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Table 1 Locus names and GenBank accession nos, forward and reverse primer sequences, repeat size, optimal annealing temperatures (T,), PCR product size, number of alleles, and observed (Hg) and expected (Hy) heterozygosities of 86 adult trees for five microsatellite loci in Platypodium elegans each were screened. Putative microsatellites were screened a second time at a lower plaque density (~200 recombinant clones per plate). Clones picked in the second screen were amplified with M13 primers and products were purified with PCR purification columns (QIAGEN). previous selective events such as seed abortion. Abortion of fertilized ovules prior to seed maturation has been documented in large numbers of angiosperms, including many tree species (Bawa & Webb 1984). Despite the selective potential of seed abortion, studies of its consequences are often limited to species with inflorescences accessible for hand-pollination experiments or embryo rescue due to a lack of tissue needed for allozyme markers. Genetic markers amplified by the polymerase chain reaction (PCR) provide an alternative to pollination studies and avoid the limitations of allozymes. PCR requires only minute amounts of DNA, making genetic analyses of embryos possible in species where a small amount of remaining tissue is recovered from aborted seeds.

Table 1 Locus names and GenBank accession nos, forward and reverse primer sequences, repeat size, optimal annealing temperatures (T,), PCR product size, number of alleles, and observed (Hg) and expected (Hy) heterozygosities of 86 adult trees for five microsatellite loci in Platypodium elegans each were screened. Putative microsatellites were screened a second time at a lower plaque density (~200 recombinant clones per plate). Clones picked in the second screen were amplified with M13 primers and products were purified with PCR purification columns (QIAGEN). previous selective events such as seed abortion. Abortion of fertilized ovules prior to seed maturation has been documented in large numbers of angiosperms, including many tree species (Bawa & Webb 1984). Despite the selective potential of seed abortion, studies of its consequences are often limited to species with inflorescences accessible for hand-pollination experiments or embryo rescue due to a lack of tissue needed for allozyme markers. Genetic markers amplified by the polymerase chain reaction (PCR) provide an alternative to pollination studies and avoid the limitations of allozymes. PCR requires only minute amounts of DNA, making genetic analyses of embryos possible in species where a small amount of remaining tissue is recovered from aborted seeds.

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Table 1 Olea europaea (GA), and (CA),, microsatellite motifs, primer sequences (fluorescent labels indicated as FAM, TET or HEX), sizes of cloned alleles and annealing temperatures (T,)
Table 1 Olea europaea (GA), and (CA),, microsatellite motifs, primer sequences (fluorescent labels indicated as FAM, TET or HEX), sizes of cloned alleles and annealing temperatures (T,)
Table 1 Polymorphic microsatellite loci in Rana lessonae (RL) and R. ridibunda (RR) ‘The full microsatellite sequences from which these primers were designed have been deposited in GenBank (Accession nos: AF195837, AF195839, AF195840, AF195841, AF195842, AF195843, AF195844, AF195845 and AF195846, respectively). tInitial annealing temperature in touchdown protocol (see text). {Heterozygosity values were calculated from one population (23-39 individuals) per primer pair whereas number of alleles and allele size range represents data from all individuals screened (n = 23-221). 3Res3 (for R. ridibunda) and Res6 exhibited null alleles for some populations.
Table 1 Polymorphic microsatellite loci in Rana lessonae (RL) and R. ridibunda (RR) ‘The full microsatellite sequences from which these primers were designed have been deposited in GenBank (Accession nos: AF195837, AF195839, AF195840, AF195841, AF195842, AF195843, AF195844, AF195845 and AF195846, respectively). tInitial annealing temperature in touchdown protocol (see text). {Heterozygosity values were calculated from one population (23-39 individuals) per primer pair whereas number of alleles and allele size range represents data from all individuals screened (n = 23-221). 3Res3 (for R. ridibunda) and Res6 exhibited null alleles for some populations.
‘Determined from sequenced clone. T °C is the optimal (touchdown) annealing temperature; H,, expected heterozygosity; Ho, observed heterozygosity
‘Determined from sequenced clone. T °C is the optimal (touchdown) annealing temperature; H,, expected heterozygosity; Ho, observed heterozygosity
*Microsatellite motifs are sequenced alleles (C. arabica var. Caturra). tEstimated among samples of 32 and 10 individuals of C. arabica and C. canephora, respectiv Table 1 Oligonucleotide primer sequences, repeat motifs, PCR product sizes, allele number, and heterozygosity level of 11 microsatellite loci isolated from Coffea arabica
*Microsatellite motifs are sequenced alleles (C. arabica var. Caturra). tEstimated among samples of 32 and 10 individuals of C. arabica and C. canephora, respectiv Table 1 Oligonucleotide primer sequences, repeat motifs, PCR product sizes, allele number, and heterozygosity level of 11 microsatellite loci isolated from Coffea arabica
*Endemic geographical distribution of species is indicated by the following letters: ‘E’ (east Africa), ‘M’ (Madagascar), ‘WC’ (west and central Africa), ‘C’ (central Africa) and ‘TY’ (India). Table 2 Cross-species amplification using primers designed for Coffea arabica. Assays producing amplification are indicated by ‘+’, no amplification is indicated by ‘’. One sample tree of each species was tested
*Endemic geographical distribution of species is indicated by the following letters: ‘E’ (east Africa), ‘M’ (Madagascar), ‘WC’ (west and central Africa), ‘C’ (central Africa) and ‘TY’ (India). Table 2 Cross-species amplification using primers designed for Coffea arabica. Assays producing amplification are indicated by ‘+’, no amplification is indicated by ‘’. One sample tree of each species was tested
T,,, optimum annealing temperature; Hy, expected heterozygosity; P, probability test for heterozygote deficiency using GENEPOP 3.1c (Rousset & Raymond 1992 Table 1 Primer sequences and characteristics of 11 Haliotis asinina microsatellite loci
T,,, optimum annealing temperature; Hy, expected heterozygosity; P, probability test for heterozygote deficiency using GENEPOP 3.1c (Rousset & Raymond 1992 Table 1 Primer sequences and characteristics of 11 Haliotis asinina microsatellite loci
T,, annealing temperature; H, expected heterozygosity; Hy, observed heterozygosity; *indicates significant deviation from Hardy— Weinberg equilibrium (P < 0.005); t,¢,§ no amplification occurred in 3, 3, 24 individuals, respectively. H, and Ho for Pde3, Pde7 and Pdel3 were calculated only among individuals amplifying at least one allele. Table 1 Characteristics of six microsatellites isolated from Pinus densiflora (n = 36 individuals)
T,, annealing temperature; H, expected heterozygosity; Hy, observed heterozygosity; *indicates significant deviation from Hardy— Weinberg equilibrium (P < 0.005); t,¢,§ no amplification occurred in 3, 3, 24 individuals, respectively. H, and Ho for Pde3, Pde7 and Pdel3 were calculated only among individuals amplifying at least one allele. Table 1 Characteristics of six microsatellites isolated from Pinus densiflora (n = 36 individuals)
Table 1 Seven polymorphic microsatellite loci in Castanopsis cuspidata. Motifs and PCR product sizes refer to sequenced alleles. Thirty: two individuals were genotyped. DDBJ accession numbers are listed under the respective loci T,, annealing temperature; H,, observed heterozygosity; H, expected heterozygosity. F, forward primer; R, reverse primer.
Table 1 Seven polymorphic microsatellite loci in Castanopsis cuspidata. Motifs and PCR product sizes refer to sequenced alleles. Thirty: two individuals were genotyped. DDBJ accession numbers are listed under the respective loci T,, annealing temperature; H,, observed heterozygosity; H, expected heterozygosity. F, forward primer; R, reverse primer.
*sequence motif found in R. vinicolor T20787; t’H’ = HEX dye, ‘F’ = fluorescein; {fragment sizes as determined by the ABI GENESCAN software are highly reproducible due to the internal size standard run in every lane, but they are relative rather than absolute and commonly include fractions of a basepair; §H, = 1 — 2P? , with P, =frequency of allele i. {An additional band of approx. 550 bp was often seen on agarose gels. This band was apparently the result of self-annealing of one of the amplified strands since the band could be re-amplified using the (GITTGCGTGGGAAGTCATTC)-primer without the (GGTACAGGGAATGCTTCG)-primer. Production of the 550 bp band from R. vinicolor extracts could be prevented by reducing the concentration of the former PCR-primer 5-10 fold. Table 1 Microsatellite loci characterized from Rhizopogon vinicolor Rhizopogon vinicolor is a false-truffle in the Boletales (Basidio- mycota) and is a common ectomycorrhizal (EM) symbiont R. vinicolor (collection no. T20787) was grown on modified Melin-NorKran’s (MMN) medium, and genomic DNA was extracted from freeze dried mycelium using the method described below. Genomic DNA was subsequently digested with Sau3AI, and fragments in the 500-1000 bp size range were ligated into the BamHI restriction site of pUC19 and cloned in Escherichia coli DH5. Transformants were detected on LB anp,x.ca Plates. White colonies were transferred to LB,,,, master plates and from there to Ny+ membranes (Millipore). Screening for mic- rosatellite sequences was performed using the AlkPhosDIRECT labelling and detection kit (Amersham Life Science) and two oligonucleotide probes, (CAC),, and (CGA),,, simultaneously at a hybridization temperature of 57.5 °C. Inserts of positive clones were amplified and sequenced with vector specific M13 forward and reverse primers. Nucleotide sequences were
*sequence motif found in R. vinicolor T20787; t’H’ = HEX dye, ‘F’ = fluorescein; {fragment sizes as determined by the ABI GENESCAN software are highly reproducible due to the internal size standard run in every lane, but they are relative rather than absolute and commonly include fractions of a basepair; §H, = 1 — 2P? , with P, =frequency of allele i. {An additional band of approx. 550 bp was often seen on agarose gels. This band was apparently the result of self-annealing of one of the amplified strands since the band could be re-amplified using the (GITTGCGTGGGAAGTCATTC)-primer without the (GGTACAGGGAATGCTTCG)-primer. Production of the 550 bp band from R. vinicolor extracts could be prevented by reducing the concentration of the former PCR-primer 5-10 fold. Table 1 Microsatellite loci characterized from Rhizopogon vinicolor Rhizopogon vinicolor is a false-truffle in the Boletales (Basidio- mycota) and is a common ectomycorrhizal (EM) symbiont R. vinicolor (collection no. T20787) was grown on modified Melin-NorKran’s (MMN) medium, and genomic DNA was extracted from freeze dried mycelium using the method described below. Genomic DNA was subsequently digested with Sau3AI, and fragments in the 500-1000 bp size range were ligated into the BamHI restriction site of pUC19 and cloned in Escherichia coli DH5. Transformants were detected on LB anp,x.ca Plates. White colonies were transferred to LB,,,, master plates and from there to Ny+ membranes (Millipore). Screening for mic- rosatellite sequences was performed using the AlkPhosDIRECT labelling and detection kit (Amersham Life Science) and two oligonucleotide probes, (CAC),, and (CGA),,, simultaneously at a hybridization temperature of 57.5 °C. Inserts of positive clones were amplified and sequenced with vector specific M13 forward and reverse primers. Nucleotide sequences were
Table 1 Primer sequences and some characteristics of polymorphic microsatellites in Helix aspersa. The number of scored individuals was 47 for Ha12, 48 for Ha9, 50 for Ha7, and 117 for the rest of the markers (#origin of individuals tested)
Table 1 Primer sequences and some characteristics of polymorphic microsatellites in Helix aspersa. The number of scored individuals was 47 for Ha12, 48 for Ha9, 50 for Ha7, and 117 for the rest of the markers (#origin of individuals tested)
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