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Identification of RGAs from a pea BAC library using BAC pools

Clarice J. Coyne1, Sarah Murray2 and Gail Timmerman-Vaughan2

1 USDA-ARS Western Regional Plant Introduction, 59 Johnson Hall, Pullman, WA 99164-6402
2
Institute of Crop and Food Research, Private Bag 4704, Christchurch, New Zealand

Abstract

A bacterial artificial chromosome (BAC) library of Pisum sativum, representing one genome equivalent (50,000 clones, average 115 kb) was constructed of partially HindIII-digested DNA from the pea germplasm line PI 269818 in the binary vector pCLD04541 (Coyne et al 2000). The library was increased to 120,000 clones, also using HindIII-digested DNA cloned into a second vector, pBeloBACII (Coyne et al 2002). High-density filters provide an economical method to probe for a gene of interest. However, 17 filters make up this 120,000 clone library so more efficient methods of probing are needed. We investigated three-plate BAC DNA pools for use in PCR-screening to rapidly identify BACs containing pea resistance gene analogs (RGAs). Seven of nine pea RGAs previously published (Timmerman-Vaughan et al 2000) were identified.

Media Summary

The pea BAC library was developed by this project as a useful genomics tool to rapidly identify clones containing plant disease resistance genes for increasing the yield of pea and other legumes.

Key Words

legumes, genomics

Introduction

One of the major constraints to fully utilizing the U.S. plant germplasm resources is the paucity of effective techniques to efficiently discover new alleles useful to crop breeding (Tanksley and McCouch 1997). Several key genomic technologies relevant to germplasm collections, however, have been developed (Borevitz and Chory 2004; Buckler and Thornsberry 2002). Information gathered from plant functional genomic approaches will be useful in identifying yield, disease and stress resistance alleles in U.S. pea germplasm accessions. The United States Department of Agriculture Agricultural Research Service’s National Plant Germplasm System (NPGS) has embraced new technologies to maximize the conservation of genetic variation for economically important traits in each collected crop species. Molecular tools will increase the efficiency in curation and utilization of collections. One such molecular tool, bacterial artificial chromosome (BAC) libraries of food legumes genomes, has the potential to provide new opportunities for the utilization and management of food legumes genetic resources (Coyne et al 2000; Rajesh et al 2004).

A BAC library of Pisum sativum, representing one genome equivalent (50,000 clones, average 115 kb) was constructed of partially HindIII-digested DNA from the pea germplasm line PI 269818 from the NPGS in the binary vector pCLD04541 (Coyne et al 2000). The library was increased to 120,000 clones, also using HindIII-digested DNA cloned into pBeloBACII (Coyne et al 2002). The P. sativum genome is estimated to be 3947 to 4397 Mbp/1C (Arumuganathan and Earle, 1993) a further 130,000 clones will eventually be added to create a the final library of 250,000 clones which we estimate will represent five to six-fold haploid genome equivalents.

Two methods of probing a BAC library for a sequence/gene of interest are high-density filters and PCR of isolated BAC clone DNA (Woo et al 1994; Green and Olsen 1990 respectively). The current library of 120,000 clones is accommodated on 17 high-density filters, with the final library taking 34 high-density filters. Here, we report the use of BAC DNA plate pools (Whisson et al 2001) as a potentially more efficient way to identify three plates to probe to identify a specific BAC clone containing the RGA sequence/genes published in pea (Timmerman-Vaughan et al 2000).

Methods

Pooling of the pea BAC clones

Three plates of 384-well clone plates were replicated onto Luria Bertani (LB) agar (Fisher Scientific) plates (150 X 15 mm) using a 384-pin replicator. Care was taken to ensure colonies did not overlap on the agar surface. The colonies were grown overnight at 37 CB.

Isolation of BAC DNA plate pools

The cells were harvested by scraping the colonies from the agar surface into 15 ml LB broth as suggested by Whisson et al (2001). The BAC DNA was isolated by alkaline lysis (Sambrook and Russell, 2001) using prepared reagents (Millipore, Bedford, MA, USA). The plate pools numbered 105, which represents DNA from of all 315 plates of the pea BAC library.

PCR of pea resistance gene analogs

To test the BAC DNA pools, primers based on the vector sequence (pBELOBACII or pCLD0451) were used for PCR. The RGA PCR reactions were set up on primers designed from published RGA sequences (Timmerman-Vaughan et al 2000). The 25 Fl reactions contained 1X PCR buffer (Roche), 200 FM of each dNTP, 0.1 FM each primer, 0.5 U Taq DNA polymerase (Roche) and approximately 50 ng DNA. Thermal cycling conditions were 40 cycles of 94 BC 1 min., 60 BC 1 min., 72 BC 1 min. followed by 72 BC 8 min. Amplified products were electrophoresed on 2% agarose gels and visualized using UV light after ethidium bromide staining of the gels.

Preparation and probing single plate filters

The rapid lysis and binding of BAC colony DNA to filters was a modification of Sambrook and Russell (2001). Each 384-well plate was doubled-spotted onto a LB agar plate and grown overnight at 37 C. Nitrocellulose filters (Hybond-C Extra, Amersham Biosciences) saturated with 5% SDS and 2XSSC were placed directly on top for colony transfer. The cells were lysed and the DNA bound by microwaving 1X for 3 min. 2X for 30 sec. and then drying the filters at 80 BC. Standard pre-hybridization and hybridization procedures were followed for probing with 32P-labeled RGA sequence (Sambrook and Russell 2001).

Results

Each BAC plate-pool DNA was quantified and tested for by PCR amplification of the vector backbone sequence (Figure 1, top panel). All tested-pools tested positive. Nine pea RGA primer pairs were used for PCR on each plate-pool. Any positives from the first PCR screening were confirmed with a second PCR test. PCR screening of 3-plate DNA pools was successful in identifying seven out of seven low copy (1-2 in the pea genome) but neither of the two RGAs with 3-4 copies per pea genome (Table 1). We were able to identify the single BAC clone containing RGA2.97 in the library by probing filters of the three plates from one of the positive pools.

Table 1. Success in screening BAC pools for R-gene analog sequences compared with the estimated copy number in the pea genome.

RGA sequence

Copy number a

Number of BAC pools containing sequence

Insert size (bp)

GenBank accession number

RGA1.1

1 b

2

540

AF123695

RGA1.5

1

2

507

AF123696

RGA2.23

1 c

2

513

AF123697

RGA2.26

3 bc

0

534

AF123698

RGA2.65

4 c

0

513

AF123699

RGA2.75

1 c

1

516

AF123700

RGA2.97

2 b

3

513

AF123701

RGA2.159

1

2

519

AF123702

RGA-G3A

2 c

5

510

AF123703

a estimated copy number determined by Southern hybridisation (Timmerman-Vaughan et al 2000).

b variable copy number was observed in different pea accessions (Timmerman-Vaughan et al 2000).

c faintly hybridising bands were observed (Timmerman-Vaughan et al 2000).

Figure 1. PCR Screening of BAC pools for the pea RGA-G3A sequence. Top panel: positive control PCR assay to detect pBelBACII backbone sequences conducted on BAC pools, PI296818 DNA (P) and pBELOBACII vector DNA (V); Bottom panel: RGA-G3A specific PCR assay showing product amplification in one BAC pool and in PI296818 DNA. M – 1 KB+ DNA ladder.

M

BAC Pools

P

V

Conclusion

The creation of three-plate BAC DNA pools for PCR screening was successful in reducing both the time and experiments needed to identify the candidate filters containing the RGA sequence of interest.

Timmerman-Vaughan (2000) has reported segregation analysis in mapping populations indicates that most of the multicopy RGAs occur in tightly linked clusters that were not resolved by recombination in the pea RIL populations examined. The exceptions are RGA2.65 and RGA2.26. RGA2.65 mapped to linkage groups III and VI, therefore occuring in distinct genomic regions. Two tightly linked RGA2.26 loci were discerned by analysing segregation. Surprisingly, our PCR assays did not succeed in identifying any BAC pools containing these sequences. This indicates the necessity to increase both the number of clones to represent the pea genome and the number of enzymes used to partially digest the pea genomic DNA during library construction.

References

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