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Sarracenia Leucophylla Lab Report

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1.Introduction

The objectives of this study were to determine the correct identity of the plant Sarracenia leucophylla using a series of molecular biology techniques. Phylogenetic analysis and genetic sequencing alignment was used to further confirm its identity in addition to construct a maximum parsimony tree. Sarracenia leucophylla, or the crimson pitcher plant, is a carnivorous plant in the Family Sarraceniaceae. The species is endemic to the Southeastern region of the United States and resides in moist, low-nutrient habitats (3 from wiki). Although the species within the genera are closely related, their geographic regions are scattered across North and South America with few current overlapping regions. Beyond determining species identity, …show more content…

leucophylla DNA was added to the master mix in rbcL and ITS2 tubes. The PCR was set to start with (1) 2 minutes at 94 ℃, (2) 30 seconds at 94 ℃ (3) 30 seconds at 51 ℃, (4) 90 seconds at 71℃ (5) 10 minutes at 71 ℃ and held at 4℃ until gel electrophoresis can be conducted. Steps 2-4 will be repeated a total of 35 times.

3.2.2 Gel Electrophoresis
To determine if our DNA sequences were properly extracted and amplified, we used gel electrophoresis. A 1% agarose gel was made using 1 gram of agarose powder per 100 ml of 1x TBE buffer. The solidified gel was placed in the electrophoresis tank containing 1x TBE buffer. Our samples were prepared by mixing gel loading solution with our PCR products (3 µL gel-loading solution, 5 µL PCR product). The samples were loaded in a selected order (table 2) and the gel was run between 30 minutes to an hour at 70-80 V (Lab 8, 2017)

Lane
Group Number
1
KB Ladder
2
(+) control
3
(-) control
4
10
5
11
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13
8
14
9
15
10
16 …show more content…

It is to be noted that for ITS2 we received the reverse primer and had to take that into account when performing sequence alignment. Both regions were aligned using MEGA 6 software with 15 other species, two outgroups, and an already defined region of rbcL and ITS2 of S. leucophylla. Alignment was using using ClustalW* (lab 9, 2017). Default parameters can be found in Taylor Dodge’s lab notebook under DNA sequencing section. The ITS region of our species was 282 bp long with 193 bp minimum overlap. The rbcL region was 496 bp long with 484 bp minimum overlap. With an adequate sequence alignment we were able to construct maximum parsimony trees for both sequences.

3.4 Phylogenetic Analysis *a more detailed protocol can be found in Lab handout 9, (lab 9, 2017).*
Using our final alignment we using MEGA to search for the most parsimonious tree for our species. ITS2 used an MP search method of tree bisection reconnection, or TBR, while rbcL used maximum branch and bound. The tree was rooted using our outgroup species seen below in figures 2 and 3. For ITS2 and rbcL, a total of 73 and 270 trees were generated, respectively. ITS2 has 325 steps and rbcL has 90 steps with all of the trees for both sequences being equally

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