Microsatellite Sequences as Useful
Genetic Markers in Sugarcane
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GM Cordeiro ([email protected]) and RJ Henry
CENTRE FOR PLANT CONSERVATION GENETICS
Southern Cross University
PO Box 157 Lismore 2480, Australia
C P C G
Sugar Research
and Development
Corporation
C
ultivated sugarcane clones (Saccharum spp.) are derived from complex
interspecific hybridisations between the species S. spontaneum (2n = 40 to 128)
and S. officinarum (2n= 60 or 80). To analyse this complex genome, and as a
complement to RFLP mapping we have investigated the potential of microsatellite
sequences [2-6bp recurring sequences grouped in 10 or more tandem repeats, also known
as Simple Sequence Repeats (SSRs) as genetic markers in sugarcane with respect to their
abundance, variability and ability to detect polymorphisms. Primer sets were designed and
Centre for
Plant Conservation Genetics
synthesised for over 100 microsatellite sequences from a 60% enriched microsatellite
library. These primers were tested on a population of sugarcane cultivars and both
heterozygosity and polymorphisms were observed. This is an indication that microsatellites
have the power to reveal co-dominantly inherited multi-allelic products of loci useful in
mapping. Microsatellite markers are likely to have many applications in sugarcane genetics
and breeding including germplasm analysis, cultivar identification, parent evaluation and
marker assisted breeding.
Introduction
Several studies involving comparisons between differing
marker systems such as RAPDs, AFLPs, RFLPs and SSRs
indicate AFLPs and SSRs to be highly reproducible techniques
between and within laboratories. The ability of SSRs to reveal
high allelic diversity is particularly useful in distinguishing
between crosses and the success of using these markers in crop
species such as barley, rice and wheat has encouraged the
testing of SSRs in sugarcane.
Allele Inheritance
Microsatellite Description
The most common (86%) repeat motifs were either dinucleotide or trinucleotide repeats (Table
1). On average, dinucleotide repeats had a much higher number (15.5) of repeats than
trinucleotide repeats (12). The maximum recorded for dinucleotides was 49 repeats. With
trinucleotide repeats, the motif TAG/CTA tended to be highly repetitive with as many as 92
repeats recorded and with an average of 39 repeats. The remaining 14% of the repeat motifs
comprised 8 tetramers and 2 pentamers of between 3 and 5 repeats, and 57 mononucleotide
repeats ranging between 8 and over 130 bases (imperfect). Only inserts with dinucleotides and
trinucleotide repeats were used for primer design. The most common repeat motif was the
TG/CA group, which reperesents over 21.7% of all microsatellites detected in S. spp (Table 2).
The TAC/GTA group represented the most common trinucleotide with 6.3% of detected
microsatelllites. Compound motifs comprised 24.9% of microsatellite containing fragments and
included a mix of dinucleotides and trinucleotides.
Table 2. Frequency of themost common
microsatellite repeat motifs in Saccharum spp.
Frequency
60·8%
30·0%
14·6%
73·3%
32·4%
24·9%
Total % is greater than 100 due to the combinations of
dinucleotides and trinucleotides in compound SSRs
*
21·7%
16·1%
10·0%
6·3%
5·2%
5·0%
3·6%
3·3%
2·3%
1·5%
14·6%
Includes Mono-, tetra-, pentanucleotides and all other
repeat motifs with frequency < 1·0%
S. spontaneum
S. officinarum
R570
Q124
Q117
From the total number of sequences, 262 could
be considered useful as they contained either
dinucleotide repeats with more than 8 repeats
motifs, or trinucleotide repeats with more than
6 repeat motifs. Of these, 57·6% had flanking
sequences that was either insufficient or too
complex to permit the design of a PCR the
design of a PCR primer pair. In total, 124
primer pairs were designed, synthesised and
tested. From this, 100 markers produced a
fragment of the expected size. This was made
up of 71 dinucleotide motifs and 29
trinucleotide motifs. Polymorphism was
observed in 91% of the markers. Of the 9
markers that were non-polymorphic, four were
dinucleotide motifs with between 9 and 30
repeats, and five were of trinucleotide motifs
of between 6 and 10 repeats.
Q117
Primer Design and Polymorphism
Figure 1. PCR products from primers of marker
SMC119CG run on an 8% acrylamide gel. Due
to the polyploid nature of sugarcane, the
number of alleles per genotype is high, in this
case, up to 12 alleles. This marker is able to
distinguish at least 3 of the 5 genotypes tested.
A combination of different markers can be used
to fingerprint sugarcane varieties and
germplasm collections.
References
Daniels & Roach (1975) Sugarcane improvement through breeding. Elsevier 7-84.
Jones et al. (1997) Molecular Breeding 3:381-390.
Sreenivasan et al. (1987) Sugarcane improvement through breeding. Elsevier 211-253.
Cross Transferability
Modern cultivated sugarcane cultivars are derived from complex hybrids between
Saccharum officinarum, S barberi and S. sinese, and the wild species S. spontaneum
and S. robustum (Sreenivasan et al. 1987). The “noble” sugar-producing cane,
Saccharum officinarum has been suggested to have evloved through hybridisation of
such species as Erianthus arundinaceus (Retz.) Jeswiet, Miscanthus, S. spontaneum
and S. robustum Brandes and Jeswiet ex Grassl (Daniels et al. 1975). Understanding
the phylogeneitc relationships between the members of the Saccharum complex,
which currently still remains uncler, could assit in the study of the evloutionary and
genome organisation in modern sugarcane and
its relatives, and also aid in breeding programs to
widen the genetic base of sugarcane.
The testing of markers on cultivated sugarcane
and their progenitor species (S. spontaneum and
S. officibarum) show that it is possible to identify
allele inheritance in cultivated canes. These
results suggest that SSR markers will be able to
provide an insight into the genome and
chromosomal evolution of sugarcane. Further
tests will be carried out on progenitor Figure 3: Observation of allele inheritance in
cultivated sugarcane from progenitor species
species such as S. barberi, S. sinense, using primer SMC371CG.
Erianthus and Miscanthus.
S. spontaneum
TG/AC
GA/CT
TA/AT
TAC/GTA
TTG/CAA
CTG/CAG
AAG/CTT
CAT/ATG
AGG/CCT
CG/GC
Others*
S. officinarum
Type of repeat
0·1%
42·7%
0·6%
R570
*
798
Q124
No insert
No microsatellite
Redundant sequences
Type of repeat*
dinucleotides
trinucleotides
other
perfect
imperfect
compound
Q117
Total Sequencing Reactions
Q117
Table 1. Microsatellite enrichment success for
Saccharum spp.
To determine the ability of the markers to reveal
allele inheritance, the forward primers of five
markers were labelled with ABI fluorescent dyes
Chitton
(TET, HEX or FAM) and tested on 2 parents and a
single progeny. The size of alleles generated as
fluorescently labelled PCR products was determined
by capillary electrophoresis on an ABI Prism 310
Saigon
genetic analyser (Applied Biosystems) based on
conditions as determined by the manufacturer.
Alleles were sized using the software program ABI
MQ72–1068
PRISM Genescan Analysis Version 2.0.2.
An example of the results obtained is found in the
Figure 2. Example of a microsatellite marker electropherogram (Figure 2). The use of capillary
[(CA/TA)29] used to distinguish between two parental electrophoresis clearly improves the resolution. This is
genotypes (Chitton x Saigon) and the observation of particularly useful in sugarcane where the high ploidy
allele inheritance in an F1 progeny (MQ72–1068).
level means that a larger number of aleles need to be
resolved. In this example, allele inheritance is clearly
observed indicating the potential for these markers to
be used in genome mapping.
Summary
These results provide a general survey of sugarcane microsatellites.
Microsatellites have the ability to detect large numbers of alleles
accurately and repeatedly. This potential has been widely accepted and
has been demonstrated to be useful in fingerprinting of such crops as
barley and wheat. Likewise, sugarcane microsatellite genotypic data
from a number of loci have the potential to provide unique allelic
profiles or DNA fingerprints and therefore be used in fingerprinting and
variety identification of commercial sugarcane cultivars. This capability
can be extended to characterising germplasm collections to determine
the degree of relatedness among individuals or groups of accessions.