Genetics and Resistance
Genetic Mapping and Chromosomal Assignment
of Magnaporthe oryzae Avirulence Genes AvrPik, AvrPiz,
and AvrPiz-t Controlling Cultivar Specificity on Rice
Chao-Xi Luo, Liang-Fen Yin, Satomi Koyanagi, Mark L. Farman, Motoaki Kusaba, and Hiroshi Yaegashi
First, second, third, fifth, and sixth authors: Faculty of Agriculture, Saga University, Saga, 840-8502, Japan; and fourth author: Department
of Plant Pathology, University of Kentucky, Lexington 40546.
Accepted for publication 9 February 2005.
ABSTRACT
Luo, C.-X., Yin, L.-F., Koyanagi, S., Farman, M. L., Kusaba, M., and seven chromosomes greater than 3.5 megabases (Mb) in size and 84R-
Yaegashi, H. 2005. Genetic mapping and chromosomal assignment of 62B possessed a small chromosome of ≈1.6 Mb. The linkage groups
Magnaporthe oryzae avirulence genes AvrPik, AvrPiz, and AvrPiz-t containing AvrPiz and AvrPiz-t were assigned to chromosomes 3 and 7,
controlling cultivar specificity on rice. Phytopathology 95:640-647. respectively. Some markers from the linkage group that contained AvrPik
hybridized with chromosome 1 and the 1.6-Mb chromosome, yet all of
A genetic map including three avirulence (Avr) genes, AvrPik, AvrPiz, the cloned RAPD markers that were closely linked to AvrPik hybridized
and AvrPiz-t, was constructed in a genetic cross of two rice field isolates, exclusively to the 1.6-Mb chromosome in 84R-62B, the parent that
84R-62B and Y93-245c-2. The chromosomal locations of the Avr genes possesses AvrPik. Thus, we conclude that AvrPik is located on the 1.6-Mb
were determined by using selected markers to probe Southern blots of the chromosome in 84R-62B.
parental chromosomes that had been separated by contour-clamped
homogenous electric fields electrophoresis. Electrophoretic karyotyping Additional keywords: Pyricularia grisea, P. oryzae, rice blast fungus.
showed that both parental isolates 84R-62B and Y93-245c-2 contained
The ascomycetous fungus Magnaporthe oryzae B. Couch that it is located just 50 bp from the telomere repeat (16). By
(formerly Magnaporthe grisea (Hebert) Barr.) (2) is the causal contrast, the AVR1-CO39, ACE1, and AVR1-MARA genes are
agent of rice blast, the most damaging disease of cultivated rice much more stable and, although they have been lost or mutated in
(Oryza sativa L.). The most effective way to control rice blast certain fungal lineages during the course of M. oryzae evolution,
disease is by growing resistant rice cultivars; however, newly they do not exhibit instability in vegetative culture (1,5,13). Sig-
developed resistant cultivars are often defeated after only a few nificantly, all of these genes reside at internal chromosomal loca-
years of commercial production due to the emergence of new tions (4,13,19). The association between chromosomal location
pathogenic variants (races) in M. oryzae populations. Creation of and stability is not perfect, however, because spontaneous PWL2
rice cultivars with durable resistance will require a firm mutants arise in vegetative culture (9), yet this gene resides in the
understanding of the processes underlying pathogenic variation in middle of chromosome 2 (21). At present, too few Avr genes have
M. oryzae. been mapped or cloned to know if PWL2 is a rare exception, or if
Blast resistance in rice generally follows a gene-for-gene re- the correlation between stability and chromosome position is
lationship (8,11,17) and, as such, is dependent on the presence in purely coincidental. It follows that a better understanding of fac-
M. oryzae of a cognate avirulence (Avr) gene. Understanding the tors affecting Avr gene mutation and loss in M. oryzae will require
genetic mechanisms underlying M. oryzae’s ability to overcome mapping, cloning, and characterization of many more genes. Here
host resistance requires molecular characterization of these Avr we describe the genetic mapping and chromosomal assignment of
genes. To date, several M. oryzae Avr genes have been genetically three M. oryzae Avr genes, AvrPik, AvrPiz, and AvrPiz-t.
mapped, and four genes (PWL2, AVR1-CO39, AVR-Pita, and
ACE1) have been cloned by chromosome walking (1,7,16,20); MATERIALS AND METHODS
and, although the gene has not yet been isolated, the locus con-
taining AVR1-MARA has been characterized at the molecular level Fungal isolates. Parental isolates, 84R-62B (MAT1-1) and
(13). Studies of the chromosomal organization of these genes Y93-245c-2 (MAT1-2), and a subset of 60 progeny used in this
have provided valuable insights into the genetic basis for patho- study have been described previously (12).
genic variability in M. oryzae (1,5,8,13,16,20). For example, AVR- DNA extraction. Each fungal isolate was grown in 50 ml of
Pita and AVR-TSUY both exhibit extremely high levels of muta- potato sucrose broth (200 ml of broth from 200 g of potato, 20 g
bility in vegetative culture, because the spontaneous virulent mu- of sucrose per liter) for 4 to 5 days at 25°C on an orbital shaker
tants of both genes were easily isolated after standard inoculation (120 rpm). Mycelia were harvested from the liquid culture by
experiments (23). Significantly, both genes mapped near telo- filtration through Kimwipe paper (Kimberly-Clark Corporation,
meres (21), and molecular characterization of AVR-Pita revealed Tokyo) and then frozen in liquid nitrogen and ground to a fine
powder with a mortar and pestle. Three- to four-hundred milli-
Corresponding author: M. Kusaba; E-mail address: grams of mycelial powder was transferred to 0.5 ml of extraction
buffer (0.5% sodium dodecyl sulfate [SDS], 100 mM LiCl,
DOI: 10.1094 / PHYTO-95-0640 100 mM EDTA, and 10 mM Tris [pH 7.5]) in a micro-centrifuge
© 2005 The American Phytopathological Society tube. After mixing, the mixture was incubated at 55°C for 30 min,
640 PHYTOPATHOLOGY
, and then centrifuged at 12,000 rpm for 15 min. The supernatant sham Biosciences UK Limited, Buckinghamshire, UK) and used
was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, as probes for hybridization. Hybridization was performed at 55°C
vol/vol/vol) and precipitated with isopropanol. The pellet was in Gene Images AlkPhos Direct hybridization buffer (Amersham
washed in 70% ethanol and resuspended in 0.5 ml of TE buffer Biosciences UK Limited) including 0.5 M NaCl and 4% blocking
(1 mM EDTA and 10 mM Tris [pH 8.0]) containing 20 ng of reagent (Amersham Biosciences UK Limited). After hybridiza-
RNase A (Nippon Gene, Toyama, Japan). After RNA digestion, tion, the membrane was washed twice in primary wash buffer
the solution was extracted twice with phenol/chloroform/isoamyl (2.0 M urea, 0.1% SDS, 50 mM NaH2PO4 [pH 7.0], 150 mM
alcohol (25:24:1, vol/vol/vol) and once with chloroform/isoamyl NaCl, 1 mM MgCl2, and 0.2% blocking reagent) at 55°C for
alcohol (24:1, vol/vol). Final supernatant was precipitated with 10 min and then washed twice in secondary wash buffer (50 mM
11/16 volumes of 20% polyethylene glycol containing 2.5 M Tris [pH 10.0], 100 mM NaCl, and 2 mM MgCl2) at room
NaCl. After washing in 70% ethanol, the DNA pellet was re- temperature for 5 min. Target DNA was detected using the CDP-
suspended in TE buffer. star reagent (Amersham International plc, Buckinghamshire, UK)
RAPD analysis. A total of 780 primers (Kit A to Z and Kit AA according to the manufacturer’s instructions.
to AM, Operon Technologies, Alameda, CA) was used in random Bal31 nuclease (New England Biolabs) digestion of genomic
amplified polymorphism DNA (RAPD) analysis. Polymerase DNA was performed as described by Farman and Leong (6). The
chain reaction (PCR) was carried out in one of two methods: with Bal31-treated DNA sample was subsequently cleaved with
primer combinations (Kit A to Z and Kit AA to AM), or single HindIII. After electrophoresis, the fractionated DNA was trans-
primers (Kit A to F) in each reaction. The amplification reaction ferred to Hybond N+ membrane and hybridized with the oligo-
was performed in a 20-µl reaction containing 0.5 units of Ex. Taq nucleotide probe (TTAGGG)10.
DNA polymerase (TaKaRa, Otsu, Japan), 1× PCR buffer provided Mapping of RFLP and RAPD markers. Telomeric restriction
by the manufacturer, 125 µM each dNTP, 30 ng of template DNA, fragments were observed as the presence of a hybridizing band of
and 0.5 µM of each primer for dual primer reactions or a given size. If a fragment was present in isolate 84R-62B, occur-
1.0 µM primer for single primer reactions. Amplification was rences of this fragment in the progeny were scored as “A”; occur-
performed in a 9600 thermocycler (Perkin-Elmer, Norwalk, CT) rences of fragments from isolate Y93-245c-2 were scored as “B”.
programmed for 3 min at 94°C, 30 cycles of 1 min at 94°C, 1 min The segregation data for each telomere consisted of a consensus
at 36°C, and 2 min at 72°C followed by 5 min at 72°C. derived by scoring fragments produced in the EcoRI and the
Amplification products were separated in a 0.8% agarose gel in HindIII digests. For progeny with inconsistent segregation of par-
0.5× Tris-borate-EDTA (TBE) buffer (44.5 mM Tris-borate and ticular telomeres due to rearrangement, data were scored as “–”.
1 mM EDTA [pH 8.0]), using a 50- to 2,500-bp DNA Marker Linkage analysis and construction of the genetic map were
(TaKaRa, Otsu, Japan) and Smart ladder (Nippon Gene, Toyama, performed using Mapmaker version 2.0 for Macintosh (E. S.
Japan) as molecular size markers. Lander, P. Green, J. Abrahamson, A. Barlaow, M. J. Daly, S. E.
Cloning of RAPD markers and sequencing. Markers of Lincoln, and L. Newburg, Whitehead Institute, Cambridge, MA).
interest were separated from other amplified fragments by agarose Parameters for map construction were a minimum logarithm of
gel electrophoresis and extracted from the gel by QIAquick Gel odds (LOD) score of 3.0 and a maximum recombination fraction
Extraction Kit (Qiagen Sciences, Germantown, MD) and cloned of 0.4. The Kosambi function was used to compute recombination
into pGEM-T Easy vector (Promega, Madison, WI). Transforma- distances in centimorgans (cM).
tion of plasmids into DH5α-competent cells (Nippon Gene, Preparation of chromosome-size DNA and contour-clamped
Toyama, Japan) was performed following the manufacturer’s in- homogenous electric fields (CHEF) electrophoresis. Protoplasts
structions. The cloned RAPD marker was sequenced using were prepared from mycelial cultures as described previously by
BigDye terminator version 3.1 cycle sequencing (Applied Bio- Tosa et al. (22). The resulting protoplasts were suspended in SE
systems, Foster, CA) according to the manufacturer’s instructions. buffer (1.0 M sorbitol, 50 mM EDTA [pH 8.0]) at a density of
Sequences were acquired using the Genetic Analyzer DNA Model approximately 1 × 109 per milliliter and were then added to an
310 (Applied Biosystems). equal volume of 1.5% low melt agarose (Bio-Rad Laboratories,
Southern hybridization analysis. Total genomic DNA was Hercules, CA) in 0.125 M EDTA (pH 7.5). The suspension was
digested with restriction enzyme and fractionated through a 0.8% mixed by pipetting and then poured into a mold to solidify into an
agarose gel in 0.5× TBE buffer. The fractionated DNA was trans- agarose/protoplast plug. The solidified plugs were poured into
ferred to an MSI nylon membrane (Osmonics, Westborough, MA) 10 ml of LET buffer (0.01 M Tris [pH 7.5], 0.45 M EDTA [pH
and fixed by UV irradiation following the manufacturer’s in- 8.0], and 7.5% β-mercaptoethanol) at 37°C for 16 to 24 h. After
structions. Plasmid clones containing RAPD markers as described washing three times in 10 ml of 0.05 M EDTA (15 min per wash),
previously and plasmid clones representing restriction fragment 10 ml of NDS buffer (0.01 M Tris [pH 7.5], 0.45 M EDTA [pH
length polymorphism (RFLP) markers in the map of Skinner et al. 8.0], and 1.0% lauryl sarcosine) containing 10 mg of proteinase K
(18) were used as probes. The membrane was hybridized over- (Invitrogen Life Technologies, Carlsbad, CA) was added and
night with biotin-labeled probes in 6× SSC (1× SSC is 0.15 M incubated at 50°C for 16 to 24 h. The plugs were then washed
NaCl plus 0.015 M sodium citrate) containing 5× Denhardt’s so- twice with 10 ml of 0.05 M EDTA for 15 min at room tem-
lution, 0.5% SDS, and 100 µg of heat denaturized salmon sperm perature before equilibrating in 10 ml of 0.05 M EDTA at room
DNA per ml at 68°C. The hybridized membrane was washed temperature for 16 to 24 h. Finally, the EDTA buffer was dis-
twice in 2× SSC, 0.1% SDS at room temperature for 5 min, and carded and 5 ml of fresh 0.05 M EDTA buffer was added, and the
then washed twice in 0.1× SSC, 0.1% SDS at 68°C for 15 min. plugs were stored at 4°C until use.
Detection of target DNA sequences was performed using the CHEF electrophoresis was performed in 0.8% Certified Mega-
NEBlot Phototope Kit (New England Biolabs, Beverly, MA). base Agarose (Bio-Rad Laboratories) using a running buffer of
Identification and characterization of telomeric restriction 0.5× TBE at 14°C. The CHEF electrophoresis was carried out in a
fragments. Total DNA of each isolate was digested with EcoRI or Bio-Rad DR III system (Bio-Rad Laboratories) with switching
HindIII and separated in 0.8 and 0.4% agarose gels in 0.5× TBE intervals of 90 min for 5 days and then 60 min for 2 days at 35 V.
buffer. The fractionated DNA was transferred to Hybond N+ Schizosaccharomyces pombe and Saccharomyces cerevisiae
membranes (Amersham Pharmacia Biotech UK Limited, whole chromosomal DNAs (Bio-Rad Laboratories) were used as
Buckinghamshire, UK) and then fixed by drying at 80°C for 2 h. size markers. After electrophoresis, the resolved chromosomes
A synthetic oligonucleotide (TTAGGG)10 was labeled with alka- were blotted to MSI nylon membranes following the protocol in
line phosphate using AlkPhos Direct Labeling Reagents (Amer- the Bio-Rad DR III system applications manual. Hybridization
Vol. 95, No. 6, 2005 641
Genetic Mapping and Chromosomal Assignment
of Magnaporthe oryzae Avirulence Genes AvrPik, AvrPiz,
and AvrPiz-t Controlling Cultivar Specificity on Rice
Chao-Xi Luo, Liang-Fen Yin, Satomi Koyanagi, Mark L. Farman, Motoaki Kusaba, and Hiroshi Yaegashi
First, second, third, fifth, and sixth authors: Faculty of Agriculture, Saga University, Saga, 840-8502, Japan; and fourth author: Department
of Plant Pathology, University of Kentucky, Lexington 40546.
Accepted for publication 9 February 2005.
ABSTRACT
Luo, C.-X., Yin, L.-F., Koyanagi, S., Farman, M. L., Kusaba, M., and seven chromosomes greater than 3.5 megabases (Mb) in size and 84R-
Yaegashi, H. 2005. Genetic mapping and chromosomal assignment of 62B possessed a small chromosome of ≈1.6 Mb. The linkage groups
Magnaporthe oryzae avirulence genes AvrPik, AvrPiz, and AvrPiz-t containing AvrPiz and AvrPiz-t were assigned to chromosomes 3 and 7,
controlling cultivar specificity on rice. Phytopathology 95:640-647. respectively. Some markers from the linkage group that contained AvrPik
hybridized with chromosome 1 and the 1.6-Mb chromosome, yet all of
A genetic map including three avirulence (Avr) genes, AvrPik, AvrPiz, the cloned RAPD markers that were closely linked to AvrPik hybridized
and AvrPiz-t, was constructed in a genetic cross of two rice field isolates, exclusively to the 1.6-Mb chromosome in 84R-62B, the parent that
84R-62B and Y93-245c-2. The chromosomal locations of the Avr genes possesses AvrPik. Thus, we conclude that AvrPik is located on the 1.6-Mb
were determined by using selected markers to probe Southern blots of the chromosome in 84R-62B.
parental chromosomes that had been separated by contour-clamped
homogenous electric fields electrophoresis. Electrophoretic karyotyping Additional keywords: Pyricularia grisea, P. oryzae, rice blast fungus.
showed that both parental isolates 84R-62B and Y93-245c-2 contained
The ascomycetous fungus Magnaporthe oryzae B. Couch that it is located just 50 bp from the telomere repeat (16). By
(formerly Magnaporthe grisea (Hebert) Barr.) (2) is the causal contrast, the AVR1-CO39, ACE1, and AVR1-MARA genes are
agent of rice blast, the most damaging disease of cultivated rice much more stable and, although they have been lost or mutated in
(Oryza sativa L.). The most effective way to control rice blast certain fungal lineages during the course of M. oryzae evolution,
disease is by growing resistant rice cultivars; however, newly they do not exhibit instability in vegetative culture (1,5,13). Sig-
developed resistant cultivars are often defeated after only a few nificantly, all of these genes reside at internal chromosomal loca-
years of commercial production due to the emergence of new tions (4,13,19). The association between chromosomal location
pathogenic variants (races) in M. oryzae populations. Creation of and stability is not perfect, however, because spontaneous PWL2
rice cultivars with durable resistance will require a firm mutants arise in vegetative culture (9), yet this gene resides in the
understanding of the processes underlying pathogenic variation in middle of chromosome 2 (21). At present, too few Avr genes have
M. oryzae. been mapped or cloned to know if PWL2 is a rare exception, or if
Blast resistance in rice generally follows a gene-for-gene re- the correlation between stability and chromosome position is
lationship (8,11,17) and, as such, is dependent on the presence in purely coincidental. It follows that a better understanding of fac-
M. oryzae of a cognate avirulence (Avr) gene. Understanding the tors affecting Avr gene mutation and loss in M. oryzae will require
genetic mechanisms underlying M. oryzae’s ability to overcome mapping, cloning, and characterization of many more genes. Here
host resistance requires molecular characterization of these Avr we describe the genetic mapping and chromosomal assignment of
genes. To date, several M. oryzae Avr genes have been genetically three M. oryzae Avr genes, AvrPik, AvrPiz, and AvrPiz-t.
mapped, and four genes (PWL2, AVR1-CO39, AVR-Pita, and
ACE1) have been cloned by chromosome walking (1,7,16,20); MATERIALS AND METHODS
and, although the gene has not yet been isolated, the locus con-
taining AVR1-MARA has been characterized at the molecular level Fungal isolates. Parental isolates, 84R-62B (MAT1-1) and
(13). Studies of the chromosomal organization of these genes Y93-245c-2 (MAT1-2), and a subset of 60 progeny used in this
have provided valuable insights into the genetic basis for patho- study have been described previously (12).
genic variability in M. oryzae (1,5,8,13,16,20). For example, AVR- DNA extraction. Each fungal isolate was grown in 50 ml of
Pita and AVR-TSUY both exhibit extremely high levels of muta- potato sucrose broth (200 ml of broth from 200 g of potato, 20 g
bility in vegetative culture, because the spontaneous virulent mu- of sucrose per liter) for 4 to 5 days at 25°C on an orbital shaker
tants of both genes were easily isolated after standard inoculation (120 rpm). Mycelia were harvested from the liquid culture by
experiments (23). Significantly, both genes mapped near telo- filtration through Kimwipe paper (Kimberly-Clark Corporation,
meres (21), and molecular characterization of AVR-Pita revealed Tokyo) and then frozen in liquid nitrogen and ground to a fine
powder with a mortar and pestle. Three- to four-hundred milli-
Corresponding author: M. Kusaba; E-mail address: grams of mycelial powder was transferred to 0.5 ml of extraction
buffer (0.5% sodium dodecyl sulfate [SDS], 100 mM LiCl,
DOI: 10.1094 / PHYTO-95-0640 100 mM EDTA, and 10 mM Tris [pH 7.5]) in a micro-centrifuge
© 2005 The American Phytopathological Society tube. After mixing, the mixture was incubated at 55°C for 30 min,
640 PHYTOPATHOLOGY
, and then centrifuged at 12,000 rpm for 15 min. The supernatant sham Biosciences UK Limited, Buckinghamshire, UK) and used
was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, as probes for hybridization. Hybridization was performed at 55°C
vol/vol/vol) and precipitated with isopropanol. The pellet was in Gene Images AlkPhos Direct hybridization buffer (Amersham
washed in 70% ethanol and resuspended in 0.5 ml of TE buffer Biosciences UK Limited) including 0.5 M NaCl and 4% blocking
(1 mM EDTA and 10 mM Tris [pH 8.0]) containing 20 ng of reagent (Amersham Biosciences UK Limited). After hybridiza-
RNase A (Nippon Gene, Toyama, Japan). After RNA digestion, tion, the membrane was washed twice in primary wash buffer
the solution was extracted twice with phenol/chloroform/isoamyl (2.0 M urea, 0.1% SDS, 50 mM NaH2PO4 [pH 7.0], 150 mM
alcohol (25:24:1, vol/vol/vol) and once with chloroform/isoamyl NaCl, 1 mM MgCl2, and 0.2% blocking reagent) at 55°C for
alcohol (24:1, vol/vol). Final supernatant was precipitated with 10 min and then washed twice in secondary wash buffer (50 mM
11/16 volumes of 20% polyethylene glycol containing 2.5 M Tris [pH 10.0], 100 mM NaCl, and 2 mM MgCl2) at room
NaCl. After washing in 70% ethanol, the DNA pellet was re- temperature for 5 min. Target DNA was detected using the CDP-
suspended in TE buffer. star reagent (Amersham International plc, Buckinghamshire, UK)
RAPD analysis. A total of 780 primers (Kit A to Z and Kit AA according to the manufacturer’s instructions.
to AM, Operon Technologies, Alameda, CA) was used in random Bal31 nuclease (New England Biolabs) digestion of genomic
amplified polymorphism DNA (RAPD) analysis. Polymerase DNA was performed as described by Farman and Leong (6). The
chain reaction (PCR) was carried out in one of two methods: with Bal31-treated DNA sample was subsequently cleaved with
primer combinations (Kit A to Z and Kit AA to AM), or single HindIII. After electrophoresis, the fractionated DNA was trans-
primers (Kit A to F) in each reaction. The amplification reaction ferred to Hybond N+ membrane and hybridized with the oligo-
was performed in a 20-µl reaction containing 0.5 units of Ex. Taq nucleotide probe (TTAGGG)10.
DNA polymerase (TaKaRa, Otsu, Japan), 1× PCR buffer provided Mapping of RFLP and RAPD markers. Telomeric restriction
by the manufacturer, 125 µM each dNTP, 30 ng of template DNA, fragments were observed as the presence of a hybridizing band of
and 0.5 µM of each primer for dual primer reactions or a given size. If a fragment was present in isolate 84R-62B, occur-
1.0 µM primer for single primer reactions. Amplification was rences of this fragment in the progeny were scored as “A”; occur-
performed in a 9600 thermocycler (Perkin-Elmer, Norwalk, CT) rences of fragments from isolate Y93-245c-2 were scored as “B”.
programmed for 3 min at 94°C, 30 cycles of 1 min at 94°C, 1 min The segregation data for each telomere consisted of a consensus
at 36°C, and 2 min at 72°C followed by 5 min at 72°C. derived by scoring fragments produced in the EcoRI and the
Amplification products were separated in a 0.8% agarose gel in HindIII digests. For progeny with inconsistent segregation of par-
0.5× Tris-borate-EDTA (TBE) buffer (44.5 mM Tris-borate and ticular telomeres due to rearrangement, data were scored as “–”.
1 mM EDTA [pH 8.0]), using a 50- to 2,500-bp DNA Marker Linkage analysis and construction of the genetic map were
(TaKaRa, Otsu, Japan) and Smart ladder (Nippon Gene, Toyama, performed using Mapmaker version 2.0 for Macintosh (E. S.
Japan) as molecular size markers. Lander, P. Green, J. Abrahamson, A. Barlaow, M. J. Daly, S. E.
Cloning of RAPD markers and sequencing. Markers of Lincoln, and L. Newburg, Whitehead Institute, Cambridge, MA).
interest were separated from other amplified fragments by agarose Parameters for map construction were a minimum logarithm of
gel electrophoresis and extracted from the gel by QIAquick Gel odds (LOD) score of 3.0 and a maximum recombination fraction
Extraction Kit (Qiagen Sciences, Germantown, MD) and cloned of 0.4. The Kosambi function was used to compute recombination
into pGEM-T Easy vector (Promega, Madison, WI). Transforma- distances in centimorgans (cM).
tion of plasmids into DH5α-competent cells (Nippon Gene, Preparation of chromosome-size DNA and contour-clamped
Toyama, Japan) was performed following the manufacturer’s in- homogenous electric fields (CHEF) electrophoresis. Protoplasts
structions. The cloned RAPD marker was sequenced using were prepared from mycelial cultures as described previously by
BigDye terminator version 3.1 cycle sequencing (Applied Bio- Tosa et al. (22). The resulting protoplasts were suspended in SE
systems, Foster, CA) according to the manufacturer’s instructions. buffer (1.0 M sorbitol, 50 mM EDTA [pH 8.0]) at a density of
Sequences were acquired using the Genetic Analyzer DNA Model approximately 1 × 109 per milliliter and were then added to an
310 (Applied Biosystems). equal volume of 1.5% low melt agarose (Bio-Rad Laboratories,
Southern hybridization analysis. Total genomic DNA was Hercules, CA) in 0.125 M EDTA (pH 7.5). The suspension was
digested with restriction enzyme and fractionated through a 0.8% mixed by pipetting and then poured into a mold to solidify into an
agarose gel in 0.5× TBE buffer. The fractionated DNA was trans- agarose/protoplast plug. The solidified plugs were poured into
ferred to an MSI nylon membrane (Osmonics, Westborough, MA) 10 ml of LET buffer (0.01 M Tris [pH 7.5], 0.45 M EDTA [pH
and fixed by UV irradiation following the manufacturer’s in- 8.0], and 7.5% β-mercaptoethanol) at 37°C for 16 to 24 h. After
structions. Plasmid clones containing RAPD markers as described washing three times in 10 ml of 0.05 M EDTA (15 min per wash),
previously and plasmid clones representing restriction fragment 10 ml of NDS buffer (0.01 M Tris [pH 7.5], 0.45 M EDTA [pH
length polymorphism (RFLP) markers in the map of Skinner et al. 8.0], and 1.0% lauryl sarcosine) containing 10 mg of proteinase K
(18) were used as probes. The membrane was hybridized over- (Invitrogen Life Technologies, Carlsbad, CA) was added and
night with biotin-labeled probes in 6× SSC (1× SSC is 0.15 M incubated at 50°C for 16 to 24 h. The plugs were then washed
NaCl plus 0.015 M sodium citrate) containing 5× Denhardt’s so- twice with 10 ml of 0.05 M EDTA for 15 min at room tem-
lution, 0.5% SDS, and 100 µg of heat denaturized salmon sperm perature before equilibrating in 10 ml of 0.05 M EDTA at room
DNA per ml at 68°C. The hybridized membrane was washed temperature for 16 to 24 h. Finally, the EDTA buffer was dis-
twice in 2× SSC, 0.1% SDS at room temperature for 5 min, and carded and 5 ml of fresh 0.05 M EDTA buffer was added, and the
then washed twice in 0.1× SSC, 0.1% SDS at 68°C for 15 min. plugs were stored at 4°C until use.
Detection of target DNA sequences was performed using the CHEF electrophoresis was performed in 0.8% Certified Mega-
NEBlot Phototope Kit (New England Biolabs, Beverly, MA). base Agarose (Bio-Rad Laboratories) using a running buffer of
Identification and characterization of telomeric restriction 0.5× TBE at 14°C. The CHEF electrophoresis was carried out in a
fragments. Total DNA of each isolate was digested with EcoRI or Bio-Rad DR III system (Bio-Rad Laboratories) with switching
HindIII and separated in 0.8 and 0.4% agarose gels in 0.5× TBE intervals of 90 min for 5 days and then 60 min for 2 days at 35 V.
buffer. The fractionated DNA was transferred to Hybond N+ Schizosaccharomyces pombe and Saccharomyces cerevisiae
membranes (Amersham Pharmacia Biotech UK Limited, whole chromosomal DNAs (Bio-Rad Laboratories) were used as
Buckinghamshire, UK) and then fixed by drying at 80°C for 2 h. size markers. After electrophoresis, the resolved chromosomes
A synthetic oligonucleotide (TTAGGG)10 was labeled with alka- were blotted to MSI nylon membranes following the protocol in
line phosphate using AlkPhos Direct Labeling Reagents (Amer- the Bio-Rad DR III system applications manual. Hybridization
Vol. 95, No. 6, 2005 641
