2004 microsatellite 甘薯长喙壳真菌 微卫星标记的发展和描述

Molecular Ecology Notes (2004) 4 , 215–218doi: 10.1111/j.1471-8286.2004.00621.x

? 2004 Blackwell Publishing Ltd

PRIMER NOTE

Development and characterization of microsatellite markers for the fungus Ceratocystis fimbriata

J. STEIM EL, C. J. B. E NG E L B R E C H T and T. C. HA RR IN GTO N

Department of Plant Pathology, Iowa State University, 351 Bessey Hall, Ames, Iowa 50011, United States

Abstract

Ceratocystis fimbriata is a serious fungal pathogen on a wide range of plants, but many cryptic species within C. fimbriata are apparently host-specialized. Anchor polymerase chain reaction (PCR) and simple sequence repeat (SSR) enriched libraries were used to develop 16 microsate llite marke rs for C. fimbriata . All marke rs we re polymorphic whe n te ste d against isolates from four host-specialized lineages of the pathogen. These markers will be valuable for phylogenetic and population genetic studies, as well as for tracking accidental introductions of host-specialized forms of the pathogen.

Keywords :Anchor PCR, Ceratocystis , Ceratocystis fimbriata , fungi, microsatellites, SSR Received 9 Deceember 2003; revision received 16 January 2004; accepted 16 January 2004

Ceratocystis fimbriata

is a fungal plant pathogen that attacks an exceptionally wide range of economically important hosts, including almond, cacao, coffee, eucalyptus, mango,sycamore, sweet potato, and taro (CAB International 2001).Several genetic lineages within the C. fimbriata complex are strongly host-specialized, and some of these lineages have caused serious epidemics after being moved beyond their native ranges by humans (Baker et al . 2003). Because of its economic importance and our studies on the genetic and ecological diversity within this species complex, it was desirable to develop genetic markers for analysing relation-ships within and among populations and for genotyping invasive strains. Santini & Capretti (2000) employed RAPDs and direct M13 primer amplification of minisatellite regions,and Barnes et al . (2001) developed PCR primers for poly-morphic regions, some with simple repeat sequences.However, we desired highly polymorphic and reproducible markers, preferably with tri- or tetra-nucleotide repeats.Here, we depict the isolation and characterization of 16microsatellite markers in four host-specialized lineages of the pathogen.

Isolates for DNA extraction were grown at 25 °C for 2 weeks in 20 mL of malt yeast extract broth (2% malt, 1%yeast). Total genomic, high molecular weight DNA was extracted from isolates as described by DeScenzo &Harrington (1994). Genomic DNA of isolate C1548 from

Theobroma cacao in Costa Rica (Baker et al . 2003) was used for identification of microsatellite loci using modifications of either an anchor PCR technique (Fisher et al . 1996) or the enrichment technique of Edwards et al . (1996). Target motifs were chosen based upon previous experience (DeScenzo & Harrington 1994) or on genome survey data (Toth et al .2000; Wostemeyer & Kreibich 2002).

For anchor PCR, four 27 bp primers of tri- or tetra-nucleotide repeat sequences with degenerative 5′-anchors were designed: DBV(CAT) 8 , D.B.H.(CAG) 8 , BBH(AAG) 8 , and BDB(GACA) 6 . The Universal Genomewalker kit (BD Biosciences, Palo Alto, CA) was utilized to further purify genomic DNA, to construct genewalking libraries, and for PCR amplification of microsatellite loci employing one of the anchor primers and the adapter primer AP1 from the kit. After a single round of PCR, products were separated by agarose electrophoresis, purified from the gel using a Geneclean II kit (BIO 101 Inc., Vista, CA), and cloned into the pGEM-T Easy Vector (Promega Inc., Madison, WI).Recombinant plasmids were extracted using the QIAprep Spin Miniprep plasmid DNA extraction kit (Qiagen Inc.,Valencia, CA) and inserts screened for size by electrophoresis after restriction by EcoR I enzyme (Invitrogen, Carlsbad,CA). Clones with inserts of the expected size were sequenced at the Iowa State University DNA Sequencing and Synthesis facility using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA). For sequences with the proper simple sequence repeat, two reverse genewalking primers were designed on the 3′ flanking region using

Correspondence: T. C. Harrington. Fax: (515)294–9420; E-mail:tcharrin@https://www.wendangku.net/doc/db11624495.html,

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oligo version 5.1 (National Biosciences Inc, Plymouth MN), and genewalking was repeated per kit instructions to obtain the upstream flanking sequences. After the two flanking regions of the microsatellite repeat were sequenced,a new forward primer was designed and fluorescently labelled with either HEX, TET, or FAM (Integrated DNA Technologies, Iowa City, IA). This labelled primer was used with one of the reverse genewalking primers to amplify the microsatellite region.

Two microsatellite-enriched libraries (CAA and CAG)were also constructed using a modified protocol of Edwards et al . (1996). Oligonucleotides of either (CAA) 10 or (CAG) 10 were bound to nylon membranes, and the PCR library (Edwards et al . 1996) of isolate C1548 was hybridized to the membrane. After a series of washings of increasing strin-gency (Edwards et al . 1996), strongly hybridizing frag-ments were eluted, reamplified (using the adaptor primers of Edwards et al . 1996), and cloned into the pGEM-T Easy Vector. Colonies with inserts were screened by hybridization with the respective 32

P-labelled oligonucleotide (DeScenzo & Harrington 1994). Plasmid DNA isolation, sequencing of positive clones, primer design and labelling were as stated for anchor PCR. When necessary, the Universal Genomewalker kit was used to obtain further flanking sequences for primer design.

PCR amplifications of all microsatellite loci were performed using a 96-well thermal cycler (PTC-100, MJ Research Inc., Watertown, Massachusetts). Cycling conditions

Table 1Loci, primers, size of PCR product, and GenBank accession numbers for microsatellite markers developed from a cacao isolate of Ceratocystis fimbriata using the anchor PCR or Edwards techniques

Locus Isolation technique Sequence motif*Primers Label Primer sequences

Actual size (bp) in isolate C1548GenBank accession no.CfAAG8Anchor PCR (AAG )11

AAG8-1F AAG8-1R TET 5′-TAG -ACA -GGG -GGT -GCG -TCA -AA

5′-TGT -CTG -CCC -TCC -ACA -TTT -GGT -CTC -TTC 187AY494859CfAAG9Anchor PCR (CAG )2 + (CAG )7AAG9-1F FAM 5′-CCT -GAA -CTG -ACA -GAG -ACA -CTT 413AY494860+ (AAG )7

AAG9-1R 5′-GCA -CCA -GCT -GTT -CCT -AAT -CGT CfCAA9

Edwards

(CAA )4(CAG )2CCAA9-F FAM

5′-GGC -TGG -TTC -ATC -ATG -ATG -TT 253

AY494861

+ (CAG )2 + CCAA9-R

5′-CTA -TGG -CAC -CTA -AGC -AAT -CT

(CAG )6 + (CAG )4+ (CAG )2(CAA )20(CAG )5

CfCAA10Edwards (CAA )4(CAG )6CCAA10-F CCAA10-R

HEX 5′-TGA -CAC -GCG -CTT -CAC -TAA -CAG 5′-TGC -ACC -ATA -CCC -AGG -GGA -CA 129AY494862CfCAA15

Edwards

(CAA )6(CAG )8 CCAA15-F TET 5′-GCT -ACA -GCA -GCC -GCA -GTG 342

AY494863

(CAA )2 + (CAA )2 CCAA15-R 5′-GAT -TGG -CGT -TAG -TGT -TAG -GT

+ (CAA )2(CAG )3(CAA )2

CfCAA38Edwards (CAG or CAA )47CAA38-1F CAA38-1R

FAM 5′-AAT -TCG -GGA -GCT -GCT -GTG -AG 5′-GAG -CCC -CAG -CCT -CAA -ACT -CA 240AY494864CfCAA80

Edwards

(CAG or CAA )43 CCAA80-F HEX 5′-ACC -CGT -CTC -GTA -TTG -GCT -AT 311

AY494865

+ (CAG )3 + (CAG )2 CCAA80-R

5′-AAT -CGT -TCG -CAT -TCA -GGT -GG

+ (CAG )2 + (CAA )3CfCAT1Anchor PCR (CAT )11 + (CAT )2DBVCAT1-1F WCAT1-1R FAM 5′-CCC -AAT -TTC -CCA -TTC -TGA -TTC

5′-AGT -ACA -GGA -TCA -ACT -ATG -GCA -TTT -CAA 272AY494866CfCAT3K Anchor PCR (CAT )6 + (CAT )2WCAT3K-1F WCAT3K-1R FAM 5′-AGG -TGC -CCT -ACA -CTT -TTT -GAA

5′-GTT -TTG -CTT -CTG -GCT -ACT -TTG -GTA -CTG 330AY494867CfCAT9X Anchor PCR (CAT )6CAT9X-1F CAT9X-2R HEX 5′-CCT -CGC -CTT -AAG -TTG -AAG -AAG

5′-GCG -GTT -GAG -GTT -GAA -GTG -TAG -AGT -GGT 273AY494868CfCAT1200Anchor PCR (CAT )7

CAT12X-1F CAT12X-1R TET 5′-ACA -AAA -GAC -GGC -ACG -CAT -ACA

5′-TGG -GGA -GAA -GTC -TGA -GTA -GAG -GGA -CAA 380AY494869CfCAG5Anchor PCR (CAG or CAA )12CAGDL2-5-1F CAGDL2-5-1R

FAM 5′-AAG -CCC -GGT -TAC -AGA -AGC -AAG

5′-GTG -CTT -GAG -TTT -GTC -CAG -GGT -TCG -GTA 342AY494870CfCAG15

Edwards

(CAG )4(CAA )2 CCAG15-F HEX 5′-GGG -CTA -GTA -GCA -GAG -TTG -G 269

AY494871

(CAG )4 + (CAG )2 CCAG15-R

5′-GCC -AAT -GTC -TTC -ACA -CCA -C

+ (CAG )3(CAA )6(CAG )9 + (CAG )2(CAA )10 + (CAG )4CfCAG900Anchor PCR (CAG )4 + (CAG )2CAG900-1F CAG900-2R HEX 5′-CTT -TGC -TAG -TCC -CCA -GTT -CCA

5′-GCG -GAC -ATG -GGA -TTG -TAA -GAG -CCT -GAG 196AY494873CfGACA60Anchor PCR (GACW )4 + GACA60-1F TET 5′-GGC -GAC -GGC -AAA -TAG -CAA -AAT

191AY494874(CACAGCA )4GACA60-1R

5′-GAT -GTG -TGG -TGC -TGT -GGT -ATG -CTG -CTG CfGACA650

Anchor PCR

(TG )4 + (CA )2 GACA6K-1F HEX 5′-AAA -CAT -CTC -GGC -AGA -ACA -GC

212

AY494875

(GACA )4 + (CCT )2

GACA6X-2R 5′-TGC -CGC -TTT -TGC -TTT -GTA -GTG -TTC -TTG

*‘+’ indicates that two simple repeats were separated by other bases.

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were an initial denaturing step of 95 °C for 95 s, fol-lowed by 35 cycles of 94 °C for 30 s, 58 °C for 1 m, and 72 °C for 30 s, with a final extension at 72 °C for 30 m. Each reac-tion (20 μL) contained 2 μL of 10X reaction buffer (500 m m KCl, 1% Triton X-100, 100 m m Tris-HCL, pH 9.0), 200 μm of each dNTP, 4 m m MgCl 2, 5.0 pmol of each primer, 0.5 U of Taq DNA polymerase (Promega Inc., Madison, WI), and 10–50 ng of RNase treated template. Before electrophoresis,the HEX, TET and FAM labelled products (0.5 μL each) from three separate reactions were combined and added to a master mix comprised of 2.4 μL formamide, 0.5 μL blue dextran, and 0.6 μL genescan -500 [TAMRA] size standard (PE Biosystems, Foster City, CA). A final volume of 1.3 μL was electrophoresed on a 4.5% acrylamide gel on an Applied Biosystems (ABI) Prism 377 DNA sequencer. Project and sample files were generated and allele sizes determined using ABI genescan software version 3.1 and genotyper software version 2.5.

Sixteen polymorphic microsatellite loci were identified (Table 1) when tested against isolates representing four host-specialized lineages within the Latin American clade of C. fimbriata (Baker et al . 2003). Number of alleles observed per locus ranged from two to 20 (Table 2). In general, more alleles were found with microsatellite loci identified by the Edwards technique than by the anchor PCR technique.

The low level of polymorphism found in the sweet potato lineage is probably due to the clonal spread of the pathogen on sweet potato storage roots from a single, unidentified population in Latin America (Baker et al . 2003). Most of the polymorphism within the sycamore and the cacao lineages was found among isolates from the southeastern USA and northwestern South America, respectively, the purported origins of these lineages.

The microsatellite markers developed here will be useful for studies of population structure and species limits within C. fimbriata . Their variability will also make them invaluable for tracking introduced strains.

Acknowledgements

This research was supported by the National Science Foundation through grant DEB-0128104. We thank Wei Chen and Nicole Wedemeyer for their technical assistance.

References

Baker CJ, Harrington TC, Krauss U, Alfenas AC (2003) Genetic variability and host specialization in the Latin American clade of Ceratocystis fimbriata . Phytopathology , 93, 1274–1284.

Barnes I, Gaur A, Burgess T, Roux J, Wingfield B, Wingfield M (2001) Microsatellite markers reflect intra-specific relationships

Table 2Microsatellite alleles, based on approximate band sizes as determined by GeneScan analysis, found in isolates of Ceratocystis fimbriata from sweet potato, sycamore and cacao. Number of isolates tested is shown in parentheses

Locus Sweet potato isolates (15)Sycamore isolates (67)

Ecuadorian–type cacao isolates (10)Costa Rican–type cacao isolates (69)CfAAG8176, 188176173188CfAAG9400409

415, 418

409

CfCAA9206270, 276, 294, 312, 368, 386, 400, 410157, 245, 254, 284184, 247, 263, 277, 280, 286, 292CfCAA10136127133130, 133CfCAA15323288, 317344323, 326, 338CfCAA38151134, 157

157227, 246, 271, 273CfCAA80305268, 291, 294, 302288, 308, 317297, 300CfCAT1257260

257260, 271, 274CfCAT3K 323323, 326, 329326

329, 332

CfCAT9X 282284/287*279/281*276/284, 276/287*CfCAT1200377391, 394, 409385, 388377CfCAG5319328

328

340

CfCAG15176317, 333, 337, 340, 343, 346, 263, 287, 308272, 275, 278353, 356, 359,365, 399CfCAG900196196193196CfGACA60190190

190190, 196CfGACA650

232

235, 256, 259, 211

211, 220, 236

276, 285, 290,295, 300, 328

*The CfCAT9X primers amplify two products for most isolates, and unique combinations of the two product sizes are scored as a single allele.

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