Published online 1 March 2007
Published in Crop Sci 47:848-850 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
Development and Mapping of PCR-Based SCAR and CAPS Markers Linked to Oil QTLs in Oat
Winson Orr and
Stephen J. Molnar*
Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Central Experimental Farm, 960 Carling Ave., Ottawa, ON, K1A 0C6 Canada. ECORC Publication Number: 03-378
* Corresponding author (molnarsj{at}agr.gc.ca).
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ABSTRACT
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Oat cultivars (Avena sativa L.) are unusually diverse in groat oil content, and breeding for this trait can increase the value of oat in human and livestock diets. Seven random amplified polymorphic DNA (RAPD) fragments closely linked or tentatively linked to quantitative trait loci (QTLs) for oil content in the ‘Terra’ x ‘Marion’ (TM) or the ‘Kanota’ x ‘Ogle’ (KO) hexaploid oat mapping populations were used to develop more robust sequence characterized amplified region (SCAR) or cleaved amplified polymorphic sequence (CAPS) markers. Three SCAR and two CAPS markers mapped on the TM population to the same map locations as the corresponding RAPD markers. One SCAR marker mapped to a homeologous TM linkage group (LG). The last two SCAR markers were mapped to regions that are currently not known to be orthologous to the original RAPD locations. All SCAR and CAPS markers were also tested in two additional high x low oil content populations. These SCAR markers have potential utility for marker-assisted selection (MAS) for high and low oil germplasm in oat breeding programs.
Abbreviations: CAPS, cleaved amplified polymorphic sequence • cM, centimorgan • DE, ‘Dal’ x ‘Exeter’ • FR, ‘Francis’ x ‘Rigodon’ • KM, ‘Kanota’ x ‘Marion’ • KO, ‘Kanota’ x ‘Ogle’ • LG, linkage group • MAS, marker-assisted selection • OM, ‘Ogle’ x ‘MAM17-5’, PCR, polymerase chain reaction • QTLs, quantitative trait loci • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • RIL, recombinant inbred line • SCAR, sequence characterized amplified region • SSR, simple sequence repeat • TM, ‘Terra’ x ‘Marion’.
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INTRODUCTION
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THE OIL CONTENT of oat (Avena sativa L.) groats (3–16%) is higher than other cereals, an important trait when breeding for food or feed applications. Oil content is inherited polygenically (Frey et al., 1975) with additive and nonadditive gene action (Brown et al., 1974) and is highly heritable (Baker and McKenzie, 1972). Using recombinant inbred line (RIL) mapping populations, researchers have identified six quantitative trait loci (QTLs) for groat oil content in ‘Terra’ x ‘Marion’ (TM) (De Koeyer et al., 2004), four in both ‘Kanota’ x ‘Ogle’ (KO) and ‘Kanota’ x ‘Marion’ (KM) (Kianian et al., 1999), and six in ‘Ogle’ x ‘MAM17-5’ (OM) (Zhu et al., 2004). Very few simple sequence repeat (SSR) markers have been mapped in oats. Random amplified polymorphic DNA (RAPD) markers have been associated with several of the oil content QTLs, but for technical reasons these markers may lack sufficient reproducibility of banding patterns for marker-assisted selection (MAS) (Penner et al., 1993a, 1993b; Hernandez et al., 1999). The development of sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers from RAPD markers not only significantly improves reproducibility but may also replace dominant markers with more informative codominant markers. Sequence characterized amplified region markers have been reported for oat (Molnar et al., 2000; Chong et al., 2004).
We describe here the development of SCAR and CAPS markers from seven RAPD markers in a targeted initiative to develop polymerase chain reaction–based markers useful for MAS for high and low oil content in oats.
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MATERIALS AND METHODS
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Plant Materials and DNA Extraction
The TM population consists of 102 recombinant inbred lines (RILs) developed by single seed descent from the cross between Terra and Marion (De Koeyer et al., 2004). The KO population is composed of 71 (O'Donoughue et al., 1995) or 141 (Wight et al., 2003) F6-derived RILs from Kanota x Ogle. The ‘Dal’ x ‘Exeter’ (DE) population includes 150 unselected RILs, and the ‘Francis’ x ‘Rigodon’ (FR) breeding population consists of 50 selected RILs. Genomic DNA was extracted from young leaves (Dellaporta et al., 1983) or leaf discs (Edwards et al., 1991).
Development of SCAR Primers from RAPD Markers
We produced RAPD products using genomic DNA from Terra, Marion, Kanota, and Ogle and analyzed them according to Penner et al. (1993b). The diagnostic RAPD polymorphic band was excised from the 2% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen Inc., Mississauga, ON). Additional cycles of polymerase chain reaction (PCR) purification(s) with the same RAPD primer were performed until the PCR product electrophoresed as a single fragment of the expected size. An aliquot of repurified DNA was cloned (TA Cloning Kit, Invitrogen Corp., Burlington, ON) and sequenced. At each border, the insert's sequence displayed the 10 bp of the original RAPD primer. Three pairs of primers (forward and reverse) ranging from 18 to 24 bases in length were designed manually. The first primer included the complete RAPD primer sequence, and the second and third primers were shifted a few bases to avoid palindromes between SCAR primers.
Analysis and Mapping of SCAR and CAPS Markers
Amplification reactions (25 µL) contained 1 unit of Taq DNA polymerase, 1 x buffer, 2.0 mM MgCl2, 200 µM of each dNTP, 50 ng genomic DNA, and 25 ng of each SCAR primer. The PTC-100 Programmable Thermal Controller (MJ Research Inc., Waltham, MA) was programmed for an initial 3 min at 94°C; 37 cycles of 1.5 min at 94°C, 2 min annealing at 62°C to 68°C, 2 min at 72°C; and a final 5 min at 72°C; cooling at 4°C. Amplified products were separated in 2% agarose gels, and amplification conditions were optimized so that the best pair of SCAR primers produced a single product of the expected size.
If the SCAR primers produced monomorphic products, a second linked polymorphism was sought by digestion with four or five cutter restriction enzymes (AluI, AvaII, CfoI, DdeI, DpnI, HaeIII, HhaI, HinfI HpaI, MboI, MseI, RsaI, Sau3AI, TaqI, ThaI). Digested products were analyzed by electrophoresis in 5% NuSieve GTG agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME) gels. Such markers are known as CAPS (Konieczny and Ausubel, 1993).
The optimal primer pairs (Table 1) were then tested on Terra, Marion, and 102 TM RILs or Kanota, Ogle, and 82 KO RILs. Mapmaker Macintosh V2.0 (Lander et al., 1987) was used for map construction with RI Self and Kosambi settings. To identify homologous relationships, linkage groups (LGs) were aligned using the C2MAPS program (Wight and Tinker, 2002).
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Table 1. Sequence characterized amplified region (SCAR) alleles, map location, primer sequences, and polymerase chain reaction (PCR) annealing temperatures.
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RESULTS AND DISCUSSION
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Genetic analysis of groat oil content in the TM RIL mapping population (hulless x covered seeded) identified six QTLs on LGs TM4_16, TM5, TM15 (2), and TM30 and associated with the unlinked marker bcd1829y (De Koeyer et al., 2004). Quantitative trait loci were also reported by Kianian et al. (1999) in KO (KO6, KO11, KO37, and an unlinked marker) and in KM (KM3, KM11, KM22, and KM5x) and by Zhu et al. (2004) in OM (OM3, OM6, OM13, OM19, OM20, and OM24) covered seeded RIL populations. Because of a limited number of common markers among these four maps, it is not clear how many of these QTLs are unique or homologous. However, the major TM15 QTL is homologous to the KM22 QTL (both are homologous to a region on KO22), and the TM unlinked (bcd1829y) QTL is homologous to the KO37 QTL (De Koeyer et al., 2004). Similarly, the KO11_41+20 and KM11_41 and OM3 LGs (all homologous to TM2) have been shown to be the location of oil QTLs (Kianian et al., 1999, Zhu et al., 2004). Five RAPD markers (acor167a, acor185, acor186, acor189, and acor198) produced using five RAPD primers (ubc167, ubc185, ubc186, ubc189 and ubc198) are linked to four TM QTLs (Table 1). A sixth RAPD marker, acor121 (TM11), is of interest because it is in a region homeologous to a region on TM15 that is linked to both oil QTLs (Fig. 1A
). A seventh RAPD marker, acor364b (KO15), is also of interest because it maps to a region that is either homologous or homeologous to a region on TM30 near an oil QTL (Fig. 1C). No other RAPD markers linked to oil QTLs exist in oat. All seven RAPD markers were used to develop SCAR or CAPS markers.
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Figure 1. Sequence characterized amplified region and cleaved amplified polymorphic sequence markers, original random amplified polymorphic DNA markers, and selected framework or reference markers mapped on either the ‘Terra’ x ‘Marion’ (TM) or ‘Kanota’ x ‘Ogle’ (KO) hexaploid oat map. Marker positions are given in centimorgans. Selected quantitative trait loci (QTLs) are shown at map locations corresponding to the statistical peak of the published QTL curve. QTLs detected in the hulless half of the TM population are marked (H).
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Three RAPD markers (acor167a, acor186, acor198) map close to each other on TM15 approximately 13 to 18 centimorgan (cM) from an oil QTL identified in the whole population and approximately 11 to 16 cM from a different but overlapping oil QTL identified in the hulless half of the population (De Koeyer et al., 2004). The two SCAR markers, ubc186s and ubc198s, mapped to the same loci as the original RAPDs (Fig. 1A). The SCAR primers derived from the 2.2kb acor167a RAPD fragment (present in ‘Terra’, absent in ‘Marion’) produced 2.2kb SCAR products from both Terra and Marion; however, digestion with HinfI, or HpaII revealed a TM polymorphism (Table 1). These two acor167-derived CAPS markers mapped to the same locus as acor167 itself (Fig. 1A). Thus a dominant RAPD marker (acor167a) was converted into a codominant CAPS marker (ubc167ts). The second CAPS marker (ubc167ms) would also be expected to be codominant; however, as only one band is visualized (Table 1), it must be scored as a dominant marker. The SCAR marker ubc121ms also mapped to TM15, although the original RAPD marker mapped to TM11 (Fig. 1A). This result is not unexpected as two restriction fragment length polymorphism (RFLP) probes (aco139, bcd327) map to loci linked to the acor121 locus on TM11 as well as to homeologous loci on TM15 (Fig. 1A). Detailed mapping of the five newly derived SCAR and CAPS markers, as well as of the RAPD markers acor167a, acor198, acor186 and RFLP markers bcd327 and aco139x (previously assigned only to intervals between framework markers), has increased the length of this LG from 29 cM (DeKoeyer et al., 2004) to 40cM (Fig. 1A). Despite relatively loose linkage to QTL peak markers, the entire LG is strongly associated with oil content by simple interval mapping (De Koeyer et al., 2004) so that these SCAR and CAP markers have potential utility for MAS. The Terra allele at the oil QTL peaking at ACC_CTA222 was associated with a 0.46% reduction of oil content in the original TM mapping population and a 1.05% reduction in the TM extended population (De Koeyer et al., 2004).
A third QTL for oil content was detected near the naked locus (N1) on TM5, tightly linked to RAPD marker acor185 (Table 1, Fig. 1B). The derived SCAR marker comapped with acor185. However, since there is a cluster of QTLs including oil content near N1, it would be difficult to select for oil content without simultaneously impacting other traits. Because there is a ß-glucan content QTL approximately 10-cM distal to cdo482x (De Koeyer et al., 2004), the ubc185s SCAR marker may have additional utility for MAS.
The fourth QTL for oil was detected on TM 4_16, tightly linked to RAPD marker acor189; however, the derived SCAR ubc189s mapped to TM8 (Table 1). There is neither an oil QTL on TM8 nor any obvious homology to TM4_16 (data not shown), suggesting that this SCAR marker maps to a locus unrelated to the RAPD.
A seventh RAPD marker, acor364b, and RFLP locus bcd115c are tightly linked on KO15 (Fig. 1C). The same bcd115 probe also mapped to the bcd115x locus on TM30 near an oil QTL identified by De Koeyer et al. (2004). SCAR marker ubc364os mapped to a region of KO11_41+20 (Fig. 1D) that is homologous to KM11 and OM3 and is the most significant genomic region influencing oil content across multiple genotypes. Also mapping here is acetyl-CoA carboxylase (accase1), which catalyzes the first step in de novo fatty acid synthesis and has a major role in determining groat oil content (Kianian et al., 1999). Although flanking markers mapped on KO11_41+20 do not give evidence of homeology of this LG with either KO15 or TM30 (Fig. 1C and D), the present results suggest that this may be the case. The hexaploid oat genome tolerates many translocations, and this may be an example of segmental homeology, a testable hypothesis as more common markers become available.
All SCAR markers were also tested in the DE RIL population, which varies for oil content (N. Tinker et al., personal communication). Three SCAR markers, ubc185s, ubc189s, and ubc364os, showed polymorphism between Dal and Exeter; however, one of these, ubc189s, is not associated with an oil QTL (Table 1). The diagnostic band of SCAR marker ubc364os was present in Dal (high oil) and in each of the 10 highest oil content RILs but absent in Exeter (low oil) and each of the 9 lowest oil content RILs. The SCAR marker ubc364os has great potential as a molecular tool for breeding high and low oil content cultivars. The other two polymorphic SCAR markers showed no such association in DE. All SCAR markers were further tested on a second RIL population derived from the cross Francis (low oil) x Rigodon (high oil) (N. Tinker et al., personal communication). Only ubc167s was polymorphic on the parents; however, it was not associated with oil content in this population based on genotyping the 11 highest and 11 lowest oil content RILs.
The parents of five mapping populations exhibited polymorphism for two SCAR markers on KO, five SCAR and two CAPS markers on TM, five SCAR and two CAPS markers on KM, three SCAR markers on DE, and one SCAR marker on FR (Table 1). Adding these robust markers to those maps may help to define homologous and homeologous relationships at oil loci and therefore the number of unique loci affecting oil content.
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ACKNOWLEDGMENTS
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This research was made possible by generous financial support from Quaker Tropicana Gatorade Canada and the Quaker Oats Company, a division of PepsiCo, USA, and from the Agriculture and Agri-Food Canada Matching Investment Initiative. We thank Charlene Wight for assistance with the computer mapping of the SCAR and CAPS markers. We thank Vern Burrows for creating the DE and FR populations and Anissa Lybaert, Nicholas Tinker, and David De Koeyer for helpful discussions and for seeds, DNA, and oil content data regarding the DE and FR populations.
Received for publication January 25, 2006.
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ABSTRACT
INTRODUCTION
