Barley Genetics Newsletter (2007) 37: 44-46

 

 

CAPS markers targeting barley Rpr1 region

 

Ling Zhang* and Andris Kleinhofs

 

Department of Crop and Soil Sciences

Washington State University, Pullman, WA, 99164-6420

*E-mail: lzhang1@wsu.edu

 

Abstract

Eight cleaved amplified polymorphic sequences (CAPS) markers were developed around Rpr1 genetic region. Eight markers were co-dominant between barley cultivars Morex and Steptoe after digestion with restriction enzymes.

 

Introduction

In barley, resistance to Puccinia graminis f. sp. tritici pathotype MCC requires the presence of at least of two host genes, Rpg1 (Brueggeman et al., 2002) and Rpr1 (Zhang et al., 2006). Mutational analysis and transcript-based cloning were used to isolate 3 candidate Rpr1 genes. These 3 candidate Rpr1 genes, HU03D17U_s_at, Contig4901_s_at and Contig7061_s_at were mapped to chromosome 4 bin 5. Screening recombinants between the three candidate genes will identify the real Rpr1 gene. Therefore, molecular markers in this region are needed.

Cleaved amplified polymorphic sequences (CAPS) markers are PCR-based markers, requires a small amount of genomic DNA, which will facilitate the screening of large numbers of genotypes at the seedling stage. 141 probesets representing a major Rpr1 eQTL served as a starting point to develop CAPS markers. Here we report the development and mapping of CAPS markers in the Rpr1 region.

 

Materials and Methods

CAPS markers development

Plant genomic DNA extraction was modified from Edwards et al. (1991); the modification added an extra-step of chloroform-isoamyl alcohol (24:1) extraction. Barley EST unigene sequences (HarvEST assembly#21; http://harvest.ucr.edu/) were used as templates for primer design. RFLP clone MWG058 was sequenced using primers T3 and T7 with the BigDye terminator system on ABI Prizm 377 DNA sequencer (Applied Biosystems) at the Bioanalytical Center, Washington State University, Pullman. A pair of primers was designed from the MWG058 sequence. All the primer pairs listed in Table 1 were used to amplify genomic DNA from the parent cultivars Morex and Steptoe. All PCRs of 20μl contained 20-50ng of genomic DNA, 0.1mMdNTP mix, 12.5 pmol of each primer, 1μl of RedTaq DNA polymerase (Sigma), and 2 μl of 10xRedTaq reaction buffer. Amplification was performed in a PTC-100 programmable thermal controller (MJ Research, Cambridge, MA) at 95°C for 4 min, followed by 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min; this was followed by 7 min at 72°C. PCR products were purified using the Gel Extraction Kit (Qiagen, Valencia, CA) and sequenced. Steptoe sequence was compared to the Morex sequence in order to identify single nucleotide polymorphisms (SNPs) that could be utilized for CAPS marker development. Sequence analysis was done by VectorNTI software (Invitrogen). SNPs were identified and restriction enzymes (New England BioLabs) were selected (Table 1). All the PCR products were digested directly using restriction enzymes correspondingly. Cleaved PCR products were then separated on 1% agarose gel.

 

Genetic mapping

The Steptoe x Morex "minimapper" population consisting of 35 selected doubled-haploid lines (DHL), was used to map the molecular markers to the barley Bin map (Kleinhofs and Graner 2002).  CAPS marker genetic order and the distance between snp_3139 and LZ2502 was estimated based on segregation data from Steptoe x Rpr1 F2 population.

 

Results and Discussion

Details of developed CAPS markers are listed in Tables 1 and Fig. 1. Molecular mapping in Steptoe x Morex population with CAPS markers showed that LZ6641, LZ13393 and LZ10152 co-segregated with LZMWG058, ABG484 and BCD453B, respectively. Markers snp_3139 (Druka, personal communication) and LZ2502 are the closest to Rpr1 delimiting the 3 markers LZ17u, LZ4901 and LZ7061 that co-segregate with Rpr1. LZ2502 and sn3139 are about 1cM apart and can be used to screen recombinants in Steptoe x rpr1 F2 population.

 

 Figure 1. Chromosome locations of eight CAPS marker in barley chr. 4 (4H).

Table 1. Summary of developed CAPS markers, primers, restriction enzymes and annotation.  All the CAPS markers were mapped to Chr 4 (4H), except LZ15227 was mapped to Chr 7 (5H).

Markers

Affymetrix Probesets

Forward Primer

Reverse Primer

Enzyme

Annotation

LZ2502

Contig

2502_at

2502F: AGCTTCAGCTTCAGGTCGAT

2502R: GAAACTTAGAACCTGAACC

HpyCH4V

putative IAA1 protein

LZ6641

Contig

6641_at

6641F: TGATTGATCCTTTGCTGTCT

6641R: CTGGAAAGCGTTCAAATGCT

AvaII

putative expressed SLT1 protein

LZ6435

Contig

6435_at

6435F:ACACCAGGAAGATCATCGAC

6435R:ACAATGGAGAACACATGGTT 

DdeI

phosphoenolpyruvate carboxy-kinase (ATP) -like protein

LZ13393

Contig

13393_at

13393F:AAGTGGACCGCGAAGCACGT

13393R:GCAGCATGTCAGGTTATACA

AvaII

hypothetical protein

LZ15227

Contig

15227_s_at

15227F:ATGGACTAATGACCCCAACA

15227R:TGCAACACACAAAGCCAGTC

AseI

microtubule associated protein

LZ1321

Contig

1321_at

1321F: CACTATCGACTTCCCGGAAT

1321R: ACTGCAATCAGGGTTCATCA

Sau3A or MboI

calmodulin

LZ10152

Contig

10152_at

10152F: AGATCTCCGGCTACGTGCTG

10152R: CGTACATCAGCTCGAAGAAA

Sau3A or MboI

putative membrane protein

LZMWG058a

 

MWG058F:  ATTCATGCATCTACCCATCTCA MWG058R: TTGGATTGGCTAGAATCCTGGA

BtsI

unknown

snp_3139b

 

3139F: AACCACGCAGCAAGCCTAT

3139R: CTCGCTTCCTCCGTCATCAT

DdeI

unknown

aLZMWG058 was developed from RFLP clone MWG058.

bsnp_3139 sequence provided by Druka A, Scottish Crop Research Institute (SCRI), Invergowrie, Dundee DD2 5DA, UK

 

 

 

 

References

Brueggeman R., Rostoks N., Kudrna D., Kilian A., Han F., Chen J., Druka A., Steffenson B., Kleinhofs A. 2002. The barley stem rust-resistance gene Rpg1 is a novel disease-resistance gene with homology to receptor kinases. Proc. Natl. Acad. Sci. USA 99: 9328-9333.

Edwards K., Johnstone C., Thompson C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19 (6):1349.

Kleinhofs A., Graner A. 2002. An integrated map of the barley genome.  In:  R. L. Phillips and I. Vasil, eds., DNA-Based Markers in Plants, 2nd Edition.  Kluwer Academic Publishers, Boston.  pp. 187-199. 

Zhang L., Fetch T., Nirmala J., Schmierer D., Brueggeman R., Steffenson B., Kleinhofs A. 2006. Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley. Theor Appl Genet 113: 847-855