Ecological Archives E091-096-A2

Andrew Olaf Shelton. 2010. The origin of female-biased sex ratios in intertidal seagrasses (Phyllospadix spp.). Ecology 91:1380–1390.

Appendix B. The DNA extraction and amplified fragment length polymorphism (AFLP) protocols.

Collection information

In May 2006, I collected leaf tissue from flowering male and female P. scouleri individuals at six sites (Appendix A). To avoid sampling cryptic, non-flowering individuals, I only collected tissue from young, actively growing leaves connected to rhizomes that produced a reproductive shoot. Each sample was separated by at least two meters to avoid sampling the same genotype multiple times. Leaves were air-dried for 24 hours before being placed in silica beads for permanent storage. DNA extraction, restriction, and amplification followed standard AFLP protocols.

TABLE B1. Number of male and female patches sampled at each site for sex-linked marker screening.


Site

Female

Male

Kydaka Pt. (KD)

12

4

Main Beach (MB)

18

15

Ozette (OZ)

2

1

Strawberry Draw (SD)

18

15

Shi Shi (SS)

20

1

Teahwhit head (TH)

11

15

Total

81

51

 

AFLP Protocol

To extract DNA from dried leaf tissue, approximately 10 mg of tissue was pulverized using a tungsten ball and a Qiagen TissueLyser machine shaking at 30 hertz for 1 minute. Following tissue disruption, genomic DNA was extracted using Qiagen Dneasy Plant Mini kits following the manufacturer’s protocol. Samples were stored in Qiagen elution buffer at 4°C during use and at – 80°C for permanent storage.

I used AFLP protocols modified from (Vos et al. 1995) and from the Wolf laboratory AFLP protocol (http://bioweb.usu.edu/wolf/aflp_protocol.htm).  Restriction and ligation of 15 µL genomic DNA were performed simultaneously for 4 hours at 37°C followed by 10 minutes at 70°C to denature enzymes.  Five U EcoRI and 1 U MseI restriction enzymes, 1 U T4 DNA ligase, 1 µL of annealed MseI adapter pair at 50µM (F: 5'-GAC GAT GAG TCC TGA G-3'; R-5'-TAC TCA GGA CTC AT-3' ), 1 µL of annealed EcoR1 adapter at 5µM (F: 5'-CTC GTA GAC TGC GTA CC-3'; R: 5'-AAT TGG TAC GCA GTC TAC-3'), 0.1 µL of 0.5M NaCl, 0.1 µL of 10mg/mL BSA, 1U T4 DNA ligase and 0.15 µL sterile H2O were added to 15 µL Genomic DNA for a total reaction volume of 20.25 µL. Following incubation each sample was diluted with 180 µL  TE(0.1 µM EDTA).

Following restriction and ligation, two rounds of amplification were conducted: a pre-selective amplification using EcoRI+A primer (5'-GAC TGC GTA CCA ATT CA-3') and MseI+C primer (5'-GAT GAG TCC TGA GTA AC-3') and a selective amplification using a two additional nucleotides on each primer. Pre-selective amplification used 5 µL diluted restricted-ligated DNA, 0.25 U of Roche Taq, 0.19mM dNTPs, 1X Roche Taq buffer, 25mM MgCl, 0.3µM of each primer, and 0.5 µL of 10mg/mL BSA in a total reaction volume of 25 µL.  Temperature profile for the preselective PCR consisted of 2 min at 72°C followed by 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 2 min. A final 10-min extension step at 60°C followed and stored at 4°C. PCR products were diluted with 175µL TE(0.1 mM EDTA). For selective amplification, two nucleotides were added to both EcoRI+A and MseI+C primers (i.e.,  EcoRI+ANN and MseI+CNN). EcoRI+ANN were labeled with a NED, VIC, 6-FAM, or PET fluorescent tags included at 0.1 µM in the selective PCR with 0.2 µM MseI+CNN, 0.5mM dNTPs, 0.38 U Roche Taq, and the remaining components in identical concentration to the pre-selective PCR. The total volume of the selective PCR reaction was 12.5 µL. The temperature profile of selective PCR consisted of 2 min at 95°C, 13 cycles of 30 s at 95°C, 30 s at 65°C reduced by 0.7°C each cycle, and 2 min of 72°C, then 26 cycles of 30 s at 95°C, 30 s at 56°C, 2 min at 72°C, and a final extension period of 30 min at 72°C before storage at 4°C. Final PCR products were run on an Applied Biosystems 3730 capillary electrophoresis sequencer with a LIZ-500 internal lane standard.

Initially eight males and eight females (two of each sex from KD, MB, SD, and TH) were screened with AFLP. A total of 94 primer pair combinations were applied to each sample with four primer pairs of different colors per lane on the ABI 3730. Chromatographs were analyzed using GeneMapper version 3.7 with a cut-off peak-height of 150. After automated scoring, the presence of each peak was verified manually. Bands smaller 60 bp in length were eliminated and the presence or absence of unambiguous bands was scored for all individuals. Bands that were present in six or more individuals of one sex and absent from the other sex were considered candidates for sex-linked makers and screened against 132 known sex individuals from all six sites (51 males and 81 females; Fig. B1). The 132 samples were conducted with a single primer pair combination in each lane. To control for amplification success, a single band present in all individuals was scored.  If the control band was less than 500 units high, amplification was assumed to have failed and the sample was run a second time.

Of seven candidate markers identified and tested, two were determined to be sex linked (Table B2). The EcoRI+ATC and MseI+CCC primer pair (hereafter Marker A) had a male-linked band at 104 bp and a control band at 103 bp. A pair of peaks at 159 and 160 bp were present in males with the EcoRI+AAC and MseI+CGG primers (hereafter Marker B) and a control band at 126 bp. For each primer pair the male-linked band was absent from females and present in 76 to 78% of males (Table B3). Sex-linked bands from the two primers were high correlated, co-occurring in nearly all of the same males. To test the repeatability of the EcoRI+ATC maker, I ran the selective PCR on 96 samples on two occasions a month apart and checked for agreement between amplification dates. Of the 73 samples (24 males and 49 females) that for which the control amplified on both occasions, all samples were assigned to be the same state (present or absent) on both occasions, indicating the markers are repeatable. Both sex-linked markers were present in at least one male from all sites sampled.

 

TABLE B2. Primer sequence, male-linked AFLP fragment size, and amplification control fragment size for Phyllospadix scouleri.


Marker

EcoR1 primer

MseI primer

Male-linked band (size in bp)

Control region (size in bp)

A

GACTGCGTAATTCATC

GATGAGTCCTGAGTAACCC

104

103

B

GACTGCGTAATTCAAC

GATGAGTCCTGAGTAACGG

159,160

126

 

TABLE B3. The accuracy of sex-linked AFLP markers.


Sex

Marker
present

Marker
absent

Proportion of samples with marker

Marker A

 

 

 

Male

40

11

0.784

Female

0

81

0

Marker B

 

 

 

Male

39

12

0.765

Female

0

81

0


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