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Published online 23 September 2005
Published in Crop Sci 45:2383-2387 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Expanding the Pool of PCR-Based Markers for Oat

J.-L. Jannink* and S. W. Gardner

Dep. of Agronomy, Iowa State Univ., 1208 Agronomy Hall, Ames, IA 50011-1010

* Corresponding author (jjannink{at}iastate.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Polymerase chain reaction (PCR)–based markers are generally more rapid and less expensive to assay than hybridization-based markers (e.g., restriction fragment length polymorphisms [RFLP]), making them useful for breeding applications, but few of such markers are available for oat (Avena sativa L.). Approaches to develop new markers cheaply include using primer pairs designed for other species or using publicly available sequence information. In this study we report on the design of 32 markers using publicly available oat sequence data and on the map locations of 20 loci from 16 markers on the oat ‘Ogle’ x ‘TAM O-301’ population.

Abbreviations: KO, ‘Kanota’ x ‘Ogle’ • OT, ‘Ogle’ x ‘TAM O-301’ • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • RIL, recombinant inbred line • SSR, simple sequence repeat • STS, sequence-tagged site


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
WHILE MANY RFLP markers are available for oat, as reviewed in Wight et al. (2003), oat suffers from a dearth of PCR-based markers. Such markers are generally more rapid and less expensive to assay, making them useful for breeding applications. Two reports have been published to date focusing directly on oat simple sequence repeat (SSR) marker development (Li et al., 2000; Pal et al., 2002), and one on SSR and intron-based PCR markers (Holland et al., 2001). These references contain primer sequences for some 121 primer pairs, of which only a total of 34 were found to be polymorphic within A. sativa. In two of these references the markers were not genetically mapped (Holland et al., 2001; Li et al., 2000). Four sequence-tagged sites (STS) and one SSR were mapped on the Ogle x TAM O-301 population (Portyanko et al., 2001), four STS and five SSR markers were mapped on the ‘Kanota’ x Ogle population (Pal et al., 2002), and two novel SSR were mapped on the Ogle x ‘MAM17-5’ population (Zhu and Kaeppler, 2003). These numbers contrast with the 568 SSR markers reported in a single publication on barley (Hordeum vulgare L.), contributing to the 325 SSR reported mapped for that species (Ramsay et al., 2000).

One approach to develop new markers for oat is to use publicly available sequence information on A. sativa to design primers to amplify sequences (Holland et al., 2001). Another approach is to use primer pairs designed for other species, such as barley, to determine if they cross-amplify polymorphic oat sequences. For this latter approach, primers designed for species that are taxonomically closely related to oat should be most useful because the sequences will then be more likely to be evolutionarily conserved. Screening of Avena species with primers designed for barley has been attempted in the past (Li et al., 2000). Perennial ryegrass (Lolium perenne L.), tall fescue (Festuca arundinacea Schreb.), and Avena, however, belong to the Poodae supertribe and are therefore more closely related to each other than any are to Hordeum (Devos and Gale, 1997). Markers developed for Lolium or Festuca may therefore cross-amplify oat sequences more effectively than markers developed for Hordeum. One study found 12% of primers designed for Lolium amplified products from oat (Jones et al., 2001). The annealing stringency used was not given. Warnke et al. (2004) also present 30 SSR from Festuca and 25 conserved grass SSR (Kantety et al., 2002) that amplified Lolium sequence. These SSR would be good candidates for cross-amplification in oat.

The objectives of this study were to design primers for SSR markers using publicly available A. sativa DNA sequences and to map selected polymorphic markers on the Ogle x TAM O-301 recombinant inbred line mapping population.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
PCR Primer Design and Screening
Eighty-eight primer pairs for PCR-based markers (SSR and also intron-polymorphism markers) were obtained from the oat and barley literature (Holland et al., 2001; Li et al., 2000; Liu et al., 1996; Pal et al., 2002). We also designed SSR primers using published A. sativa sequences available from the National Center for Biotechnology Information. A file of the 703 A. sativa sequences available in GenBank (available at www.ncbi.nlm.nih.gov) as of 13 Nov. 2001 was downloaded. Each sequence was examined for SSR with repeat motifs of length 2, 3, or 4 having at least three repetitions of the motif. Of the GenBank sequences, 107 had at least one SSR. For each sequence with such SSR, the Primer 3 package was used to design primers that flanked at least one of the SSR in a sequence. Acceptable primers were forced to have a GC clamp of at least one base, were of length 16 to 24 with an optimum length of 20. Using SSR that had a total length of the repeated motifs of at least 12 base pairs in addition to these criteria, we designed 32 primer pairs surrounding putative SSR. In addition, we obtained sequences for one primer pair that had been developed by Pal et al. (2002), but had been omitted from that publication (L. Domier, personal communication, 2001). In total, we screened 121 primer pairs for their ability to generate marker information.

The 88 SSR primer pairs obtained from the literature were initially screened on DNA from two lines from a high oil selection program (Frey and Holland, 1999). On the basis of the clarity of amplicons from that screening we selected 18 primer pairs to screen with the 32 sequence-based primer pairs by amplifying the DNA from oat lines previously used to develop mapping populations: Ogle, TAM O-301, Kanota, and ‘Marion’ (Groh et al., 2001; Pal et al., 2002; Portyanko et al., 2001). Leaf tissue from three-week-old seedlings of 10 plants per line were harvested and bulked. Tissue was lyophilized then ground, and DNA extracted using the CTAB method (Saghai-Maroof et al., 1984). DNA was diluted to a uniform 100 µg mL–1 concentration using its absorbance of 260-nm wavelength light. PCR amplification was performed using 5 µL of DNA from each line loaded into a 96-well PCR plate. Ten microliters of polymerase solution {0.7 µL water, 3.0 µL Betaine-SCR (5x), 1.5 µL Bovine Serum Albumin, 1.5 µL 10x PCR buffer [100 mM Tris-HCl (pH 8.3) + 500 mM KCl], 1.5 µL MgCl2 (25 mM), 0.6 µL dNTPs (2.5 mM each), 0.2 µL TAQ Polymerase (5 units µL–1), 0.5 µL left primer, and 0.5 µL right primer} were pipetted into each well of the plate, and one drop of mineral oil was added to the wells. The plate was subjected to an initial hot start of 95°C for 1 min. Two cycles of 94°C for one minute, 65°C for 1 min, and 72°C for 2 min were performed. The same double cycle was then repeated 10 more times with a 1° decrease in the annealing temperature at each repetition. The final cycle (94°C for 1 min, 55°C for 1 min, and 72°C for 2 min) was repeated 20 times, followed by a 4°C cooling step. Amplification products were run on 4% (w/v) metaphor agarose in 1x TBE with a 50-bp ladder molecular weight standard. Metaphor agarose gels of the amplification products were photographed with Alphaimager software (Alpha Innotech Co., San Leandro, CA) and scored with ProScan software (DNA Proscan Inc., Nashville, TN).

Locus Mapping
We mapped 20 loci from 16 markers that were polymorphic between Ogle and TAM O-301 on a restricted set of the Ogle x TAM (OT) recombinant inbred line (RIL) population using the program MapPop (Vision et al., 2000). We imported the OT framework map (Portyanko et al., 2001) into MapPop using the command LOADFRAME and used the SAMPLEMAX command to select 30 RIL from the 136 RIL in the original RIL population. With this sample, MapPop predicted marker placement within a 5-cM interval and, in the worst case, within a 23-cM interval. Fluorescent-labeled PCR was conducted on the two parents and the 30 selected RIL using PCR and genotyping procedures described above. Markers were added to the framework map using the MapPop commands LOADNEW and ADDNEW. Output from the ADDNEW command was used to determine the approximate position of the PCR marker locus and the previously mapped framework marker nearest to it.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Amplification of Sequences Using Primers Designed from GenBank Accessions
We screened 32 SSR markers designed from A. sativa sequences available in GenBank on four oat inbred lines that have been used in developing mapping populations: TAM O-301, Ogle, Kanota, and Marion. All but two markers successfully amplified oat sequences, though only 10 markers were polymorphic across the mapping parents (Table 1). Holland et al. (2001), who also designed SSR markers using GenBank sequences, found 3 out of 14 markers to be polymorphic on 22 different A. sativa cultivars. This fraction is not significantly different from the fraction that we found. Note that the amplicon number and sizes reported in Table 1 are conservative. That is, we did not report faint bands, nor did we classify as distinct amplicons that we were not sure were of different lengths based on metaphor agarose gel separation. Further evaluation of these SSR using polyacrylamide separation might reveal further polymorphism than we report here. As for Holland et al. (2001), several of the SSR primers we designed generated more than one amplicon. Because A. sativa is an allohexaploid, it seems likely that the primers anneal to homologous locations in the different base genomes. Null alleles were frequently observed along with amplicon length polymorphisms. The presence of null alleles was also found to be common by Holland et al. (2001).


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  		Table 1. Primer sequences and polymorphism of amplicons from four oat mapping parents of SSR primers designed from A. sativa sequences available in GenBank.{dagger}

 
For both Avenin and Astavea markers, alleles differed by between 20 and 25 base pairs rather than by 2 to 3 base pairs, as is common for SSR markers. The large base-pair difference between alleles was not observed for Astaveb. These markers were designed from avenin sequences. Avenins are prolamin seed storage proteins that are characterized by repeated blocks of short peptide motifs (Shewry and Halford, 2002). The cDNA from which Avenin was constructed, M83381, contains two amino acid repeat regions, one with a repeat motif of seven amino acids and the other with a repeat of nine amino acids. A variable number of repeats of these motifs could explain the allelic differences observed here. The fact that the length differences between the alleles are not multiples of three (as would be expected if the differences were due to differences in the amino acid sequence of the protein product) could be due to error in the measurement of amplicon lengths.

Five primer pairs were designed from receptor-like kinases, Pc68LrkA to Pc68LrkC4. "Pc68" prefixes these sequence names not because they are putative clones of the disease resistance locus but because they were isolated from the oat experimental line Pc68 (Cheng and Armstrong, 2002).

SSR Marker Map Locations
The program MapPop (Vision et al., 2000) allowed us to map 20 loci to 11 linkage groups on the OT mapping population using a restricted set of 30 RIL (Table 2). One of these markers (Rast1-4) had previously been mapped using the full set of RIL (Portyanko et al., 2001), and we redid this mapping using MapPop simply to build our confidence in the MapPop procedure. MapPop placed this marker on linkage group OT11 close to cdo244, as had been observed by Portyanko et al. (2001). Of the other loci, 15 had previously not been mapped, and four had previously been mapped in the KO mapping population (Pal et al., 2002). Of those four, three mapped to the OT linkage group homologous to the KO linkage group where they mapped. The fourth mapped to a linkage group that was either homoeologous or homologous (homology between all OT and KO linkage groups is not yet determined). Given these consistent mappings, these four markers could further contribute to determining consensus segments between the OT and KO maps.


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  		Table 2. Map positions of 20 loci in the Ogle x TAM O-301 (OT) mapping population determined using a restricted set of 30 RIL.

 
Three avenin loci were mapped (Table 2): Astavea.314 and Astaveb.371 from this study and Avenin.240 from Holland et al. (2001). Astavea.314 mapped to OT29, which is homologous to KO4 (Portyanko et al., 2001) where avnA maps (Wight et al., 2003). Avenin.240 mapped to OT8, which is homologous to KO6 where avnC maps. In contrast, MapPop unambigously placed Astaveb.371 on OT32, which is homologous to KO17, which is not where avnB maps. AvnB maps to KO5 (Wight et al., 2003). The SSR primers designed from Astaveb may be amplifying a different sequence than that for which they were designed. There is a small region of homeology between OT32 and KO5 at the cdo949 marker close to which Astaveb.371 maps (Portyanko et al., 2001). This region could be due to a translocation that carries some sequence from the avnB locus. Astavea.315 and Avenin.240 are PCR-based markers that appear to reliably identify the avnA and avnC loci. Further evaluation of Astaveb.371 needs to be done.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
As had been found previously, primers designed using oat sequence data reliably produce amplicons, but these are not frequently polymorphic (Holland et al., 2001). It appears that SSR sequences are generally less polymorphic in oat than in other species, regardless of whether the sequences originated from hybridization-enriched libraries as in Li et al. (2000) and Pal et al. (2002), or from analysis of deposited sequences as in Holland et al. (2001) and this study.


   ACKNOWLEDGMENTS
 
We thank Wendy Woodman-Clikeman and Michael Lee for their help in the lab. We thank Les Domier for communicating SSR primer sequences to us before their publication. This research was supported by funds from the Quaker Oats Company, the Iowa State University Plant Sciences Institute, the Raymond F. Baker Center for Plant Breeding, the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 3571, and by the Hatch Act and State of Iowa.

Received for publication April 8, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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G. Hu, E.W. Jackson, and J. M. Bonman
Expansion of PCR-based Marker Resources in Oat by Surveying Genome-Derived SSR Markers from Barley and Wheat
Crop Sci., September 1, 2007; 47(5): 2004 - 2012.
[Abstract] [Full Text] [PDF]

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