Molecular Ecology Notes (2006)
doi: 10.1111/j.1471-8286.2006.01611.x
PRIMER NOTE Blackwell Publishing Ltd
Seventy microsatellite markers from Persea americana Miller (avocado) expressed sequence tags J A M E S W . B O R R O N E ,* R A Y M O N D J . S C H N E L L ,* H E L E N A . V I O L I † and R A N D Y C . P L O E T Z † *United States Department of Agriculture, Subtropical Horticulture Research Station, National Germplasm Repository, 13601 Old Cutler Road, Miami, FL 33158, USA, †Department of Plant Pathology, University of Florida, Institute of Food and Agricultural Sciences, Tropical Research and Education Center, 18905 SW 280th Street, Homestead, FL 33031, USA
Abstract Expressed sequence tags for Persea americana Mill. were investigated to expand upon the number of informative microsatellite markers available for avocado. Seventy informative loci were discovered using 24 P. americana var. americana Mill. accessions. The number of alleles detected ranged from two to 17 and averaged 7.1 alleles per locus. These primers successfully amplified products in different varieties of P. americana, hybrids and a related species, Persea schiedeana. These primers will be useful for characterizing germplasm, determining genetic relationships of cultivated accessions, and for marker-assisted development of root rot-tolerant P. americana var. americana rootstock material. Keywords: expressed sequence tag libraries, Persea americana Miller, simple sequence repeat, unigene Received 23 August 2006; revision accepted 15 October 2006 Persea americana Mill. is an evergreen tree native to Mesoamerica. A member of the subgenus Persea, P. americana is a polymorphic species consisting of several varieties, or subspecies, adapted to geographically distinct microenvironments. These include wild varieties [var. floccosa Mez, var. steyermarkii Allen, var. nubigena (Williams) Kopp] and three economically important botanical varieties also known as ‘races’ [var. americana Mill. ‘West Indian’, var. guatemalensis Williams ‘Guatemalan’, var. drymifolia (Schlecht. and Cham.) Blake ‘Mexican’] (Scora & Bergh 1990). Currently, there is a limited set of microsatellite markers for P. americana (Lavi et al. 1994; Sharon et al. 1997; Ashworth et al. 2004), some of which do not consistently amplify in all varieties (Ashworth & Clegg 2003;Ashworth et al. 2004). Only 14 of 35 microsatellites were suitable to fingerprint diverse collections of P. americana (Schnell et al. 2003). To increase the number of informative microsatellite markers, publicly available Persea americana expressed sequence tags (ESTs) were screened. Data-mining microsatellites from ESTs has proven effective for generating markers for fingerprinting, genetic mapping and comparative mapping among species (Varshney et al. 2005). A P. americana unigene set consisting of 6183 unigenes from 8735 ESTs (Build 4, November 2004) was obtained from Correspondence: Raymond J. Schnell, Fax: 305-969-6410; E-mail:
[email protected] © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd
the Plant Genome Network (PGN, http://pgn.cornell.edu/) (Albert et al. 2005). Microsatellites were identified by using the Cotton Microsatellite Database SSR Server (Clemson University Genomics Institute; www.mainlab.clemson.edu/ cgi-bin/cmd/cmd_ssr). The minimum number of repeats were ≥ 7 for di-, ≥ 5 for tri-, ≥ 4 for tetra-, ≥ 3 for penta- and ≥ 3 for hexanucleotide. Polymerase chain reaction (PCR) primers were designed using prime in Genetics Computer Group (GCG) (Wisconsin Package Version 10.2, Accelrys). Fluorescently labelled (6-FAM or HEX) and unlabelled PCR primers were purchased from Sigma-Genosys. PCRs were conducted with PTC-225 thermalcyclers (MJ Research). Each reaction contained 0.1 µm forward primer, 0.1 µm reverse primer, 200 µm dNTPs, 10 ng of bovine serum albumin, 1× PCR buffer with 1.5 mm MgCl2, 0.5 U of Taq polymerase (New England Biolabs), and approximately 5 ng of DNA in a total volume of 10 µL. Thermalcycler conditions were: 94 °C, 2 min; (94 °C, 20 s; 60 °C, 45 s; 72 °C, 1 min) × 5; (94 °C, 20 s; 56 °C, 45 s; 72 °C for 1 min) × 35; 72 °C, 10 min. Post-PCR, samples were ethanol-precipitated and diluted 1:5 or 1:20 with sterile H2O. Capillary electrophoresis was performed on the ABI PRISM 3730 Genetic Analyser with LIZ-labelled GeneScan-600HD as the internal molecular size standard (Applied Biosystems, Inc.). Alleles were scored using genemapper version 3.7 (Applied Biosystems). Descriptive statistics (Table 1) were generated with gda version 1.1 (Lewis & Zaykin 2001). Tests for
2 PRIMER NOTE Table 1 Characteristics of 70 microsatellite loci developed from ESTs and tested on Persea americana var. americana (West Indian) accessions
Locus SHRSPa001 SHRSPa003 SHRSPa004 SHRSPa005 SHRSPa006 SHRSPa009 SHRSPa010 SHRSPa012 SHRSPa014 SHRSPa015 SHRSPa016 SHRSPa017 SHRSPa018 SHRSPa021 SHRSPa022 SHRSPa023 SHRSPa026 SHRSPa027 SHRSPa028 SHRSPa029 SHRSPa030 SHRSPa031 SHRSPa033 SHRSPa034 SHRSPa036 SHRSPa038 SHRSPa039
Accession no.†
Primer sequence (5′−3′)
Repeat(s)
CK743300 (209399) CV001225 (209321) DT594699 (211122) CV458262 (210203) CV004514 (209916) CK754670 (211730) CV003544 (214064) CK758907 (209266) CK764212 (209344) CO995346 (212428) CK761914 (213168) CK761933 (213929) CK761074 (212265) CO995394 (212496) DT593051 (215378) CK750255 (210963) CV003665 (210330) CV001989 (209253) CK750182 (210620) CV460746 (209553) CV459605 (212051) CK748429 (214523) CK753888 (209972) CK749396 (211416) CK760992 (210285) CK751968 (215311) CO997138 (213290)
F: CGAGCATTGCTTTCCATATCC R: ATGTCTTTCTTCCTGTTCCCTTTC F: ATCAGTCGGTGTGCAGAAC R: CACCAGCTCCTGCAAATA F: GCGTTCCTTTTCATTCTATTCG R: GCTGATCCGCCATTCTC F: AACAGAAGGAGAAGAAGCG R: CTCAAACACCTGCGATACT F: GGAGCGAGAAGAGCAGCCGA R: CTGCCACGCCCATCTGCTTAC F: ACCCAATCAACAAACAAACCC R: CGTTTCCCCAATCCATTTCT F: CGAAGAAGGATAGTCTGAAAACCC R: GAGGAAGGATCGGAAGAGAGG F: AACCCTAACGGATTTCAACTAC R: ACGGTATAGCTCCTTCCATTC F: CCCAAAACCACCTATCCCT R: GTGCCGCCGATGATAAG F: GCTGCTGCTATTGAGAAGGA R: CCACCGAAGAGGCAAAGA F: CTGAGGAATGAATGGAACTGAA R: GAGAAAAACCCATCTATGTCG F: GTCCTTCGTCAAGAAGAGAA R: GTATCCGAAAACGAGAAGTC F: CTGGTCCCAAAATTGAAGA R: AGCACAACTGACATGAAACA F: ACACCCAACAGATGTGGATAGATAAG R: CAGATGAAAAGAACATGGCATTGA F: TTCGGGCGGTTGGAT R: AGCATGGAAGGTCATTACATC F: GTGAAAATCATCTCTCCCTATG R: CACTTAACCAAGTACCAGCG F: CTCTCACCTCACCTCACCTGCT R: GGTCCATCATCTCCCTCGTGTA F: CACCTTGTCTTGGTCGCCTTC R: TGGTGTTCTTCGTCCGACATCTT F: GGACATCCAGATTCTCAGCAT R: CAGCATAAACATAAGTCGCATAC F: AGGGTTTTAGGCCCAGAAG R: GTGTATTCTCTTGTAACCACCTC F: GAGGAAAACCAGAAACCAGAG R: GAGATTACGAACGAGGAAAG F: AAACAACACTTTGATGGTGG R: AGGTTTCTGATTGTCCGAG F: TGCCAAGTGAGACTCAATTCC R: CAAATCCTTCCACTGACCAAC F: CCAACCCCTCGTCTCAAC R: CGAAAATCCCACAAAGTATCAG F: TTCCATCTCCCAAATCTCC R: GATTGTCTTCCCCGTTAGG F: GAAGAAATCCGCCATAATCGT R: CACATCAGAGAAAGAAACCTAAACC F: GGCGACTGACTTTGGCATA R: AATCCATGACTGAAAACTGCTG
(CT)2T(TC)7 (GA)9(GAA)2GATG(GA)3 (AG)2GCC(GA)19 AGAGG(AGA)5GAGGA(ATGGAT)2 (GAG)7(AG)8TTG(GA)2AG AAA(CAAC)2(CTCTC)4CTT(TC)2N13(TC)2N9 (GAAG)2(GAA)2GGG(GAA)4 (AAC)7 (CT)2(TTC)2N6(TC)2N7(GAA)7GA(TC)2AAGA(TC)2 (CCTTCT)3 (GAGAAG)3N41TCTGGT(TCTG)2TGA(GT)4(TG)2 (CT)2(AT)2(TC)2N10(TC)2G(CTT)2N4(TC)2 CT(TC)13G(CT)5A(CT)2(TC)2N5(GTC)2 GAAGAGAA(GAAGA)3AAA TC(TA)2CA(CT)12 (AGC)2(TCTT)2TC(TCA)3N4(CAT)2N4(AG)3(AAG)6 CGC(GGAGCC)3GGAGCACC(CGG)2N111(TGA)4 (GA)2(TA)3A(AT)12TT(TA)2(AG)2 GA(GATA)2(GA)6–9 (AG)7(AAG)4 (GT)5G(GA)2 (AGA)4AGG(AGA)7(AAG)2AGGGAAAA (AG)3(AAAGG)2C(CGTT)6(TG)2TT(TTC)3 (TGG)2(CGG)4TGG (CT)14 (AT)2(CT)9TC(AT)2 (AG)6CA(AG)2(GA)4 (ATC)8(AT)2 (TAG)3N15(TA)3N6(TC)2N7(AT)2N12(CT)8(C)2
© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd
PRIMER NOTE 3 Table 1 Continued
Locus SHRSPa040 SHRSPa043 SHRSPa044 SHRSPa046 SHRSPa049 SHRSPa052 SHRSPa053 SHRSPa055 SHRSPa056 SHRSPa057 SHRSPa060 SHRSPa061 SHRSPa062 SHRSPa065 SHRSPa067 SHRSPa068 SHRSPa073 SHRSPa076 SHRSPa077 SHRSPa081 SHRSPa082 SHRSPa085 SHRSPa086 SHRSPa088 SHRSPa089 SHRSPa090 SHRSPa091 SHRSPa093
Accession no.†
Primer sequence (5′−3′)
Repeat(s)
CO997192 (209525) CO996515 (213336) CK750214 (210093) CK758779 (211119) CV005371 (210005) CK767024 (213993) CV461047 (209863) CK759476 (213097) CK751704 (209294) CK746481 (212087) DT592477 (210290) CK753113 (212010) CK761843 (212906) CK754635 (211701) CK762811 (209309) CK751792 (210574) CK761985 (214215) CK759435 (209426) CK754758 (209944) CK749691 (209566) CV460219 (213064) CO996399 (213288) CO997868 (209895) CK755655 (209318) CK762720 (213422) CV458387 (213525) CV002414 (215312) CO997558 (214102)
F: AACGACACGTCAAAAACCCT R: TCGCCATTAAAGTTGCAGAGA F: TCACTGCTCTCTTCTTGCCC R: ATCTATTGCCCTCTTGTACTCACT F: GCCAACGAGGGTCAGATCAA R: CGCAAACCAACCGCACA F: CCCCAAAAACCCTTCCTTCC R: CAACGCCATAAAAACCCGAT F: CGACTTGCCTAGCGTTCA R: CGACGAAAACCCTAACAGA F: CCTGCCCTAAAAACGGAGT R: GGTAATGGTCTTCCCAGTTAG F: GATGTTGTCAATGATCTCAAGGG R: GCAAAACAAATACTGCAAGGC F: TCTCTTCATCAACTCGACTGC R: AACGGTATCCAAACGCTAAT F: GGTTTTCCAATTCTCTCTATCCCC R: TTGATGCCTGCTTCCGTGTG F: GCAAGGCATTACGATGTCA R: CTCTAGTGGACAAAATCGACAA F: TCCAAGCCCGTCACCATCG R: GCCAAACACACCAGCACCCA F: CTAAACAAAGTGCCTAAGTCCAAACAG R: CAAAAAATCACACAACTCACCCC F: GCCAAGCTCGACAAGATATGA R: TCTTCCACAACCAGCAAATG F: GATGTCGTCTATGCCCTCAAG R: TCTCAAATTCTCACACTATGCTCC F: ATGTGTTCTACTTCTCCTCCAA R: CATTCCAGCCATGATTCC F: TGTTTCGCTCTCTAACTTCCAT R: CGCAAAAGCACACCTCTG F: CTGCTTTTCCCACTGCTC R: CCAGAACAAACTGAACAACAA F: TTCCATTTCGATCTCACTCTCTC R: CTTCAATATCGCTCCCAAACTC F: GGAACAGAGGAGGACAAGGG R: GCTGCTGCTTTTGCGTCT F: GGGCTTCAATTCAATCCAATCC R: TCTTCAGCACGCCACGAGTCT F: TCCGAAACATCAAACACCAACA R: CCGAACAAACACGAATCAGAGA F: GTGAGTGGGAGATGAAGAAAGA R: CTGGGTGGAAGATTACATGCT F: CCTTCAGGAGCAACCCACA R: TACAGGAGACTGCTACAAACA F: AGTTAGGGTTTGGAAAAGAATCAGTT R: GAGAGAGAATCGTCGGAATCGT F: GCAGAAGCCACCTCAGAAC R: CAAACAACAGATGAGCGGA F: GGCTTTCCTTCCCTGAC R: GCTGGTGACTGATTCCGT F: CCAAATGAACCATCATCAACG R: CAAAACACCTCCAAAACAACC F: CGACGAAAACAGCAAGAGGAG R: TCAATCAACCACAAAGGGACTATG
(CT)14G(CT)3N17(TC)5(TA)2(TC)2
© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd
(TC)2GCA(TC)14(TG)2N6(CAAA)2 (CTT)3(TTTTAT)4 (TC)6ATT(TC)14TG(TCTT)3CTCC (TA)3TT(TC)4TAN11(CT)10(C)2A(TC)2 (GAGAA)2G(GAA)6TCGGA(AG)3 (TGCCT)4 CC(TTCT)2(TTA)2CAA(CT)16TT(CT)2 (CT)14 (CT)16 GATCA(GATCT)3GG(TTA)2N11(TGATC)3 (GAAAT)3 (TAGATA)2(GAA)2TTTC(CT)12 (GTAG)6TATT(GTAG)6TATT(GTAG)5 (GGTTCT)3(CT)2(CTT)3(TCT)2N38(AG)2N4(GT)2 (T)7G(T)5G(T)3GC(TG)7(T)9 T(CTT)2CCTATC(CT)14 (AG)7AA(AG)2 AGAA(AG)4(AAGGAG)3AAGA (AAGAGG)4 (C)2(GA)7 CC(CT)7T (AG)8N19(TG)2(TTG)2 (TG)3N9(AG)10C(TG)2GA (CT)8N4(GC)2N8(TCT)3(TCC)2CA(ATC)3(TTC)4 (CT)14A(TG)3(CG)2 (CT)7CAATC(CT)3 (TTC)2(TATC)5TT(TC)2 (TC)22
4 PRIMER NOTE Table 1 Continued
Locus SHRSPa098 SHRSPa099 SHRSPa100 SHRSPa101 SHRSPa102 SHRSPa103 SHRSPa104 SHRSPa105 SHRSPa106 SHRSPa107 SHRSPa109 SHRSPa110 SHRSPa111 SHRSPa114 SHRSPa115
Accession no.†
Primer sequence (5′−3′)
Repeat(s)
CV459152 (210414) CK759537 (213153) CK758785 (209793) CK759394 (209406) DT590788 (209349) DT591571 (209459) CK754784 (213891) CK753881 (213652) CK756206 (209536) CV002973 (212385) CK757900 (214863) CK758825 (209780) CK752490 (209724) CK752754 (210869) CV004661 (210773)
F: CACAGACAGATATTCTCTCTCTCTC R: GTGGCTAATACCATTCCTTGA F: TCATCCCAATTCCCACCTTC R: AGCGGCGGATTTTAGCG F: ATCAACCCCATCATCTCTTC R: CGCCATCACAAATCTAACAC F: GATCTCTTTCGACTCTTTCTCTC R: CTCAGCATCCTTCCTTCTAAC F: GGCACAAACCCTACAAATACCA R: TCTTCTTGAGTCGCAGCAGC F: TGGCAAACAAAAGGAAGC R: GTGGCAAGAAATCACGGA F: GCTGATGATGCTTCCTAACAAC R: GAAAAGGCGAGCGTCCA F: GCATACTCTCGGCTCTTACA R: ACATGATACTAGCTCCCCCA F: CTTTCAACGAATCGCAAGT R: CGATAAACTCGGACCACG F: CGCAGTCTTCAATGATACCA R: CCCCCCTTCACTTCCAA F: TTCCAGCTACTACTCCTCCAGT R: AAGGAGGTGAGCCGAATG F: ACGGACCCAAACCCTGATG R: AGGACCTTCCACGGACACAC F: ACCTTTCAATCTCTTCCCAAAC R: CCGCTCCAATCCAACCA F: TGCCAGATGACAGTTTTTCC R: ACAGCACATAAGTTCAACTCAGA F: AGCAAAGCAAACGGACAGGT R: TTATGGCACTGAAAACGGAAAG
(CT)11(CCCT)2(CT)2CCCT(ACA)2 (AGA)9A(AG)2 (CTT)3TC(CT)2N7(GA)9C(AG)2CAG (AGA)5 A(GAA)6AG (AG)8 (AAG)11AAAA(TCC)3 (CCCA)2T(TTC)2(TC)10 (TTCTC)2(TC)6TA(G)3CT(TC)3GCTTCGT(CTC)2 (AT)4N4(AC)3TA(AC)2(CT)2(TG)2(AGA)2AA(TG)2 TAT(TC)8 (TA)2(CTT)2TTG(CT)7CCN31(AG)2N6(GAA)14GAT (TG)11 (AG)8G(GA)5 (TC)20TG(CG)3 G(GT)2(TA)3N7(TA)3(GT)2(TA)3GAG(TC)7(TA)6 (TAT)2TTC
Locus
n
Size range
No of alleles
HE
HO
f
LD‡
SHRSPa001 SHRSPa003 SHRSPa004 SHRSPa005 SHRSPa006 SHRSPa009 SHRSPa010 SHRSPa012 SHRSPa014 SHRSPa015 SHRSPa016 SHRSPa017 SHRSPa018 SHRSPa021 SHRSPa022 SHRSPa023 SHRSPa026 SHRSPa027 SHRSPa028 SHRSPa029 SHRSPa030
24 22 18 23 23 24 24 23 24 23 21 24 24 24 22 23 20 22 24 24 23
68 –75 74 –102 58 – 82 84 – 87 70 –110 128 –155 125 –140 135 –141 144 –156 136 –160 121–147 339–359 154 –191 181–187 160 –196 177–189 83 – 95 87–108 83 – 88 94 –119 75 – 88
3 8 5 2 13 8 5 4 3 5 8 6 10 3 4 7 7 8 3 7 4
0.423 0.492 0.717 0.085 0.826 0.659 0.690 0.345 0.566 0.667 0.688 0.649 0.797 0.669 0.532 0.770 0.692 0.608 0.159 0.801 0.312
0.458 0.455 0.500 0.000* 0.522*++ 0.500 0.583 0.304 0.625 0.609 0.381*++ 0.292*++ 0.333*++ 0.500 0.364* 0.739 0.200*++ 0.500* 0.167 0.750 0.174*+
− 0.086 0.077 0.309 1.000 0.374 0.245 0.157 0.120 − 0.106 0.089 0.452 0.556 0.587 0.257 0.321 0.041 0.716 0.181 − 0.051 0.065 0.448
3, 39 1, 9, 10, 12, 14, 15, 28, 36, 39, 62, 67, 73, 81, 90, 103, 105, 106 10, 62 — — 3, 10, 14, 15, 23, 33, 39, 57, 73, 81, 86, 88, 102, 107 3, 4, 9, 55, 62, 68, 73, 81, 86, 88, 90, 102, 110 3 3, 9, 29, 36, 62, 76 3, 9, 28, 40, 81, 90 — — — 28, 36, 40, 44, 73, 90, 106, 110 — 9, 33, 39, 57, 61, 81 — — 3, 15, 21, 67, 86, 90, 106 14, 57 —
© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd
PRIMER NOTE 5 Table 1 Continued
Locus
n
Size range
No of alleles
HE
HO
f
LD‡
SHRSPa031 SHRSPa033 SHRSPa034 SHRSPa036 SHRSPa038 SHRSPa039 SHRSPa040 SHRSPa043 SHRSPa044 SHRSPa046 SHRSPa049 SHRSPa052 SHRSPa053 SHRSPa055 SHRSPa056 SHRSPa057 SHRSPa060 SHRSPa061 SHRSPa062 SHRSPa065 SHRSPa067 SHRSPa068 SHRSPa073 SHRSPa076 SHRSPa077 SHRSPa081 SHRSPa082 SHRSPa085 SHRSPa086 SHRSPa088 SHRSPa089 SHRSPa090 SHRSPa091 SHRSPa093 SHRSPa098 SHRSPa099 SHRSPa100 SHRSPa101 SHRSPa102 SHRSPa103 SHRSPa104 SHRSPa105 SHRSPa106 SHRSPa107 SHRSPa109 SHRSPa110 SHRSPa111 SHRSPa114 SHRSPa115 Mean
24 24 23 24 24 22 24 24 23 24 24 24 24 23 24 24 24 24 23 24 24 24 24 24 24 24 24 24 24 22 24 22 24 24 24 23 23 23 24 24 24 23 23 24 24 21 24 24 22
83 – 89 90 –116 102–122 115 –121 105 –118 106 –117 103 –115 148 –190 168 –183 336 –370 90 –117 99 –102 91–116 81–127 86 –122 182–213 193 –213 206 –210 201–299 199 –239 240 –249 240 –288 117–130 121–133 123 –135 204 –264 221–238 221–225 226 –233 237–247 288 –321 289 –303 291–295 283 –331 74 –113 56 – 89 129 –153 95 –114 93 –120 96 –107 78 –110 140 –160 143 –170 147–179 132–162 148 –150 142–171 146 –184 159 –174
2 8 6 3 4 6 3 12 8 8 8 2 4 17 15 14 6 4 11 7 5 13 5 4 3 10 7 3 6 6 16 5 2 11 11 3 11 5 8 6 8 8 9 11 16 2 15 11 7 7.1
0.223 0.735 0.742 0.294 0.691 0.607 0.082 0.886 0.420 0.634 0.491 0.082 0.160 0.860 0.825 0.842 0.578 0.233 0.840 0.847 0.501 0.866 0.337 0.198 0.590 0.759 0.688 0.582 0.631 0.293 0.853 0.390 0.311 0.802 0.825 0.127 0.745 0.651 0.723 0.653 0.762 0.735 0.809 0.860 0.910 0.285 0.902 0.820 0.667 0.593
0.250 0.708 0.348*++ 0.333 0.625 0.500 0.083 0.792* 0.391 0.417*+ 0.375* 0.083 0.125* 0.913 0.667*+ 0.667+ 0.417 0.167 0.739 0.625+ 0.500 0.708+ 0.333 0.125 0.667 0.625 0.458*+ 0.458* 0.583 0.318 0.708*+ 0.364 0.208 0.667* 0.667* 0.130 0.478*++ 0.391*++ 0.708 0.625 0.708 0.609 0.739 0.708+ 0.792* 0.238 0.458*++ 0.583*++ 0.409*++ 0.474
− 0.122 0.037 0.537 − 0.136 0.097 0.179 − 0.011 0.108 0.070 0.348 0.240 − 0.022 0.225 − 0.063 0.196 0.212 0.283 0.290 0.122 0.266 0.002 0.185 0.011 0.373 − 0.134 0.180 0.339 0.217 0.077 − 0.089 0.172 0.069 0.335 0.172 0.196 − 0.031 0.363 0.405 0.021 0.044 0.071 0.175 0.088 0.179 0.132 0.167 0.497 0.293 0.392 0.205
91 9, 23, 57, 81, 107 — 3, 14, 21, 62, 88, 90, 103 40, 57 1, 3, 9, 23, 57, 81, 91, 103, 106 15, 21, 38 — 21, 60, 65, 68, 86 — — 73, 102 — 10, 88, 102, 104 — 9, 23, 29, 33, 38, 39, 61, 81 44, 102, 105, 110 23, 57, 102, 105 3, 4, 10, 14, 36, 90 44 3, 28, 106 10, 44, 81, 102, 106 9, 10, 21, 52, 76, 81 3, 14, 73 none 3, 9, 10, 15, 23, 33, 39, 57, 68, 73, 86 — — 9, 10, 28, 44, 68, 81 9, 10, 36, 55, 90 — 3, 10, 15, 21, 28, 36, 62, 88 31, 39 — — none — — 9, 10, 52, 55, 60, 61, 68 3, 36, 39, 105 3, 55, 110 3, 60, 61, 103 3, 9, 21, 28, 39, 67, 68, 110 9, 33 — 10, 21, 60, 104, 106 — — —
n, number of accessions successfully genotyped out of 24; HE, expected heterozygosity; HO, observed heterozygosity; f, estimate of fixation index; LD, linkage disequilibrium; *departs significantly from HWE at P < 0.05; evidence of null alleles detected at (+) P < 0.05 and (++) P < 0.01; †GenBank Accession no. given for an EST that contains both primer sites and the microsatellite region. The number in parentheses is the PGN number for the unigene; ‡SHRSPa# given for loci showing significant LD (P > 0.05), excluding loci that significantly depart from HWE.
© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd
6 PRIMER NOTE Table 2 Results of amplifications on additional Persea americana varieties and Persea schiedeana Species
Race
n
Loci*
P
A
Ap
HE
HO
f
P. americana var. drymifolia P. americana var. guatemalensis P. americana P. americana var. nubigena P. schiedeana
Mexican Guatemalan hybrids — —
2 2 8 2 1
70 (100%) 70 (100%) 70 (100%) 70 (100%) 55 (79%) Mean
0.714 0.771 0.986 0.899 0.415 0.757
2.24 2.29 5.16 2.55 1.42 2.73
2.74 2.67 5.22 2.73 2.00 3.07
0.462 0.545 0.691 0.676 0.415 0.574
0.420 0.514 0.673 0.710 0.415 0.561
0.139 0.083 0.029 − 0.079 0.000 0.036
n, number of accessions genotyped; P, proportion of polymorphic alleles; A, mean number of alleles per locus; Ap mean number of alleles per polymorphic locus; HE, expected heterozygosity; HO, observed heterozygosity; f, estimate of fixation index; *number of loci successfully amplified, percentage given in parentheses.
Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were performed using genepop 3.4 (Raymond & Rousset 1995). Tests for null alleles were performed using micro-checker 2.2.3 (van Oosterhout et al. 2004). Twenty-four P. americana var. americana (West Indian) accessions maintained as part of the USDA National Germplasm Repository were assayed and are a subset of the accessions described in Schnell et al. (2003). The number of alleles/locus ranged from two (five loci) to 17 (one locus) with a mean of 7.1 and median of 6.5 alleles/locus. Twenty-six loci departed significantly from HWE (P < 0.05). Overall, the observed heterozygosity was lower than the expected heterozygosity (Table 1) and was consistent with that described by Schnell et al. (2003). The observed departure from HWE is likely due to the nature of the material assayed, although 20 loci did show evidence for the potential of ‘null’ alleles at P < 0.05 (Table 1). Forty-two of 44 loci in HWE showed significant LD (P < 0.05) with at least one other locus. The primer sets were also tested upon a limited number of other varieties and hybrids of P. americana, as well as an individual representative of another species, Persea schiedeana Nees. Contrary to microsatellite markers previously developed (Sharon et al. 1997; Ashworth & Clegg 2003; Ashworth et al. 2004), these primers consistently amplified a single locus in all P. americana varieties tested (Table 2). Although limited to a single sample of P. schiedeana, 55 of the 70 primer sets amplified a locus that migrated within the expected size range. The development of 70 polymorphic markers increases the number of informative microsatellite markers available for P. americana by 50%. As these markers successfully amplify all P. americana varieties they will be useful for determining the genetic relationships, exploring potential pedigrees and estimating the genetic background of cultivated accessions of P. americana.
Acknowledgements This research was supported by a USDA-CSREES T-STAR grant ‘Development of avocado rootstocks via molecular and conven-
tional means’. We would like to thank Dr D. Stottlemyer of the University of California, Riverside for collecting and sending leaf material from the South Coast Field Station. A portion of the material reported here was presented at the 2006 Joint Annual Meeting of the Florida State Horticulture Society and the Soil and Crop Science Society of Florida, Krome Memorial Section in Tampa, Florida.
References Albert VA, Soltis DE, Carlson JE et al. (2005) Floral gene resources from basal angiosperms for comparative genomics research. BMC Plant Biology, 5, 5. doi:10.1186/1471-2229-5-5. Ashworth VETM, Clegg MT (2003) Microsatellite markers in avocado (Persea americana Mill.): genealogical relationships among cultivated avocado genotypes. Journal of Heredity, 94, 407– 415. Ashworth VETM, Kobayashi MC, De La Cruz M, Clegg MT (2004) Microsatellite markers in avocado (Persea americana Mill.): development of dinucleotide and trinucleotide markers. Scientia Horticuturae, 101, 255–267. Lavi U, Akkaya M, Bhagwat A, Lahav E, Cregan PB (1994) Methodology of generation and characteristics of simple sequence repeat DNA markers in avocado (Persea americana M.). Euphytica, 80, 171–177. Lewis PO, Zaykin D (2001) Genetic Data Analysis: Computer Program for the Analysis of Allelic Data, Version 1.1. Free program distributed by the authors over the internet http:// hydrodictyon.eeb.uconn.edu/people/plewis/software.php. van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes, 4, 535–538. Raymond M, Rousset F (1995) genepop (Version 1.2): population genetics software for exact test and ecumenicism. Journal of Heredity, 86, 248–249. Schnell RJ, Brown JS, Olano CT et al. (2003) Evaluation of avocado germplasm using microsatellite markers. Journal of the American Society for Horticultural Science, 128, 881–889. Scora RW, Bergh B (1990) The origins and taxonomy of avocado (Persea americana) Mill. Lauraceae. Acta Horticulturae, 275, 387–394. Sharon D, Cregan PB, Mhameed S et al. (1997) An integrated genetic linkage map of avocado. Theoretical and Applied Genetics, 95, 911–921. Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends in Biotechnology, 23, 48–55. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd
