⬘)a
Microsatellite compositionb
NED-CAGAACCAACATACTTTCTACGG
ATGTGCAAAAACTTGATGTGG
NED-CGTCATAAACCGTGCTTGTG
GGCCACTTTAAGGCTGTCAC
(AG)16
MgCl2 (mM)
(AT )2(AC)18(AT )4
3.25
6FAM-GAAATCATTGGAAGAACATATACTACA
TTGCTCCGTATTTTCCAGCT
6FAM-TTTTGCTCCATTTTTTTGCC
TTTATTAATTTCGTTTAGCTTCCG
HEX-TTCGAGATCCCCTGTTCTGA
CATATTCCAGTCAAACCAAACG
HEX-ATTTTTCCCTTGGTGGTCCT
ACATGATAGAAATATAAACCAAACACG
NED-CGATTTTACTGCTTCAGACTTG
TACCATCTCACGGATCCACA
NED-TGAAGAGCTCGTTGTTGCAC
CGAAAGAGACAAGCAATGCA
(AG)31
(GA)17
( TG)21
(GA)22
(GA)10
(CT )21
2.50
6FAM-AAAGGAAATGGTCATTGGCA
AGTACCCGCCATTTCAAGTG
HEX-ATCCAAACCATTGCACCACT
GTGTACGTTGAGAAGTCATCTGC
NED-GTGTTGTCATCGGCACTGAC
TGCAATGGTTCAGTGGTGAT
(AG)12
(GT )2AT(GT )12
(AG)4AC(AG)10
2.50
6FAM-GAGTTTGGGGGCTGTGTATG
GGGGTGCTTATGTCAGACGT
HEX-CGCTCTTGGTCATGGATTTT
GCCAAGCTCACTGGAAGAAC
(GT )18
( TG)10
2.50
HEX-AGAGATGCAATGGGATCGAC
ATGTGATAATGCGGGGAATG
NED-GCAAAAACGAGTTGACCCAT
CCTGGTTTTCAAGCCTGTTC
(CA)12
( TC)20
3.25
HEX-ACTTTGAGATATTGTTATTCTACCCG
TCGAACAATTACGAATCAAATG
NED-ATATTTTATTCTTTTAATAGCTTTATTCCC
TTGGAAAAACCCATGGTGAT
(GA)14
6FAM-GGATACGTAGGCCTCCACATATTC
GCAGAAAAGTGTTTAACTTGCGACAG
6FAM-CCGAAATATACTTGTCATCTAAACG
CCCCCGGACTAATTTTTCAA
6FAM-GAAAAACCAAAAAGGAAAATCG
CTCCCTCTCTCTAACCGGCT
HEX-GCGCAGATATTATTATCACAGC
TATATAAATTTGCATCAGTTGGC
HEX-TGTAACTGAGCAGCCGTACG
GCCAAAGCAGAGTGAGATCC
HEX-CCTTCTCTGACACTCTGCCC
TGTTTCTCTTCTTTTCCTTATACTTTT
NED-GCCGAAACTAAAACGTCCAA
TCCTTACTCACTAAGCAGCCG
NED-AGTGGGTTGGACTTTCATGC
CACGGGCTTTTTTTTTTTCA
—
(AC)16
3.00
Individual Primers Not Used in Multiplex PCR Bins
BNL3888
GCCCACTTTGCCTCTTACAG
AGCTTTTCCCCTTTCACCAT
BNL1434
AAATTCAAGAATCAAAAAACAACA
TTATGCCAAAGTATATGGAGTAACG
BNL1597
GGGCTTTCCGATACTGAACA
CCTGCAATAAGGCGTTCAAT
BNL2496
TCGAAATGAATTTAGATGACCA
TCCTTTTTTTTGTACTTCTCTTGC
BNL3558
AAGCAAATCATGATGAACATACG
TGCGAAGAGTAGCTCTGCTG
(AG)15
(AG)11
—
(CA)13
(CA)16
(AG)11
( TC)6T(AC)15G(CA)2
( TG)15
2.5
(AG)13
2.5
(GA)13
2.5
(GA)15
2.5
(AC)11
2.5
were performed in 10 ␮l volumes containing 10 ng of cotton template DNA, 1⫻ Perkin-Elmer PCR Buffer II (10 mM Tris-HCl,
pH 8.3, 50 mM KCl), 2.5–3.5 mM MgCl2, 0.2
mM dNTPs, 0.15 ␮M of each single primer,
0.4 unit of AmpliTaq Gold (Perkin-Elmer/
Applied Biosystems). The actual MgCl2
concentration for each PCR single or multiplex bin is shown in Table 1. Dye-labeled
‘‘forward’’ and unlabeled ‘‘reverse’’ primers were used in all multiplex PCR reactions. Temperature cycling was conducted
on a GeneAmp PCR System 9600 (PerkinElmer/Applied Biosystems). The amplification profile consisted of an initial period
of DNA denaturation and AmpliTaq Gold
activation at 95⬚C for 12 min, followed by
40 cycles of 93⬚C (step 1) for 15 s, 55⬚C
(step 2) for 30 s, and 72⬚C (step 3) for 1
min. The ramp time was 42 s to step 1, 36
s to step 2, and 40 s to step 3. After 40
cycles, the extension temperature of 72⬚C
was held for 6 min. Multiplex PCR products were diluted 5- to 10-fold before preparing the loading samples.
Electrophoresis and Detection
Samples containing 1.0 ␮l of diluted multiplex PCR products and 1.0 ␮l loading
cocktail [60% formamide, 17% GeneScan
350 ROX (6-carboxy-x-rhodamine) internal
lane size standard (Perkin-Elmer/Applied
Biosystems), 23% accompanied loading
buffer] were heated at 92⬚C for 2 min,
placed on ice, then loaded on a 5% Long
Ranger ( FMC BioProducts, Rockland,
ME) denaturing acrylamide gel (6 M urea).
Each gel was used twice. Samples were
electrophoresed on an automatic DNA sequencer (Perkin-Elmer/Applied Biosystems, model 377) in 1⫻ TBE running buffer (89 mM Tris, 89 mM borate, 2.5 mM
EDTA pH 8.3) at a constant voltage (1680
V) and a constant temperature of 45⬚C for
1 h 15 min. Short gels (12 cm well-to-read)
were used with a square-tooth comb. Fragment size data was automatically collected with GeneScan version 2.0.2 (PerkinElmer/Applied Biosystem). The fragment
with the highest fluorescent intensity was
scored in amplified SSRs that showed
band stuttering. The ‘‘local Southern’’ algorithm ( Elder and Southern 1987) was
used to automatically determine fragment
size.
Single primer-pair PCR reactions involved unlabeled primers, thus they were
scored on high-resolution agarose gels.
Each of these undiluted PCR products
were mixed with 5 ␮l of 6⫻ loading buffer
and loaded directly on a 3.5% (w/v)
MetaPhore ( FMC BioProducts, Rockland
Liu et al • Cotton Microsatellite Loci 329
Table 1. Continued.
Microsatellite
name
BNL3359
BNL2553
BNL3646
BNL3008
BNL448
Flanking primer sequence (5⬘→3⬘)a
Microsatellite compositionb
MgCl2 (mM)
TTGTTGTTGGGAATGATGGA
TGACCCTTCACCGACTTTCT
GGGTCAAAAGTGGAAAACGA
GCCCACAGGAAAACAAAAAA
CCCAATACGAGGAGAGCACA
TCGAAAATGGGGGAGAGAG
ATCTCAGCTTTAAACATATAATAGAGGG
TAAAATGAAGGCCATCAGGC
GCAGCTTGCTTTTCTGCTTC
ACGCAAGCTTGGTCAATACC
(AG)16
2.5
(GA)10
2.5
( TC)14
2.5
(GA)13
2.5
(CT )13
2.5
Upper sequence is forward-labeled primer, lower is the reverse primer.
In TM1, composition unavailable for CML primers.
c
Bins 2A and 2B were combined after PCR and multiloaded on the gel.
d
Bins 4A and 4B were combined after PCR and multiloaded on the gel.
a
b
ME) gel. The 20 cm gel was prepared with
1⫻ TBE buffer (89 mM Tris, 89 mM borate,
2 mM EDTA, pH 8.3) according to the manufacturer’s instructions and run in 1⫻ TBE
buffer at 100 V for 6 h.
A size standard (pBR322 DNA–MspI digest) was loaded in the first, middle, and
last lane of each gel. After electrophoresis,
the gel was stained in 0.5 ␮g/ml ethidium
bromide for 30 min and destained in water
for 15 min. Gels were photographed with
an AlphaImager 950 Gel Documentation
System (Alpha Innotech Corp., San Leandro, CA). SSR bands were accurately sized
with ProRFLP version 2.17 ( DNA ProScan
Inc., Nashville, TN).
Results
Sixty-five SSR primer pairs were used to
amplify SSR marker loci in the cotton parental and aneuploid stocks ( Table 1).
Multiplex PCR and automated detection of
amplified products were optimized for 55
of the SSR primer pairs. Thirteen multiplex
PCR bins were developed with an average
of four SSR primer pairs per bin. Bins 2A
and 2B were combined after PCR as were
bins 4A and 4B prior to gel loading. Seventy-one SSR loci were identified in this
experiment ( Table 2). The SSR allele fragments amplified ranged in size from 101 to
270 bp. All marker loci segregated normally as codominant loci in a TM1/
NM24016 F2 cotton genetic mapping population ( Liu 1999).
This method of genotyping yielded unambiguous and precise fragment detection
of SSR alleles and their chromosome location (Figure 1). The SSR markers were codominant, producing a TM-1 allele and a 379 allele in the euploid TM-1/3-79 F1. Markers
were found on all of the monosomic stocks
except for chromosomes 1 and 4. Forty-two
330 The Journal of Heredity 2000:91(4)
markers were localized to either a chromosome or a chromosome arm in the aneuploid stocks. This suggested that the remaining SSR markers with undesignated
locations in Table 2 were likely on chromosomes where aneuploid lines were not available (i.e., chromosomes 8, 11, 13, 19, 21, 23,
or 24). Primer pairs BNL3408, BNL3599,
BNL1414, BNL1440, CML66, and BNL686
each amplified two loci. When two loci were
observed they were labeled in a consecutive
fashion, beginning with the locus possessing
the smallest alleles, that is, BNL686-1 and
BNL686-2. Figure 1 illustrates the amplified
products for both loci amplified by BNL686.
Possibly BNL1440 amplified two homologous SSR loci; BNL1440-1 on chromosome 25
and BNL1440-2 on chromosome 6. Reinisch
et al. (1994) also reported duplicate RFLP
loci on these two putative homologues.
BNL3408 amplified one SSR loci on 3sh and
one on 17Lo, which may be homologues, although there is no other supporting evidence. As more aneuploid cotton stocks are
developed, the potential exists for further
identifying homologues with duplicate SSR
marker loci.
Surprisingly, only 30 SSR loci could be
unambiguously assigned to diploid subgenome A or D. Nineteen were localized
to the A genome and 11 were on the D
genome. The other SSR primer pairs either
failed to amplify in either diploid or amplified a significantly different size product
from that found in TM-1 or 3-79. This subgenome assignment should be considered
preliminary since homoplasy could bias
comparison of electromorphs in the tetraploids with the putative diploid subgenomes. Microsatellite size homoplasy
was reported by Doyle et al. (1998) for
chloroplast markers in wild perennial relatives of soybean. Viard et al. (1998) detected size homoplasy in sequenced elec-
tromorphs of SSRs of Apis mellifera,
Bombus terrestris, and Bulinus truncatus.
Further experiments are under way in our
laboratory to sequence the different amplified products in the diploids to ensure
they contain the appropriate SSR. Of interest, of the SSR loci assigned to the A or D
genomes ( Table 2), there are no conflicts
with the assignments to chromosomes
based on the monosomics and monotelodisomics. A-genome-specific markers were
assigned to chromosomes 1–13 and D genome markers to chromosomes 14–16. For
chromosomes 2, 3, 6, 9, 10, 12, 18, 20, and
25 markers were localized to chromosome
arms based on the monotelodisomics, the
observed results agreed with that from
the monosomics for the same chromosomes. That is, there were no conflicts in
data derived from the two sets of aneuploids.
Discussion
The relatively large number of chromosomes complicates cotton genetic map
construction, along with the propensity
for extensive recombination per homologous chromosome arm and polyploid-associated redundancy (Stelly 1993). Linkage mapping with DNA markers in cotton
may yield multiple linkage groups for individual chromosomes in the early stages
of map construction ( Liu 1999). This is a
function of the number of markers and the
recombination ‘‘size’’ of the chromosomes. The forty-two SSR marker loci localized to cotton chromosomes and/or
chromosome arms in this experiment will
aid in anchoring present and future linkage groups to individual chromosomes.
This is the first report of the assignment
of SSR markers to cotton chromosomes.
The SSRs in this experiment were scattered over the various cotton chromosomes with no apparent clustering pattern. At least one SSR marker was
assigned to each of 14 different cotton
chromosomes and 32 markers were localized to 12 different chromosome arms.
The number of SSR markers remaining to
be assigned to cotton chromosomes is a
function of the lack of a complete monosomic series in cotton. Data from wheat
suggest that microsatellites are not localized to specific chromosome regions, but
are found in centromeric and interstitial
as well as telomeric regions (Röder et al.
1998a).
Based on tests with the 26-chromosome
species, genomic affinities (A versus D)
could only be deduced for 30 of the 71 SSR
Table 2. Seventy-one SSR loci amplified in cotton aneuploid stocks
a
b
SSR locus
Mutiplex
bin
Chromosome
location
Allele sizea
Genomeb
BNL1434
BNL3971
BNL1053
BNL3441
BNL3408-1
BNL3556
BNL3995
BNL3255
BNL3359
BNL1064
BNL1440-2
CML60
CML66-1
BNL1597
BNL686-2
BNL1414-1
BNL2590
BNL3563
BNL2960
BNL1665
BNL3895
BNL256
BNL1679
BNL3599-2
BNL3065
BNL3408-2
BNL3955
BNL2496
BNL3479
BNL1721
CML63
BNL2544
BNL3558
BNL2553
BNL3646
BNL3008
BNL448
BNL3792
BNL169
BNL3103
BNL1440-1
BNL3482
BNL3888
BNL3442
BNL3084
BNL3599-1
BNL3816
BNL3627
BNL252
BNL3902
BNL3649
BNL3452
BNL2572
BNL2634
BNL3147
BNL1672
BNL1317
BNL3279
BNL840
BNL1350
BNL786
BNL3449
BNL3090
BNL686-1
BNL2895
BNL3034
BNL3383
CML66-2
CML43
BNL1059
BNL1414-2
—
4A
2A
5
7
1
10
4B
—
2A
11
5
11
—
6
2A
11
1
2B
5
8
6
2B
3
2A
7
9
—
11
4B
5
11
—
—
—
—
—
6
2B
10
11
3
—
1
1
3
4A
5
6
8
9
11
1
1
1
2A
2A
3
3
3
4A
5
6
6
6
7
7
11
11
11
2A
2
2Lo
3
3sh
3sh
5sh
5sh
5sh
6
6sh
6sh
7Lo
7Lo
7
9sh
9Lo
9Lo
10Lo
10Lo
10Lo
10Lo
10Lo
12
12sh
16
17Lo
17Lo
17Lo
18Lo
18Lo
18sh
18sh
18
20
20
20
20Lo
20Lo
20sh
25sh
25sh
26Lo
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
236/250
146/122
194/176
214/206
132/136
138/134
198/190
232/210
212/218
145/153
262/270
125/119
132/128
201/207
185/189
138/128
190/206
230/248
150/186
162/150
188/200
208/188
164/190
216/212
192/182
146/174
171/195
112/130
248/256
188/176
138/126
219/207
216/212
196/200
166/155
133/141
205/203
235/215
198/214
190/206
241/245
140/146
182/194
132/146
164/160
193/179
203/187
184/176
179/169
194/174
183/193
192/198
254/240
254/264
165/155
107/101
191/209
126/108
165/155
197/207
117/107
150/134
215/227
158/144
210/226
158/174
191/177
140/162
116/112
218/230
157/165
A1(234)
A1(146)
—
—
—
A1(130)
A1(190)
—
A1(202)
A1(147)
—
—
—
A1(193)
—
A1(128)
—
A1(230)
A1(150)
A1(150)
—
—
A1(190)
—
—
—
—
D5(120)
—
D5(176)
D5(126)
D5(219)
D5(214)
D5(200)
—
D5(149)
—
—
D5(200)
—
—
—
A1(186)
A1(132)
D5(160)
D5(193)
A1(203)
A1(188)
A1(179)
D5(174)
A1(183)
A1(198)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Denotes bp size of TM-1 allele fragment and 3-79 allele fragment, respectively.
Subgenome designation, with A1 for G. herbaceum (PI408780) and D5 for G. ramondii (PI530947). Size of amplified
product in the diploid is denoted in parentheses.
loci in this study. The proportion of SSR
loci that could be assigned to a subgenome by testing against the 26-chromosome
species, approximately 42%, is similar to
that reported for RFLPs by Reinisch et al.
(1994). In their experiment, 115 of 276
fragments yielded definitive information.
In this study, more SSR markers were assigned to the A genome than to the D genome. As more loci are assembled into genetic linkage groups, it will become
increasingly easy to infer the genome of
origin for individual SSR loci, particularly
for those on chromosomally identified
linkage groups. Higher numbers of markers might be expected from the A than D
subgenomes, given that the former is nearly twice as large physically (Reinisch et al.
1994). Moreover, at this early stage of testing, the likelihood of assignment may differ between the subgenomes according to
their relatedness to extant A and D genomes of 26-chromosome species. Jiang et
al. (1998) speculated that the D subgenome in 52-chromosome cotton has diverged
further from its ancestral genome during
polyploidization than has the A subgenome from its ancestral genome. Such differences could affect primer annealing in extant A- and D-genome species with
AD-derived primers; if so, it would be
more difficult to determine genome origin
of markers on the D subgenome of 52chromosome cotton by comparing to contemporary G. raimondii ( D5D5). With assembly into linkage groups, however, the
heritage will become evident.
In contrast to RFLPs, most cotton microsatellite locus primer pairs were found to
amplify only one locus, specific either to
the A or D genome. In just six cases, the
SSR primer pairs amplified fragments from
two loci that were independent, based on
linkage analysis in a mapping population
( Liu 1999). Microsatellite primer pairs
BNL3408 and BNL1440 seemed to amplify
homologous SSR loci. BNL3408 amplified
loci on chromosome 3(A) and 17( D) while
BNL1440 amplified loci on 6(A) and 25( D).
A high priority should be placed on developing more comprehensive cytogenetic
stocks in cotton, since chromosomal identification is fundamental to establishing a
good genetic map. Nevertheless, the reported SSRs collectively provide a framework of codominant markers that can be
used in building a consensus genetic map
of cotton. However, many more SSR markers are needed to achieve genomewide
coverage of the cotton genome.
The PCR basis of SSR analysis renders it
amenable to scale-up and thus use for
Liu et al • Cotton Microsatellite Loci 331
marker-assisted selection in large breeding programs. Incorporation of these SSR
loci into existing genetic maps will facilitate assignment of linkage groups to cotton chromosomes. The SSR markers can
be scored in a very efficient manner using
multiplex PCR bins and semiautomated genotyping. On the ABI377 automated DNA
sequencer with 36-lane capacity, six gels
can be run in a single day. With an average
of four SSR markers scored in each bin,
this translates into approximately 864
data points per machine-day. The high
throughput capacity is best illustrated by
multiplex PCR bin 11 in this experiment
that contains 8 SSR primer pairs that amplify 10 polymorphic SSR loci. At the rate
of detection just mentioned, this SSR bin
would yield 2160 data points per machineday.
New methods for discovering microsatellites by screening bacterial artificial
chromosomes ( BACs) with labeled oligonucleotides show great promise to increase the number of SSRs in rice ( Zhang
et al. 1996) and soybeans (Cregan et al.
1999). This has the potential to populate
regions of the genome currently without
SSR markers developed previously by nontargeted methods of marker development.
Similar procedures in cotton are warranted since several high-quality BAC libraries
are available and more are continually being developed. The important aspect of
deriving future cotton SSRs from BAC libraries is the opportunity to efficiently integrate the genetic and physical maps of
cotton.
complex traits (Paterson AH, ed). Boca Raton, FL: CRC
Press; 13–29.
marker-1. Description of a genetic standard for Gossypium hirsutum L. Crop Sci 10:670–671.
Brubaker CL, Paterson AH, and Wendel JF, 1999. Comparative genetic mapping of allotetraploid cotton and
its diploid progenitors. Genome 42:184–203.
Liu S, 1999. Cotton genetic mapping and QTL analysis
using simple sequence repeat markers and the identification of their chromosome locations (Masters thesis). Las Cruces, NM: New Mexico State University.
Jiang C, Wright RJ, El-Zik KM, and Paterson AH, 1998.
Polyploid formation created unique avenues for response to selection in Gossypium (cotton). Proc Natl
Acad Sci USA 95:4419–4424.
Zhang HB, Choi S, Woo SS, Li Z, and Wing RA, 1996.
Construction and characterization of two rice bacterial
artificial chromosome libraries from the parents of a
permanent recombinant inbred mapping population.
Mol Breed 2:11–14.
References
Kimber G, 1961. Basis of the diploid-like meiotic behavior of polyploid cotton. Nature 191:98–99.
Received July 10, 1999
Accepted December 31, 1999
Kohel RJ, Richmond TR, and Lewis CF, 1970. Texas
Corresponding Editor: Susan Gabay-Laughnan
Burow MD and Blake TK, 1998. Molecular tools for the
study of complex traits. In: Molecular dissection of
332 The Journal of Heredity 2000:91(4)
Chen X, Temnykh S, Xu Y, Cho YG, and McCough SR,
1997. Development of a microsatellite framework map
providing genome-wide coverage in rice (Oryza sativa
L.). Theor Appl Genet 95:553–567.
Cregan PB, Mudge J, Fickus EW, Marek LF, Danesh D,
Denny R, Shoemaker RC, Matthews BF, Jarvik T, and
Young ND, 1999. Targeted isolation of simple sequence
repeat markers through the use of bacterial artificial
chromosomes. Theor Appl Genet 98:919–928.
Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal
A, Millasseau P, Marc S, Hazan J, Seboun E, Lathrop M,
Gyapay G, Morissette J, and Welssenbach J, 1996. A
comprehensive genetic map of the human genome
based on 5,264 microsatellites. Nature 380:152–154.
Dietrich WF, Miller J, Steen R, Merchant MA, Damronboles D, Husain Z, Dredge R, Daly MJ, Ingalls KA,
O’Connor TJ, Evans CA, DeAngelis MM, Levinson DM,
Kruglyak L, Goodman N, Copeland NG, Jenkins NA,
Hawkins TL, Stein L, Page DC, and Lander ES. 1996. A
comprehensive genetic map of the mouse genome. Nature 380:149–152.
Diwan N and Cregan PB, 1997. Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers
to assay genetic variation in soybean. Theor Appl Genet 95:723–733.
Doyle JJ, Morgante M, Tingey SV and Powell W, 1998.
Size homoplasy in chloroplast microsatellites of wild
perennial relatives of soybean (Glycine subgenus Glycine). Mol Biol Evol 15:215–218.
Elder JK and Southern EM, 1987. Computer-aided analysis of one-dimensional restriction fragment gels. In:
Nucleic acid and protein sequence analysis—a practical approach ( Bishop MJ and Rawlings CJ, eds). Oxford: IRL Press; 165–172.
Endrizzi JE, Turcotte EL, and Kohel RJ, 1985. Genetics,
cytology and evolution of Gossypium. Adv Genet 23:
271–375.
Goldstein DB and Pollock DD, 1997. Launching microsatellites: a review of mutation processes and methods
of phylogenetic inference. J Hered 88:335–342.
Mitchell SE, Kresovich S, Jester CA, Hernandez CJ,
Szewc-McFadden AK, 1997. Application of multiplex
PCR and fluorescence-based, semi-automated allele sizing technology for genotyping plant genetic resources.
Crop Sci 37:617–624.
Reinisch AJ, Dong J, Brubaker CL, Stelly DM, Wendel
JF, and Paterson AF, 1994. A detailed RFLP map of cotton, Gossypium hirsutum ⫻ Gossypium barbadense:
chromosome organization and evolution in a disomic
polyploid genome. Genetics 138:829–847.
Röder MS, Korzun V, Gill BS, and Ganal MW, 1998a. The
physical mapping of microsatellite markers in wheat.
Genome 41:278–283.
Röder MS, Korzun V, Wendehake K, Plaschke J, Tixier
MH, Leroy P, and Ganal MW, 1998b. A microsatellite
map of wheat. Genetics 149:2007–2023.
Saha S and Stelly DM, 1994. Chromosomal location of
Phosphoglucomutase 7 locus in Gossypium hirsutum. J
Hered 85:35–39.
Schmidt T and Heslop-Harrison JS, 1998. Genomes,
genes and junk: the large-scale organization of plant
chromosomes. Trends Plant Sci 3:195–198.
Schuler GD, 1998. Electronic PCR: bridging the gap between genome mapping and genome sequencing.
Trends Biotechnol 16:456–459.
Shappley ZW, Jenkins JN, Meredith WR, and McCarty
JC Jr, 1998. An RFLP linkage map of upland cotton, Gossypium hirsutum L. Theor Appl Genet 97:756–761.
Stelly DM, 1993. Interfacing cytogenetics with the cotton genome mapping effort. In: Beltwide Cotton Conference, January 10–14, 1993, New Orleans, LA ( Herber
DJ and Richter DA, eds). Memphis, TN: National Cotton
Council of America; 1545–1550.
Viard F, Franck P, Dubois MP, Estoup A, and Jarne P,
1998. Variation of microsatellite size homoplasy across
electromorphs, loci, and populations in 3 invertebrate
species. J Mol Evol 47:42–51.
Yu K, Park SJ, and Poysa V, 1999. Abundance and variation of microsatellite DNA sequences in beans (Phaseolus and Vigna). Genome 42:27–34.