Microsatellite primers for the fungi <i>Rhizopogon kretzerae</i> and <i>R. salebrosus</i> (Rhizopogonaceae) from 454 shotgun pyrosequencing

Keywords: conservation genetics, GS-FLX Titanium, microsatellites, rare fungi, Rhizopogon kretzerae, Rhizopogon subgenus Amylopogon

Premise of the study: Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) are ectomycorrhizal fungi symbiotic with pines and the mycoheterotrophic plant Pterospora andromedea (Ericaceae). Microsatellite loci will allow population genetic study of fungal hosts to P. andromedea . 
• Methods and Results: Shotgun pyrosequencing of R. kretzerae DNA resulted in primer development of 23 perfect microsatellite loci and screened across two populations each for R. kretzerae and R. salebrosus . Twelve loci were polymorphic in R. kretzerae populations, and 11 loci cross-amplifi ed in R. salebrosus populations. For R. kretzerae and R. salebrosus , number of alleles was one to eight and one to nine, respectively, and observed heterozygosity ranged from 0.00-0.57 and 0.00-0.70, respectively. • Conclusions: These are the fi rst microsatellite loci developed for any species within Rhizopogon subgenus Amylopogon. These microsatellite loci will be used in conservation genetic studies of rare to endangered eastern populations and to compare plant and fungal population genetic structure at different hierarchical levels. 

Rhizopogon Fr. (Rhizopogonaceae) is a fungal genus of species that form ectomycorrhizal symbioses with conifers and are pivotal components of temperate forest ecosystems ( Molina et al., 1999 ). Four species ( R. kretzerae Grubisha, Dowie & Mill., R. salebrosus A. H. Sm., R. ellenae A. H. Sm., and R. arctostaphyli A. H. Sm.) in Rhizopogon subgenus Amylopogon (A. H. Sm.) Grubisha & Trappe are obligately ectomycorrhizal with several pine species and are the symbiotic mycobiont host to the mycoheterotrophic plant, Pterospora andromedea Nutt. (Ericaceae;Bidartondo and Bruns, 2002 ;Dowie et al., 2011Dowie et al., , 2012 ; ;Hazard et al., 2012 ;Grubisha et al., 2014 ), a North American endemic divided into eastern and western populations ( Bakshi, 1959 ). Pterospora andromedea primarily associates with two closely related Rhizopogon species: western P. andromedea are symbiotic primarily with R. salebrosus while eastern populations are found with R. kretzerae ( Grubisha et al., 2014 ). Rhizopogon kretzerae is currently only known from collections of P. andromedea roots and only found in association with Pinus strobus L. ( Hazard et al., 2012 ;Grubisha et al., 2014 ). Eastern P. andromedea populations have undergone recent decreases in population size and possible local extinction from a variety of anthropogenic factors ( Schori, 2002 ). 
Copopulation genetic studies of the fungal host and mycoheterotrophic plant will (1) provide information about levels of genetic variation and population structure at different hierarchical levels in plant and fungal populations, and (2) be important in developing conservation management plans for the rare to endangered eastern populations of both species. While microsatellite loci have been previously developed for distantly related species in three of the fi ve Rhizopogon subgenera ( Kretzer et al., 2000( Kretzer et al., , 2004 ; ;Grubisha et al., 2005 ), the microsatellite loci reported here, which were developed from R. kretzerae and R. salebrosus , are the fi rst for any species in Rhizopogon subgenus Amylopogon . 

METHODS AND RESULTS

Genomic DNA (gDNA) was isolated from 60 mg of silica gel-dried pure cultures of R. kretzerae isolate Miller207 (Michigan; Table 1 ) and R. salebrosus isolate Miller322 (Washington ; 46.20545 ° N, 117.7704 ° W) separately using the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). These two cultures were obtained from P. andromedea monotropoid roots following Grubisha et al. (2014) . Each culture was ground in liquid nitrogen to a fi ne powder. Twice the recommended volume of buffer AP1, RNaseA, and buffer AP2 were used. Library construction and 454 shotgun pyrosequencing were performed by the Advanced Genetic Technologies Centre (AGTC) at the University of Kentucky (Lexington, Kentucky, USA). Libraries for each species were prepared using the GS FLX+/XL+ Titanium Rapid Library Preparation Kit (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). Instead of nebulizing, gDNA was sheared using a Bioruptor NGS (UCD-600TS; Diagenode, Denville, New Jersey, USA) by sonicating at 4 ° C with six cycles of 5 s on and 90 s off. Shotgun 454 pyrosequencing was performed on 25% of a Perfect microsatellite repeats were identifi ed from the assembled contigs using MSATCOMMANDER version 1.0.8 ( Faircloth, 2008 ) for R. kretzerae and R. salebrosus , yielding 43 and 21 dinucleotides with at least six repeat units, 137 and 48 trinucleotides with at least fi ve repeat units, 34 and 12 tetranucleotides with at least four repeat units, fi ve and 11 pentanucleotides with at least four repeat units, and fi ve and 11 hexanucleotides with at least four repeat units, respectively. Within MSATCOMMANDER, Primer3 ( Rozen and Skaletsky, 2000 ) was used to generate primers for all loci using default parameters except that difference in acceptable primer melting temperature was 2 ° C with an optimum of 60 ° C; maximum poly X was set to three; GC clamp = yes; and a target PCR product size of 90-210 bp. Selection of loci for initial screening was based on Primer3 output tabulated in a spreadsheet by MSATCOMMANDER, and those that had no lowercase letters in the primer sequences that would refl ect nucleotide mismatches across reads in assembled contigs. Forty-eight loci (38 identifi ed from R. kretzerae and 10 from R. salebrosus ) were initially screened across four samples (two R. kretzerae , two R. salebrosus ; Table 1 ) for positive PCR products using agarose gel electrophoresis. Loci were PCR amplifi ed using 0.1 μ M of each forward and reverse primer in a 10-μ L reaction volume that included 1 × CoralLoad PCR Buffer with 1.5 mM MgCl 2 (QIAGEN), 200 μ M each dNTP, 2.5 units Taq DNA Polymerase (QIAGEN), and 1.0 μ L of 1 : 10 diluted genomic DNA. Thermocycler parameters were 94 ° C for 3 min, 35 cycles of 94 ° C for 30 s, 55 ° C for 30 s, and 72 ° C for 30 s, followed by a fi nal extension for 10 min at 72 ° C. PCR products were visualized in a 2% agarose gel stained with GelRed (Phenix Research Products, Candler, North Carolina, USA) on a UV transilluminator. Of the 48 loci assessed, 27 loci (23 from R. kretzerae and four from R. salebrosus ) yielded no more than one or two bright PCR product bands within the approximate expected size range (target size + 40 bp, -10 bp) and no PCR products outside the expected size range. The lower threshold was set to target size -10 bp due to the relatively low number of repeats. 
These 27 loci were screened in 65 isolates from two populations each for R. kretzerae ( n = 14 and n = 11; Table 1 ) and R. salebrosus ( n = 20 both; Table 1 ), and for cross-amplifi cation in two distantly related Rhizopogon subgenus Amylopogon species that are also hosts for P. andromedea : R. arctostaphyli ( n = 2 for both; Table 1 ) and R. ellenae ( n = 2 for both; Table 1 ). Pterospora andromedea ectomycorrhizal roots were collected and fungal DNA was isolated as previously described ( Dowie et al., 2012 ;Grubisha et al., 2014 ). Due to the extremely small sample of rootball collected, voucher specimens in herbaria were not made except for samples from Michigan (DAOM 242738) and Quebec (DAOM 242739) that were previously deposited at the Canadian National Mycological Herbarium ( Grubisha et al., 2014 ). The forward primer was 5 ′ end-labeled with one of four dyes in the ABI DS-33 dye set ( Table 2 ) . Loci were PCR amplifi ed in multiplex reactions using the QIAGEN Multiplex PCR Kit in a 10-μ L volume with 1 × QIAGEN Multiplex PCR Master Mix, 50 nM each primer, 0.25-0.5 × Q-solution, and 1.0 μ L of 1 : 10 diluted genomic DNA. Touchdown thermocycler conditions were: 95 ° C for 15 min; 10 cycles of 94 ° C for 30 s, 67 ° C for 90 s, decreasing 1 ° C each cycle, and 72 ° C for 30 s; 25 cycles of 94 ° C for 30 s, 57 ° C for 90 s, 72 ° C for 30 s; with a fi nal extension of 45 min at 60 ° C. Primers with a higher or lower touchdown annealing profi le are listed in Table 2 . Fragment analysis was conducted using the GeneScan 500 LIZ Size Standard (Applied Biosystems, Foster City, California, USA) on an ABI 3730 DNA Analyzer (Applied Biosystems) by the Biotechnology Resource Center (BRC) at Cornell University. Allele sizes were called manually using the Microsatellite Plugin in Geneious version R6.1.5 ( Drummond et al., 2011 ). Loci selected for the fi nal set were PCR amplifi ed independently to verify allele size ranges. 
Of the 23 loci developed in R. kretzerae and screened with fl uorescently labeled primers in R. kretzerae populations, 12 loci were polymorphic ( Table 1 ), seven loci were monomorphic, and four loci had excessive stutter. In R. salebrosus populations, 21 of the 23 loci developed in R. kretzerae successfully PCR amplifi ed and 11 loci were polymorphic, four of which were monomorphic in R. kretzerae populations ( Table 1 ). The four loci isolated in R. salebrosus did not PCR amplify in R. kretzerae populations screened. In R. salebrosus populations, only one locus (Rsal2) was polymorphic ( Table 1 ). A few loci management plans for eastern P. andromedea . We are also conducting copopulation genetic studies to determine if this fungal host and mycoheterotrophic plant share similar patterns of population genetic structure and variation at different hierarchical levels. Cross-amplifi cation of loci isolated in R. kretzerae with R. salebrosus and two distantly related Rhizopogon subgenus Amylopogon species suggests they may be useful in studies of other species in this subgenus. cross-amplifi ed in R. arctostaphyli and R. ellenae isolates and may prove to be useful when screened at a larger scale ( Table 1 ). Locus Rkre19 had stutter problems in both R. kretzerae and R. salebrosus but was polymorphic in R. arctostaphyli and R. ellenae ( Table 1 ). Number and size range of alleles, and observed ( H o ) and expected heterozygosities ( H e ) were calculated in GenAlEx version 6.5 ( Peakall andSmouse, 2006 , 2012 ; Table 1 ). When both populations for each species were considered ( R. kretzerae , n = 25; R. salebrosus , n = 40), for the loci isolated in R. kretzerae (Rkre3-Rkre38; Table 1 ), there were one to eight alleles, and H e ranged from 0.00 to 0.64 in R. kretzerae populations ( Table 1 ); and there were one to nine alleles and H e values of 0.00 to 0.76 in R. salebrosus populations ( Table 1 ). For locus Rsal2 there were one to three alleles, and H e ranged from 0.00 to 0.10. Scoring errors due to stuttering, large allele dropout, and null alleles were checked using MICROCHECKER 2. 2.3 ( van Oosterhout et al., 2004 ). For R. kretzerae populations, two loci showed signs of possible null alleles (Rkre5 in Quebec and Rkre37 in Michigan). In the combined R. salebrosus populations, possible null alleles and scoring errors due to stuttering were detected at four loci (Rkre3, Rkre17, Rkre27, Rkre34). 

CONCLUSIONS

These are the fi rst microsatellite loci developed for any species in Rhizopogon subgenus Amylopogon . Presently we are using these loci to assess the conservation genetic status of R. kretzerae , which will be important for developing conservation 



  no PCR amplifi cation; A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; I = does not amplify in all isolates; M = monomorphic; n = sample size; P = polymorphic; Ra = R. arctostaphyli ; Re = R. ellenae ; S = stutter. a Geographic coordinates for the populations are: Oregon = 44.86375 




  . S. 1959 . Ecology and morphology of Pterospora andromedea. Botanical Gazette 120 : 203 -217 . BIDARTONDO , M. I. , AND T. D. BRUNS . 2002 . Fine-level mycorrhizal specifi city in the Monotropoideae (Ericaceae): Specifi city for fungal species groups. Molecular Ecology 11 : 557 -569 . DOWIE , N. J. , J. J. HEMENWAY , S. M. TROWBRIDGE , AND S. L. MILLER . 2011 . Mycobiont overlap between two mycoheterotrophic genera of Monotropoideae ( Pterospora andromedea and Sarcodes sanguinea ) found in the Greater Yellowstone Ecosystem. Symbiosis 54 : 29 -36 . DOWIE , N. J. , J. J. HEMENWAY , AND S. L. MILLER . 2012 . Identity, genetic lineages and putative hybrids of an obligate mycobiont associated with the mycoheterotrophic plant Pterospora andromedea in the south-central Rocky Mountains. Fungal Ecology 5 : 137 -146 . 


TABLE 1 .

 1 Genetic properties of 17 microsatellite loci developed from Rhizopogon kretzerae 


	and R. ellenae .
	and one locus from R. salebrosus , and potential applicability in R. arctostaphyli



  Titanium GS-FLX (454 Life Sciences, a Roche Company) yielding a total of 152,007 reads with an average length of 414 bp for R. kretzerae and a total of 92,252 reads with an average length of 517 bp for R. salebrosus . At the AGTC, raw sequence reads were initially processed using prinseq-lite.pl (http:// prinseq.sourceforge.net/) and involved removing all (1) reads with >2% length 


	: Oregon = 44.49815 ° N, 120.38706 ° N, ° W; Washington = 46.24940	plate on a with uncalled bases (N), (2) duplicate reads, (3) reads with Q < 17, (4) reads too short in length [average length -2(standard deviation)], and (5) reads too long in length [average length + 2(standard deviation)]. Processed reads were as-sembled de novo using Newbler version 2.8 (454 Life Sciences, a Roche Com-pany) at the AGTC, which produced 10,190 contigs for R. kretzerae (average contig size = 1502 bp, N50 = 1701 bp) and 5178 contigs for R. salebrosus (aver-age contig size = 1302 bp, N50 = 1428 bp).
	° N, 118.36720 ° W.	
	117.76921 ° W. Rhizopogon ellenae : Wyoming = 45.08520 ° N, 109.35103 ° W; Washington = 44.78683	

° N, 118.40122 ° W; Wyoming = 41.18207 ° N, 106.13342 ° W; Michigan = 46.6427 ° N, 89.1787 ° W; and Quebec 45.4947 ° N, 76.3786 ° W. Due to the rare to endangered status of the eastern populations, locations are approximate. Rhizopogon arctostaphyli 
TABLE 2 .

 2 Characteristics of 12 polymorphic and fi ve monomorphic microsatellite loci developed in Rhizopogon kretzerae and one polymorphic locus developed in R. salebrosus . Rkre = isolated from R. kretzerae ; Rsal = isolated from R. salebrosus . 


							GenBank
	Locus a	5 ′ end-labeled dye	Primer sequences (5 ′ -3 ′ )	Repeat motif	Allele size range (bp)	T a ( ° C)	accession no.
	Rkre3	NED	F: TACCTTGCCCACTATCGACG	(TGC) 5	200-221	60	KJ443650
			R: TAATCGATGGACCCGCTGAG				
	Rkre5	VIC	F: AGACCAATACGCGTGAACAC	(CG) 6	179-183	57	KJ443651
			R: CAGAGCAATCCGAACACGAG				
	Rkre6	PET	F: CCGTGACAATCCAACTGCAC	(ACG) 6	218-221	57	KJ443652
			R: GGCAACACTGAATCTGGAGC				
	Rkre14	VIC	F: AATGCTGGACTTCTTGCACG	(AG) 6	192-196	60	KJ443653
			R: TGGGCATCATGGTGTAGGAC				
	Rkre15	6-FAM	F: TGTTTGGTCGTGATCTTGTAGC	(AT) 7	169-181	52	KJ443654
			R: ACCTTCGTAGTCCAGTGCTG				
	Rkre17	PET	F: AAGCAATAAAGGAATCTGCAGC	(AT) 11	176	57	KJ443659
			R: CCGGATGTCTAACACTGGTC				
	Rkre18	6-FAM	F: TCAATGACCGGTCCACTCTC	(ATGC) 5	120-128	60	KJ443655
			R: GTCAGTAGGTGTATTCTTGCTGG				
	Rkre19	PET	F: GATGCAAGGCTCGTTGTCTC	(TCT) 10	202	57	KJ443660
			R: GCAAGTTCTGGAGCAAGGAC				
	Rkre20	VIC	F: TGCTTCTCAAACAACCGAGG	(ACC) 6	114	57	KJ443661
			R: GGTGGTAGCGATTGAGGGAC				
	Rkre23	PET	F: ACTTTGATGCAGGCGAGAAC	(AGC) 5	129-132	57	KJ443656
			R: TGACCGTCACAAATCGTTCC				
	Rkre24	NED	F: TCAGGGTTGGACTCGTGAAG	(AGC) 7	117-132	52	KJ443657
			R: CGCGCCTGAAGTTTGGAC				
	Rkre27	6-FAM	F: ATGGAGGATGAGAGTGCGG	(ACG) 6	108-111	57	KJ443658
			R: CGTCGTGACATGAGTGCTTG				
	Rkre29	PET	F: AGGTTGGCTGTGCATGAATG	(AT) 6	188	57	KJ443662
			R: ACACTAGCCTGCTTCCTCAG				
	Rkre34	PET	F: TATAGACTCGGACGCACACC	(CA) 10	119-131	57	KJ443663
			R: AGGTTCCCGTGAGTGATATCG				
	Rkre36	NED	F: AAGCAGGGTACAGACTCACC	(TC) 11	173-215	57	KJ443664
			R: TCTGGATCGTTTGCAATGCC				
	Rkre37	VIC	F: TGGTGGTGGGTATGAGTTGG	(TGG) 9	183-217	52	KJ443665
			R: TTTGTCAAAGCAGGCTACGC				
	Rkre38	NED	F: ATGCAGGTAGACGGGACTTG	(GTG) 6	209	57	KJ443666
			R: TACTCGTCGCTACAGTCCG				
	Rsal2	NED	F: TGTCCTCAAGAGATCCGCAG	(TCG) 5	110-119	57	KJ443667
			R: AGATTCATCGAAACCGCACG				
	Note : T a = annealing temperature.					

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Funding for this research has been provided by the National Science Foundation (DEB-1050315 to M.R.K. and DEB-1050292 to S.L.M.).
Lisa C Grubisha, Jessica D Brewer, Nicholas J Dowie, Steven L Miller, Steven M Trowbridge, AND Matthew R Klooster
Microsatellite primers for the fungi <i>Rhizopogon kretzerae</i> and <i>R. salebrosus</i> (Rhizopogonaceae) from 454 shotgun pyrosequencing