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Published online 1 September 2007
Published in Crop Sci 47:2021-2026 (2007)
© 2007 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Marker-Assisted Selection of Columbia Root-Knot Nematode Resistance Introgressed from Solanum bulbocastanum

L.-H. Zhanga, H. Mojtahedib, H. Kuangc, B. Bakerc and C. R. Brownb,*

a IAREC, Washington State Univ., 24106 N. Bunn Rd., Prosser, WA 99350
b USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350
c USDA-ARS and Univ. of California-Berkeley, Plant Gene Expression Center, 800 Buchanan St., Albany, CA 94710

* Corresponding author (cbrown{at}pars.ars.usda.gov).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Columbia root-knot nematode (Meloidogyne chitwoodi Golden et al.) is a serious pest that reduces tuber quality of potato (Solanum tuberosum L.) in the U.S. Northwest and other parts of the world. A gene, RMc1(blb), derived from the Mexican wild species Solanum bulbocastanum Dunal, encodes resistance to this pest. An F1 mapping population with >250 individuals generated from an intraspecific cross between resistant and susceptible clones of S. bulbocastanum, SB22 and PT29, respectively, was used for marker screening and genetic linkage analysis. One amplified fragment length polymorphism marker and five sequence tagged site (STS) markers cosegregated with RMc1(blb). The five STS markers were developed from bacterial artificial chromosome (BAC) end sequences of BAC clones that were derived from another wild species, S. demissum Lindl, and contained homologs of resistance gene N against tobacco mosaic virus. These markers were tested on families that were part of the introgression of RMc1(blb) into advanced breeding lines in BC5. The utility of an efficient alternative to greenhouse and field phenotypic screening was demonstrated. The results of this study confirm that molecular markers closely linked to RMc1(blb) will assist in a selection program, reducing expense and time involved in root-knot nematode screening.

Abbreviations: BAC, bacterial artificial chromosome • cM, centimorgan • CRN, Columbia root-knot nematode • PCR, polymerase chain reaction • RF, reproductive factor • STS, sequence tagged site

Marker-Assisted Selection of Columbia Root-Knot Nematode Resistance Introgressed from Solanum bulbocastanum

L.-H. Zhanga, H. Mojtahedib, H. Kuangc, B. Bakerc and C. R. Brownb,*

a IAREC, Washington State Univ., 24106 N. Bunn Rd., Prosser, WA 99350
b USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350
c USDA-ARS and Univ. of California-Berkeley, Plant Gene Expression Center, 800 Buchanan St., Albany, CA 94710

* Corresponding author (cbrown{at}pars.ars.usda.gov).

The Columbia root-knot nematode (Meloidogyne chitwoodi Golden et al.) is a serious pest that reduces tuber quality of potato (Solanum tuberosum L.) in the U.S. Northwest and other parts of the world. A gene, RMc1(blb), derived from the Mexican wild species Solanum bulbocastanum Dunal, encodes resistance to this pest. An F1 mapping population with >250 individuals generated from an intraspecific cross between resistant and susceptible clones of S. bulbocastanum, SB22 and PT29, respectively, was used for marker screening and genetic linkage analysis. One amplified fragment length polymorphism marker and five sequence tagged site (STS) markers cosegregated with RMc1(blb). The five STS markers were developed from bacterial artificial chromosome (BAC) end sequences of BAC clones that were derived from another wild species, S. demissum Lindl, and contained homologs of resistance gene N against tobacco mosaic virus. These markers were tested on families that were part of the introgression of RMc1(blb) into advanced breeding lines in BC5. The utility of an efficient alternative to greenhouse and field phenotypic screening was demonstrated. The results of this study confirm that molecular markers closely linked to RMc1(blb) will assist in a selection program, reducing expense and time involved in root-knot nematode screening.

Abbreviations: BAC, bacterial artificial chromosome • cM, centimorgan • CRN, Columbia root-knot nematode • PCR, polymerase chain reaction • RF, reproductive factor • STS, sequence tagged site


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE COLUMBIA root-knot nematode is a serious pest of potato in the U.S. Pacific Northwest and other parts of the world (Evans and Trudgill, 1992; Brown and Mojtahedi, 2005). The nematodes enter the tubers, mature, deposit eggs, and cause brown spots that reduce the tuber quality. With severe infection, galls also develop on the tuber surface (Brown et al., 1995). Columbia root-knot nematode (CRN) is controlled by soil fumigation, which is costly and environmentally hazardous (Brown et al., 2003). Through crop rotation, the nematode population can be reduced, but due to the low damage threshold on potato, this is not a viable alternative for a commercial grower. Thus, developing cultivars resistant to this pest presents an attractive alternative to fumigation and crop rotation.

Resistance to CRN was discovered in several indigenous wild Solanum species from Mexico and the U.S. Southwest (Brown et al., 2003). These species include S. bulbocastanum, S. hougasii Correll, and S. fendleri A. Gray. The gene RMc1(blb), controlling resistance to CRN, was identified in S. bulbocastanum from Mexico and used in potato germplasm development. By using restriction fragment length polymorphism (RFLP), RMc1(blb) was mapped on the upper arm of Chromosome 11 (Brown et al., 1995, 1996). A genetic locus at the same location on Chromosome 11 was identified in S. hougasii that conditions resistance to CRN (Brown et al., 1999). A susceptible S. bulbocastanum clone, PT 29, was used as the susceptible parent in a cross with SB22, a resistant clone.

To date, many disease-resistance genes have been mapped in potato. Some of these genes are clustered in "hot spots" in the potato genome (Solomon-Blackburn and Barker, 2001a, 2001b). Thus, selection for several diseases at once may be possible without screening for each disease individually, whether marker-assisted or phenotypic selection is used. Throughout the history of potato breeding and natural selection for disease resistance, it is probable that some advantage has already accrued from the fact that some resistance genes occur in these clusters. Resistance to potato wart (Synchytrium endobioticum) attributed to a single factor, Sen1, and a quantitative trait locus for resistance to potato leafroll virus have been also mapped to this arm of Chromosome 11 (Hehl et al., 1999; Marczewski et al., 2001). In addition, the upper arm of Chromosome 11 has been found to harbor genes for resistance to potato virus Y and potato virus A derived from the wild species S. stoloniferum and the Andean cultivated S. tuberosum L. subsp. andigenum (Juz. & Bukasov) Hawkes (Brigneti et al., 1997; Hämäläinen et al., 1997, 1998). Following mapping, several disease resistance genes in potato have been or are being cloned (Solomon-Blackburn and Barker, 2001a, 2001b; Gebhardt and Valkonen, 2001).

Molecular markers linked to resistance genes can be used for marker-assisted selection. A polymerase chain reaction (PCR)-based assay has been developed for marker-assisted selection of root-knot nematode resistance (Rmc1) in potato (ver der Voort et al., 1999). The closest markers to Rmc1, however, are localized 4 centimorgans (cM) away from the gene. The goal of this study was to find molecular markers closely linked to RMc1(blb) suitable for identifying the RMc1(blb) locus in breeding lines in our ongoing potato crop improvement program. Localizing the resistance gene will also facilitate eventual gene cloning. The specific objective of this experiment was to test the utility of five STS markers cosegregating with RMc1(blb) on the breeding lines generated for introgressing RMc1(blb) into cultivated potato to facilitate breeding efforts.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
We used a S. bulbocastanum clone proven to be resistant to CRN reproduction (SB22), and PT29, a susceptible genotype (Brown et al., 1996, 2003). An F1 mapping population of >250 individuals generated from an intraspecific cross between SB22 and PT29 and was used for marker screening and genetic linkage analysis. This population segregates with a ratio of 1:1 for resistant/susceptible, suggesting parental genotypes of heterozygous resistant and homozygous susceptible. Bulked segregant analysis was used to determine closely linked markers in the distal end of potato Chromosome 11 for tagging the RMc1(blb) locus. Two DNA pools of F1 individuals (SB22 x PT29) were constructed based on phenotypic screening. Later, marker panels were tested on 18 BC5 breeding lines (Fig. 1 ) with a total of 180 individuals to identify marker segregation. These panels were also tested on 14 selected independent breeding clones descended from SB22 (Table 1 ).


Figure 1
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  		Figure 1. Scheme of introgression of Meloidogyne chitwoodi resistance into a tetroploid gene pool. SB22 = resistant; CBP233 = F1 (through protoplast fusion with R4), A84118–3, tetraploid breeding lines.

 

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  		Table 1. The resistance to Meloidgyne chitwoodi race 1 (WAMC1) and genotyping results from the five sequence tagged site markers on the selected breeding clones.

 
Nematode Resistance Tests
The CRN host race 1 (WAMC1) (Mojtahedi et al., 1988) was maintained on ‘Rutger’ tomato (Lycopersicon esculentum Mill.) plants at the USDA-ARS (Prosser, WA) glasshouse facilities. The eggs were extracted from infected tomato roots (Hussey and Barker, 1973). Resistance of test plants to CRN-WAMC1 was based on nematode efficiency to reproduce at 55 d after inoculating with 5000 eggs (reproductive factor [RF] = final egg count/initial egg inoculum, 5000). An entry was rated resistant if RF ≤ 0.1 (non- to poor host), and susceptible if RF ≥ 1 (suitable host) (O'ostinbrink, 1966; Sasser et al., 1984). For each genotype, three replicate resistance tests were performed. In each test, the roots were weighed before egg extraction to ascertain that a low nematode egg count was not related to poor root growth of test plants. Rutger tomato host plants were also included in each test to ascertain the aggressiveness of the inoculum used. ‘Thor’ alfalfa (Medicago sativa L.) and ‘California Wonder’ pepper (Capsicum annuum L. var. annuum) plants were included to prove that the inoculum was free from CRN race 2 and Meloidogyne hapla. Columbia root-knot nematode race 2 colonizes alfalfa but not pepper. Both alfalfa and pepper are suitable hosts of M. hapla.

DNA Extraction and Polymerase Chain Reaction Conditions
The DNA was extracted from the same plants evaluated for resistance to nematode in the greenhouse. Using a Fast DNA Spin Kit (following the manufacture's instructions, MP Biomedical, Irvine, CA), approximately 200 mg of young leaf tissue was harvested and placed in spin tubes. The PCR primers were selected from the BAC end sequences using the computer program Primer 3 (Rozen and Skaletsky, 2000). A potato BAC library (Kuang et al., 2005) constructed from the hexaploid wild potato, S. demissum (PI 161729), was probed with the conserved regions of Nl (N-like) resistance genes. The N gene of tobacco (Nicotiana tabacum L.) encodes resistance to the tobacco mosaic virus, which was cloned from tobacco and sequenced (Whitham et al., 1994, 1996). Oligonucleotide primers were designed based on the Nl sequence and PCR products were amplified from genomic DNA of S. demissum using these primers. The PCR products were used as a probe to hybridize high-density filters containing BAC clones of S. demissum. Insert ends of 240 positive BAC clones (BAC ends) were obtained. Oligonucleotide primers were designed based on these BAC end sequences and were used to screen the F1 population segregating RMc1(blb). The ends of the inserts in the identified BAC clones were sequenced and these sequences were used to develop PCR primers, which were assessed for linkage to RMc1(blb).

The PCR reactions were performed in a 10-µL volume containing 5 µL of 2x CLP TAQ master mix (a final Mg concentration of 1.5 mmol L–1; CLP, San Diego, CA), 0.5 µmol L–1 of each primer (forward and reverse, Integrated DNA Technologies, Coralville, IA), and 10 ng of DNA templates. The PCR was performed in a PTC-200 thermocycler (MJ Research, Watertown, MA), set to the following program: 3 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at annealing temperature (Ta), 1 min 30 s at 72°C, and a final extension step of 10 min at 72°C. After PCR amplification, the PCR products were separated on a 2% agarose gel in 0.5x TBE (Tris–borate–EDTA) buffer and visualized by ethidium bromide staining. In addition, one of the PCR products was separated on a nondenaturing 6% polyacrylamide gel at a constant power of 0.5 W. After electrophoresis, the banding pattern was visualized using Ag staining.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Six Markers Cosegregate with RMc1(blb)
The F1 mapping population showed a 1:1 segregation for resistant/susceptible phenotypes, suggesting parental genotypes of heterozygous resistant and homozygous susceptible. Bulked segregant analysis revealed that a total of 43 markers that mapped on the upper arm of Chromosome 11 of potato and tomato were used in this study. The markers most closely linked with the RMc1(blb) locus are cleaved amplified polymorphic sequence (CAPS) markers M33 and M39, which were originally developed for fine mapping resistance to potato virus Y (Brigneti et al., 1997). Markers M33 and M39 were 2.4 and 3.8 cM from RMc1(blb), respectively. By screening 576 amplified fragment length polymorphism (AFLP) primer combinations, one AFLP marker, EAGGMCTA213, was found to cosegregate with the RMc1(blb) resistance gene (Fig. 2 ).


Figure 2
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  		Figure 2. Potato Chromosome 11 of the parent SB22 and the markers tightly linked to the Columbia root-knot nematode resistance gene RMc1(blb) on the distal end of the upper arm of potato Chromosome 11. Distance is measured in centimorgans.

 
Besides the AFLP marker, five STS markers also cosegregated with RMc1(blb) (Fig. 2). A Toll-interleukin receptor type R gene (N-like gene = Nl) was shown previously to be closely linked to Sen1, which is near RMc1(blb) (Hehl et al., 1999). Five markers (Table 2 ) developed from five independent BAC clones were found to cosegregate with the RMc1(blb) locus: PCR products produced amplicons from all resistant plants but not from any susceptible plants.


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  		Table 2. Primers used for the marker-assisted selection.

 
Sequence Tagged Site Markers vs. Resistance Phenotypes
The high RF value of CRN on tomato indicated that the inocula used in these tests were infective. Lack of reproduction of the nematode on alfalfa and pepper proved that the inocula were indeed CRN race 1 (i.e., WAMC1). The screening results in the BC5 showed there were 82 individuals (52%) that were non- to poor hosts (resistant with RF ≤ 0.1), and 75 individuals (48%) that were suitable hosts (susceptible with RF ≥ 1). The female parents of the BC5 were resistant to CRN race 1, and all the STS markers amplified PCR products from the female parents, as expected. At the same time, all of the male parents were susceptible and did not amplify PCR products using any of the five STS markers.

To demonstrate the usefulness of these markers in the breeding program, 180 plants of 18 BC5 families plus their maternal and paternal parents were screened and classified individually for nematode infection and genotyped using the above five STS markers cosegregating with RMc1(blb). All resistant plants showed PCR products using each of the five STS markers, while all susceptible plants did not have PCR products for any of the five STS markers (Fig. 3 ). Among 14 selected breeding clones for introgressing RMc1(blb) into cultivated potato, the expected PCR amplification bands associated with RMc1(blb) were shown in all 11 resistant clones, and not shown in three susceptible clones (Table 2). The genotyping results for 176 individuals (out of a total of 180 tested progenies and parents) matched perfectly with the phenotyping results in the greenhouse. We concluded that there was 100% accuracy involved using these five STS markers for selecting potato harboring CRN resistance, that is, the RMc1(blb) locus (Table 3 ).


Figure 3
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  		Figure 3. Segregation of sequence tagged site (STS) markers (a) 39E18 and (b) 193I9 in BC5 breeding lines derived from the cross between a backcross resistant parent and susceptible cultivated potato. Individuals with RMc1(blb) nematode resistance locus contain a band of ~400 base pairs (bp) in 39E18 and ~300 bp in 193I9. R = resistant, S = susceptible.

 

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  		Table 3. Segregation of host screening and polymerase chain reaction (PCR) markers in evaluated BC5 progenies.

 
Nematode Resistance Screening
Of the157 progenies screened with five STS markers for the presence of the RMc1(blb) locus, 82 plants were positive and 75 plants were negative. The results from both host screening and PCR screening showed that the 1:1 segregation ratio fit the model of a single dominant gene present in the heterozygous state in the RMc1(blb) parents, PA99N82–4 and PA99N88–2 (significance of {chi}2 test on the value {alpha} = 0.05, P = 0.5764, Table 3).


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
From previous studies, the resistance gene RMc1(blb) was known to be located on Chromosome 11; however, the exact location of the gene is still unknown. The closest markers used in the study still are several centimorgans away from the target gene (Brown et al., 1996; van der Voort et al., 1999). To facilitate the map-based cloning efforts, we are developing a high-resolution mapping for the RMc1(blb) locus. The upper arm of Chromosome 11 is a "hot-spot" of resistance genes. There are several resistance genes, including Sen1 and Ryadg, mapped to this region (Hehl et al., 1999; Brigneti et al., 1997; Hämäläinen et al., 1997, 1998). Furthermore, Sen1 and Ryadg are both syntenic with the N locus in tobacco for resistance to tobacco mosaic virus (Gebhardt and Valkonen, 2001). Therefore, the N homologous sequences and markers developed for the CRN resistance could benefit other R genes on the distal end of Chromosome 11.

There are also markers developed in this region by earlier potato and tomato researchers. The RFLP probe sequences, BAC sequences, and STS, SCAR, CAP, and SSR markers that were mapped to the region were assessed to find more markers closely linked to the gene. Van der Voort et al. (1999) developed markers for this CRN resistance in 29 potato clones of a BC3 population derived from a cross between S. tuberosum and S. bulbocastanum. The two closest markers, however, CT182 and M39b, were still 4 cM away from Rmc1. After screening 43 markers that were mapped previously on the upper arm of Chromosome 11, there were only two markers that segregated in the mapping population. The genetic linkage map in Fig. 2 shows that M39 is 3.5 cM away from the RMc1(blb) locus. A rapid and efficient approach was needed for detecting more closely linked DNA markers and to expedite plant gene isolation by positional cloning and the construction of high-density genetic and physical maps of the RMc1(blb).

Originally, SB22, which is reproductively isolated from cultivated potato, was hybridized with cultivated potato by protoplast fusion (Austin et al., 1993). This was the most expedient means of introgressing the resistance gene into advanced breeding populations. It also provided the BC2 generation mapping capability as the S. bulbocastanum genome assorted and recombined randomly. "Fine mapping" of RMc1(blb) will be accomplished in the intra S. bulbocastanum resistant x susceptible population. It is difficult and time consuming to introgress a resistance gene into an old cultivar by means of traditional breeding while retaining the desirable characteristics of the old cultivar. The outcome, in a highly heterozygous crop, does not recover the recurrent parent phenotype, but rather an approximation of it. Marker-assisted selection may be a very valuable tool not only for mapping efforts, but also for introgressing RMc1(blb) and other disease resistance genes. Potential benefits include accuracy and the ability to perform the selection at very early seedling stages.

Wild potato species play a very important role in germplasm enhancement and resistant introgression. A late blight resistance gene, RB, was found in S. bulbocastanum. The parental line PT 29, which was the susceptible parent in this study for the introgression of the CRN resistance gene, was used as the resistance source for RB gene mapping and cloning efforts (Song et al., 2003). The PCR-based markers for marker-assisted selection were also developed for tracking the RB gene in breeding populations (Colton et al., 2006).

Our results indicate that it is possible to use a sequence homology of known resistance genes to identify candidate BACs containing resistance gene analogs and develop markers from another wild potato species. A saturated map of the RMc1(blb) locus region facilitated a map-based cloning approach of the gene responsible for the resistance to the Columbia root-knot nematode. Tightly linked STS markers demonstrated their practicality for marker-assisted breeding by differentiating between resistant and susceptible clones of a set of diverse breeding lines. Marker-assisted selection for disease resistance in potato has been applied to the Ryadg gene (Hämäläinen et al., 1997). The most likely practical use for marker-assisted selection for disease resistance in potato seems to be the introgression of resistance genes from wild species or otherwise poor parents, and also selecting seedlings for several traits simultaneously. In the short term, marker-assisted selection might be practical for conventional potato programs, but is likely to become a more viable option with further improvements in the technology and development of amplification protocols that detect several important genes in one test.


   ACKNOWLEDGMENTS
 
This research is a part of the Potato Genome Project funded by National Science Foundation Grant no. DBI-9975866.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication January 3, 2007.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





This Article
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