Characterization of Microsatellites and Development of - Angelfire

12 Pages·2000·0.26 MB·English

Plant Molecular Biology Reporter 18: 5–16, 2000.
© 2000 International Society for Plant Molecular Biology. Printed in Canada.
Commentary
Characterization of Microsatellites and
Development of Chromosome Specific STMS
Markers in Bread Wheat
RAJEEV K. VARSHNEY, ALOK KUMAR, HARINDRA S. BALYAN,
JOY K. ROY, MANOJ PRASAD and PUSHPENDRA K. GUPTA*
Molecular Biology Laboratory, Department of Agricultural Botany, Ch. Charan Singh
University, Meerut- 250 004 (U.P.), India
Abstract.Microsatellites,orsimplesequencerepeats(SSRs,)havebecomethemarkersof
choice for genetic studies with many crop species including wheat. Currently an interna-
tional effort is underway to enrich the repertoire of available sequence tagged micro-
satellite site (STMS) markers in wheat. As a part of this effort, we have sequenced
43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones con-
tained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide re-
peats)wereclassifiedas19simpleperfect,18simpleimperfectand4compoundimperfect
types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16
microsatellites could be designed, since the flanking sequences of the others were either
too short or were otherwise not suitable for designing the microsatellite specific primers.
Microsatellite loci of the expected size and polymorphism were successfully amplified
from15ofthese16primerpairsusingthreewheatvarieties.14locidetectedby12outof
the 15 functional primer pairs were assigned to 11 specific chromosomes.
Key words: Bread wheat, chromosome assignment, microsatellite, molecular marker,
polymorphism
Abbreviations: STMS, sequence-tagged-microsatellite-site; WMC, Wheat Microsatellite
Consortium. Varshney et al. Microsatellite characterization and marker
development 16
Introduction
In plant systems, microsatellites or simple sequence repeats (SSRs) have become
popular as DNA molecular markers. These sequences are abundant, ubiquitous,
hypervariable, co-dominant and have been used for a variety of studies including
genome mapping in several animal, insect, and plant species (Gupta et al., 1996;
Gupta and Varshney, 2000). Particularly in wheat, microsatellites are now the
markers of choice (R‘der et al., 1995, 1998; Plaschke et al., 1995; Ma et al.,
1996; Bryan et al., 1997; Stephenson et al., 1998). Serious efforts are being made
through an international Wheat Microsatellite Consortium (WMC) to enrich the
repertoire of wheat microsatellite markers. So far, the partner members (~35) of
*Authorforcorrespondence.e-mail:[email protected];fax:[phone];ph:91-121-768195.
6 Varshney et al.
WMC have developed more than 500 microsatellite markers in addition to the
more than 325 such markers already mapped (R‘der et al., 1998; Stephenson et
al., 1998). As the representative WMC member from India, we have sequenced
43 of 48 clones from a microsatellite enriched genomic library produced by Ed-
wards et al. (1996) and have characterized 41 different microsatellites. Using se-
quences of these microsatellite-containing clones, we have designed primers for
16 such microsatellites for use as sequence-tagged-microsatellite-site (STMS)
markers. This communication reports the nature, potential application and chro-
mosomal assignment of these STMS markers in bread wheat.
Materials and Methods
Genomic clones
48 wheat genomic DNA clones belonging to a microsatellite rich genomic DNA
library were received from AGROGENE, France (Edwards et al., 1996). The li-
brary had been developed and enriched for 10 microsatellites using repeat motifs
that included four dinucleotide repeats [(GA) , (GT) , (AT) , (GC) ], three
15 15 15 15
trinucleotide repeats [(CAA) , (ATT) , (GCC) ] and three tetranucleotide re-
10 10 10
peats [(GATA) , (ATAG) , (CATA) ].
10 10 10
Sequencing and characterization of microsatellites
The above clones were sequenced using Sequenase Version 2.0 DNA Sequencing
Kit from United States Biochemicals (USB) (Sambrook et al., 1989). A few
clones were also sequenced at the Indian Institute of Sciences (IISc), Bangalore,
India using an automatic sequence analyzer system ABI PRISM, Model 377, Ver-
sion 2.1.1.
Designing and synthesis of PCR primers
PCRprimersformicrosatellitesweredesignedusingcomputersoftwarePrimerPick
of AGROGENE, France. These primers were synthesized commercially by
Genosys, U.K.
PCRs for polymorphisms and chromosome assignments
The PCR amplification protocols used for the study of polymorphism and for
chromosomalassignmentsaredescribedelsewhere(Prasadetal.,1999,2000;Roy
et al., 1999).
Results and Discussion
Characterization of microsatellites
Wewereabletosequence43outofthe48clonesfromthemicrosatelliteenriched
genomic library. One sample lacked an insert, 8 clones contained no micro-
satellites, 27 clones contained each a single microsatellite and 7 clones contained
twodifferentmicrosatelliteseach.Therefore,atotalof34(71%)clonescontained
microsatellites.
Microsatellite characterization and marker development 7
Types and relative frequencies of microsatellites
Themicrosatelliteswereclassifiedassimpleorcompoundandeachclasswasfur-
ther subdivided into perfect and imperfect types (Tables 1, 2), following a slight
modification of the classification by Weber (1990). The 41 microsatellites con-
tained mono-, di- or tri-nucleotide repeat motifs only, even though the genomic
library had been enriched for three different tetranucleotide repeats also. Micro-
satellites representing simple perfect and simple imperfect repeat families were
predominant. Only 9.76% were represented by compound imperfect repeat fami-
lies and no compound perfect repeat was available. It can also be seen that the
dinucleotide repeats were more abundant (70%) than other types of
microsatellites. This agrees with earlier reports suggesting an abundance of
dinucleotide repeats in plant systems (Wang et al., 1994) including wheat (Ma et
al., 1996; Bryan et al., 1997). Further, although the parent library was enriched
for four different dinucleotide repeats, nearly 50% of the dinucleotide
microsatellites were represented by (GT) . In contrast, screening of wheat
n
genomic libraries for dinucleotide, trinucleotide and tetranucleotide repeats pro-
duced a greater abundance of (GA) instead of (GT) (R‘der et al., 1995; Ma et
n n
al., 1996; Bryan et al., 1997). This suggested that our sample of clones may not
be representative of the whole genome. Furthermore, not a single (AT)
n
microsatellite was recovered, despite the fact that (AT) along with three other
n
dinucleotide repeats was used to enrich the library and that (AT) microsatellites
n
were more abundant in sequence databases (Condit and Hubbell, 1991;
Lagercrantzetal.,1993;MorganteandOliveri,1993;Wangetal.,1994).Thisab-
sence of (AT) may, therefore, be more apparent than real, since the library was
n
enriched by hybridization, and this may not detect (AT) motifs due to secondary
n
configuration resulting from intra-strand pairing between A and T nucleotides.
For the same reason perhaps, information on the abundance of (AT) is not avail-
n
able for many plant species including wheat. Therefore, (GT) and (GA)
n n
dinucleotiderepeatsmayprovetobemoreusefulinwheatduetotheirabundance
and/or ease of their isolation relative to (AT) microsatellites, which are abundant
n
in plants, but create difficulty in hybridization based assays.
In this study, no microsatellite with a tetranucleotide repeat motif was re-
covered and only 8 (~20%) trinucleotide microsatellites including 5 simple per-
fectand3simpleimperfectrepeatswereobtained.Thisfrequencyoftrinucleotide
repeats is lower than the 38% trinucleotide repeats reported earlier for wheat (Ma
et al., 1996). This difference may be partly attributed to the relatively small sam-
ple of microsatellites examined (41 vs 146). Nevertheless, the isolation of
trinucleotide repeats in wheat is important since these repeats have been shown to
be highly polymorphic and stably inherited in the human genome (Edwards et al.,
1991). Furthermore, detection of polymorphisms involving trinucleotide micro-
satellites may be easier compared to that involving dinucleotide repeats, due to
the presence of an extra base pair in the repeat unit (Hearne et al., 1992). Also,
tetranucleotide repeats in the wheat genome may be rare; none were recovered in
this study or in an earlier study (Ma et al., 1996).
8 Varshney et al.
Variation in repeat number in microsatellites
Individual microsatellites were also analyzed for number of repeats (Tables 1, 2).
Although the average repeat number in mononucleotide imperfect microsatellites
varied from 15 to 39, the number of contiguous repeats never exceeded 6. Similar
variation in repeat number was also observed in the simple di- and trinucleotide
repeats (Table 1). In compound imperfect microsatellites, with mainly dinucle-
otide repeats, the range of contiguous repeats was 3–15, although the range of to-
tal repeats (with interruptions) was 17–47 (Table 2). This agrees with earlier
studies where the range of repeat numbers in various wheat microsatellites varied
between 4 and 64 (R‘der et al., 1995; Ma et al., 1996).
Development of primers
In this study, sequences for 41 clones carrying microsatellites were available for
designing primers. However, primers for only 16 (39%) microsatellites (Table 3)
could be synthesized, including 9 simple perfect repeats, 6 simple imperfect re-
peats, and a solitary trinucleotide simple perfect repeat. For the remaining 25
(61%) microsatellites, primer pairs could not be designed due to one or more of
the following reasons: (1) microsatellite sequences were too short, (2) microsatel-
lites were too close to the cloning site, or (3) the flanking sequences were not
unique. Similarly, in earlier studies also, primer pairs could be successfully de-
signedforonly21%(Maetal.,1996),for68.9%(Bryanetal.,1997)andfor54%
(Röder et al., 1998) of the microsatellites.
Functionality of primer pairs
Functionalprimerpairsarethosewhichamplifiedafragmentofthesizepredicted
by its sequence. By corollary, the non-functional primer pairs amplify either a
large number of fragments (resulting in a smear on a gel), a fragment of the
wrong size, or nothing. In this study, 15 primer pairs were functional and one
(WMC260) was non-functional. This high proportion of functional primers
(~93%) is much higher than the ~30% functional primers previously reported
(R‘der et al., 1995, 1998; Bryan et al., 1997) in studies in which primer pairs
weredesignedfromclonesselectedfromarandomlibrary.Inanotherstudy,when
232 primer pairs from a microsatellite enriched library were examined, 72% were
found to be functional primers (Prasad et al., 1999; Roy et al., 1999). This high
frequency may be attributed to the use of enriched libraries.
Detection of polymorphism
To check their potential for detection of polymorphism, the 15 functional STMS
primer pairs were used to amplify DNA from Chinese Spring and a mixture of
three wheat varieties (Brigadier, Amigo and Soissons). Fourteen of these primer
pairs (87.5%) detected polymorphism on PAGE only and one primer pair,
WMC262 detected polymorphism by PAGE and as well with agarose gel. These
results are in agreement with those of R‘der et al. (1998), who showed that 80%
of the STMS primers (294 primers were tried) detected polymorphism between
the two parents of ITMIpop used for mapping. These results, and others from our
laboratory (Prasad et al., 1999, 2000; Roy et al., 1999) suggest that the STMS
Microsatellite characterization and marker development 9
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Table1.Summaryofth Characteristicsofmicrosatellites TypeofsimplerepeatPerfectImperfect Numberofrepeats1–56–1011–1516–20>20Totalnumberofrepeats–rangeContiguousrepeats–mean DevelopmentofprimersNo.ofmicrosatellitesforwhichPCRprimerswerepreparedFlankingsequencestooshortornotsuitablefordesigningPCRprimers
10 Varshney et al.
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Microsatellite characterization and marker development 11
Table 2.Summaryof thesequencedcompoundmicrosatellites.
Description (AG)-(TG) (CT)-(CA) (TC)-(CT) (GT)-(GA)
No. of microsatellite 1 1 1 1
Total no. of repeats 27 42 17 47
Repeat range (contiguous 4–15 5–13 3–11 4–13
repeats)
No. of microsatellites for which 1 – – –
PCR primers were prepared
Flanking sequences too short or – 1 1 1
not suitable for designing PCR
primers
markers will be useful for a variety of studies involving detection of polymor-
phism.
In a separate study, we evaluated 20 WMC primers using template DNA
from 55 wheat genotypes (Prasad et al., 2000). These were selected from those
not useful for tagging genes/QTLs for protein content and pre-harvest sprouting
tolerance (Prasad et al., 1999; Roy et al., 1999) and included two of the 15 func-
tionalprimerpairsreportedinthepresentstudy.TheprimerpairWMC254ampli-
fied locus Xwmc254-4B localized on 4B. On this locus, 12 alleles with
polymormphic information content (PIC) of 0.85 were detected (Figure 1A). The
otherprimerpair,WMC256,amplifiedtwoloci(Xwmc256-6AwithPIC=0.41and
Xwmc256-6D with PIC= 0) (Figure 1B). It may be noted that the PIC values for
these two STMS markers fall within the range of the following PIC values re-
ported in three earlier studies on wheat: 0-0.72 (R‘der et al., 1995), or 0.21-0.81
(Ma et al., 1996) or 0.29-0.79 (Plaschke et al., 1995). These results, taken to-
gether with our data, suggest that the STMS markers in wheat differ by the level
ofpolymorphismeachmaydetect.Thisfactaloneunderlinestheneedforexpand-
ingtherepertoireofmicrosatellitemarkersforapplicationsinwheatbreedingand
other studies.
Chromosomal assignment of microsatellite loci
ThefifteenprimerpairsgivingamplifiedproductswerealsousedtoamplifyDNA
fromthe21nullisomic-tetrasomicstocksinordertolocalizethelocitoachromo-
some. Three primer pairs (WMC252, WMC253 and WMC258), each produced
amplified product of the same size from all 21 stocks, suggesting their presence
on more than one chromosome and no polymorphism between loci on different
chromosomes, which made it difficult to assign them to specific chromosomes.
SimilarresultswerealsoreportedinwheatbyPlaschkeetal.(1996).Theremain-
ing 12 STMS primer pairs amplified a total of 14 microsatellite loci, which could
be assigned to 11 chromosomes including 5 chromosomes of the A (1A, 2A, 3A,
4A and 6A) and 4 chromosomes of the B (2B, 4B, 5B and 6B) and two chromo-
somesoftheD(3Dand6D)genome.Thisinformationwasusedforassigningap-
propriate symbols to the loci (Table 3) according to the Rules of Nomenclature
for DNA markers (McIntosh et al., 1998), as approved earlier at the 7th Interna-
tional Wheat Genetics Symposium held at Cambridge (Hart and Gale, 1988). It
may be noted that two or more fragments amplified with a single primer pair and
12 Varshney et al.
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Microsatellite characterization and marker development 13
localized on a single chromosome were considered to belong to a single locus, al-
though these may very well be due to duplicate or triplicate loci (Figure 2A). In
this manner, 14 microsatellite loci, amplified by 12 primer pairs, could be as-
signedto11specificchromosomes.TheprimerpairWMC262amplifiedthesame
two products in 19 of the 21 nulli-tetras, but in the two remaining lines (nulli 4A,
nulli 5B) only one of these two products was amplified. In another case
(WMC256), out of the three amplified products of different sizes observed in
each of the 19 nulli-tetras, two products of different sizes were obtained in one
line (nulli 6D) and the solitary third product was observed in the remaining
nullisomic-tetrasomic line (nulli 6A) (Figure 2B). This facilitated assignment of
two loci for each of these two markers to two different chromosomes.
Out of the 12 primer pairs for which the corresponding loci could be
assigned to specific chromosomes, 10 primer pairs (WMC250, WMC251,
WMC254, WMC255, WMC257, WMC259, WMC261, WMC263, WMC264 and
WMC265) each amplified only a single locus and two primer pairs (WMC256
and WMC262) each amplified two loci on two different chromosomes (Table 3).
Thus,the10primerpairs,eachamplifyingasinglelocusshowedhighlocusspec-
ificity and could be of value when chromosome specificity is desired. The two
lociamplifiedbyWMC256andassignedto6Aand6Dchromosomes(Figure2B)
could be homoeologous loci. On the other hand, the two loci amplified by
WMC262 and assigned to 4A and 5B may represent a case of interchromosomal
duplication involving non-homoeologous chromosomes or a case of homoeo-
logous segments transferred to non-homoeologous chromosomes. Microsatellite
loci amplified by three remaining primer pairs, which could not be assigned to
specific chromosomes may also have monomorphic homoeoloci, so that out of 15
primer pairs, 5 (33%) do have homoeoloci. It may be noted that, in comparison
to the frequency of homoeologous RFLP loci (Anderson et al., 1992) the fre-
quency of homoeologous microsatellite loci in this study was rather low. This
may be due to differences in the flanking sequences of homoeologous micro-
satellite loci brought about during evolution, thus not permitting amplification of
null alleles at homoeologous loci using the same primer pair. In contrast to this,
the RFLP probes hybridize to sequences showing up to 80% homology and thus
are able to detect homoeologous loci in high frequency, despite some degree of
mismatch (Anderson et al., 1992).
Conclusions
In this study we designed sequences of 15 functional primer pairs which can be
used as locus- specific multiallelic STMS molecular markers in wheat for a vari-
ety of studies including genome mapping, gene tagging, germplasm characteriza-
tion and genetic diversity studies. This adds to a rich repertoire of wheat STMS
markers already mapped, and those soon to be mapped by other members of
WMC. These will eventually become available in the public domain. In our expe-
rience, even this new set of 15 primer pairs would suffice to examine genetic di-
versity and to characterize germplasm in wheat (Prasad et al., 2000). A larger set
of primer pairs may be needed for gene tagging. The markers assigned in this
study to specific chromosomes, along with already mapped markers, may also
14 Varshney et al.
Table 3. Microsatellite marker, type of repeat, primer sequences, annealing temperature, expected
PCRproductsize,andlocus nomenclature.
Expected
Primer sequences Anneal. product Locus
S. No. Marker Repeat L:5¢fi 3¢R:5¢ fi 3¢ Temp. (°C) size (bp) designation**
1. WMC250 (GA) ATGCTTATGGAC 61 113 Xwmc250-6D
18
CGTGACAGAGAG
TGCAAAGGCGCC
GATTTATG
2. WMC251 (GT) — GAAGAGTGAGTG 51 142 Xwmc251-4B
4
(GT) — TGCAAAAGCGAG
17
(GT) AGTGATGGCGTA
7
GATT
3. WMC252* (AC) TCACTATGCTCA 61 108 –
15
CTCTTCGGCTCTC
CTCGTGTTCCTG
ATATGCT
4. WMC253* (AC) TCACCGATTGTA 61 138 –
13
AATAGCACCAGA
TAAATCACTTCG
GGTGAGGG
5. WMC254 (AC) — AGTAATCTGGTC 51 193 Xwmc254-1A
28
(AC) CTCTCTTCTTCTA
7
GGTAATCTCCGA
GTGCACTTCAT
6. WMC255 (GT) — TCGAGGCGCGTG 61 76 Xwmc255-3D
4
(GT) — GATAACCACCTT
7
(GT) GCATATATGACT
5
GAGC
7. WMC256 (CA) CCAAATCTTCGA 61 117 Xwmc256-6A
12
ACAAGAACCCAC Xwmc256-6D
CGATCGATGGTG
TATACTGA
8. WMC257 (GA) — GGCTACACATGC 51 329 Xwmc257-2B
14
(GA) ATACCTCTCGTA
7
GTGGGTGAATTT
CGGA
9. WMC258* (GT) GCGATGTCAGAT 61 293 –
26
ATCCGAAAGGAC
CAGGACACCAGA
ACAGCAAT
10. WMC259 (AG) — GTCGGAGCTGAC 51 179 Xwmc259-6B
4
(TG) — TTGATTACATTG
8
(TG) CACAGTAGATAG
15
CTAGCGGT
11. WMC260= (GA) CAAGATCGTGCC 51 111 –
26
AAATCAAGGTAC
CAGTAGCTATGT
GTTAGATCTC
12. WMC261 (GT) GATGTGCATGTG 61 110 Xwmc261-2A
11
TGCATGTGAATC
TCAAAAGTAAAA
GAGGGTCACAGA
ATAACCTAAA
13. WMC262 (AG) GCTTTAACAAAG 61 198 Xwmc262-4A
29
ATCCAAGTGGCA Xwmc262-5B
TGTAAACATCCA
AACAAAGTCGAA
CG