Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

Lucas, Marília Souza; Carvalho, Carolina da Silva; Hypolito, Giovane Böerner; Côrtes, Marina Corrêa

doi:10.1590/2236-8906-94/2018

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Article • Hoehnea 46 (02) • 2019 • https://doi.org/10.1590/2236-8906-94/2018 copy

Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

Protocolo otimizado para isolar DNA genômico de alta qualidade em diferentes tecidos de uma espécie de palmeira

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

The application of molecular techniques to tackle ecological and evolutionary questions requires genomic DNA in good quality and quantity. The quality of the isolated DNA, however, can be influenced by the tissue type and the way the sample was conserved and manipulated. Therefore, customizing protocols to improve the DNA isolation and locus amplification is crucial. We optimized a cheap and manual protocol of DNA extraction and microsatellites amplification using five different tissues of a palm species of the brazilian Atlantic Forest. We successfully extracted DNA from all five tissue types. Leaf, stem, and endocarp of non-dispersed seeds presented the highest rates of successful DNA extraction and microsatellite amplification; whereas root, endocarp of dispersed seeds, and embryo showed the lowest quality and quantity. Based on these results, we discussed the implications of using different tissues for studies about seed dispersal, pollination, and population genetics.

Keywords:
DNA extraction; Euterpe edulis; microsatellite; tropical palm

Authorship

Marília Souza Lucas ³3. Corresponding author: marilia_souza2@hotmail.com

. Universidade do Estado de São Paulo, Instituto de Biociências, Departamento de Ecologia, Av 24 A,1515, 13506-900 Rio Claro, SP, BrasilUniversidade do Estado de São PauloBrazilRio Claro, SP, Brazil. Universidade do Estado de São Paulo, Instituto de Biociências, Departamento de Ecologia, Av 24 A,1515, 13506-900 Rio Claro, SP, Brasil

Carolina da Silva Carvalho

. Instituto Tecnológico Vale, R. Boaventura da Silva, 955 - Nazaré, 66055-090 Belém, PA, BrasilInstituto Tecnológico ValeBrasilBelém, PA, Brasil. Instituto Tecnológico Vale, R. Boaventura da Silva, 955 - Nazaré, 66055-090 Belém, PA, Brasil

Giovane Böerner Hypolito

Marina Corrêa Côrtes

3. Corresponding author: marilia_souza2@hotmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (2)
Tables (3)

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Figure 1
Tissue types from the palm tree Euterpe edulis. a. Adult tree. b. Leaf. c. Stem stalk. d. Root. e. Root detail. f. Ripe fruit. g. Fruit without pulp (endocarp of not dispersed seeds). h. Endocarp of dispersed seeds. i. Seed embryo.

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Figure 2
Electrophoresis gel with the results of Euterpe edulis genomic DNA extraction from five plant tissue types using two DNA extraction protocols: leaf, stem, root, non-dispersed seed endocarp and embryo using the general protocol, and dispersed seed endocarp using the endocarp protocol (see text). The Low Mass ladder (Invitrogen) was used for DNA quantification; the bands refer to 100 ng/μL, 60 ng/μL, 40 ng/μL, 20 ng/μL, 10 ng/ μL and 5 ng/μL, from top to bottom, respectively.

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Table 1
Microsatellite molecular markers, respective primers sequences and expected variation of loci size in number of base pairs of the palm Euterpe edulis. Markers were originally described in Gaiotto et al. (2001Gaiotto, F.A., Brodani, R.P.V, & Grattapaguia, D. 2001. Microsatellite markers for heart of palm - Euterpe edulis and E. oleracea Mart. (Arecaceae). Molecular Ecology, Notes, 1: 86-88.).

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Table 2
Amount of reagents and annealing temperature for optimized PCR of seven microsatellite loci for dispersed seed endocarps (Endocarp) and stem, leaf, root, non-dispersed seed endocarp and embryo (General) of the palm Euterpe edulis. The values next to each reagent correspond to its stocked concentration. The amount of all reagents is given in microliters (µL). The reactions were carried out in a final total volume of 13 µL.

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Table 3
Rate of DNA extraction success (proportion of samples that presented bands in the agarose gel), average DNA amount (± standard deviation), and rate of microsatellite amplification success (average, minimum and maximum number of loci that were successfully amplified) from 10 samples for each of the five tissue types of the palm Euterpe edulis.

Figure 1 Tissue types from the palm tree Euterpe edulis. a. Adult tree. b. Leaf. c. Stem stalk. d. Root. e. Root detail. f. Ripe fruit. g. Fruit without pulp (endocarp of not dispersed seeds). h. Endocarp of dispersed seeds. i. Seed embryo.

Figure 2 Electrophoresis gel with the results of Euterpe edulis genomic DNA extraction from five plant tissue types using two DNA extraction protocols: leaf, stem, root, non-dispersed seed endocarp and embryo using the general protocol, and dispersed seed endocarp using the endocarp protocol (see text). The Low Mass ladder (Invitrogen) was used for DNA quantification; the bands refer to 100 ng/μL, 60 ng/μL, 40 ng/μL, 20 ng/μL, 10 ng/ μL and 5 ng/μL, from top to bottom, respectively.

Table 1 Microsatellite molecular markers, respective primers sequences and expected variation of loci size in number of base pairs of the palm Euterpe edulis. Markers were originally described in **Gaiotto et al. (2001**Gaiotto, F.A., Brodani, R.P.V, & Grattapaguia, D. 2001. Microsatellite markers for heart of palm - Euterpe edulis and E. oleracea Mart. (Arecaceae). Molecular Ecology, Notes, 1: 86-88.).

Primer	Sequence of primers (5'3')	Variation of allele size (bp)
EE3	F: TTCgCgCACACTgAgAg R: ggTAgCgTTgATTggTCC	194-210
EE 23	F: gTTCTgCgATTCATACTCCTg R: TACgAACCAAgATggAgCAA	100-132
EE25	F:CggATCCTgAgACTgAATTg R:CACACAgATTCAgAgCACA	156-190
EE45	F: AAAgAAATTggCgTgACATC R: AACCAgTCTTCTCCCTCTCg	70-154
EE47	F:CgAAATCAATggTTTCAgTg R:AATTATTgTTgTgggCAgC	214-246
EE52	F:TTCTgTggAgAgTCAATCATC R:AATCTgACAAggCCTCAAC	230-260

Table 2 Amount of reagents and annealing temperature for optimized PCR of seven microsatellite loci for dispersed seed endocarps (Endocarp) and stem, leaf, root, non-dispersed seed endocarp and embryo (General) of the palm Euterpe edulis. The values next to each reagent correspond to its stocked concentration. The amount of all reagents is given in microliters (µL). The reactions were carried out in a final total volume of 13 µL.

Reagent	EE3		EE23		EE25		EE45		EE47		EE52		EE54
Reagent	Endocarp	General	Endocarp	General	Endocarp	General	Endocarp	General	Endocarp	General	Endocarp	General	Endocarp	General
DNA (2,5 ng)	3	3	3	3	3	2	3	3	3	3	3	3	3	3
Buffer (10x)	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3
DNTP (10mM)	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32
Primer F (0.9 uM)	2.88	2.88	2.88	2.88	2.88	1.8	2.88	2.88	2.88	2.88	2.88	2.88	2.88	2.88
Primer R (0.9 uM)	2.88	2.88	2.88	2.88	2.88	1.8	2.88	2.88	2.88	2.88	2.88	2.88	2.88	2.88
Taq (5u)	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
BSA (62.5 mg/mL)	0.65	0.65	0.65	0.65	0.65	0.52	0.65	0.65	0.65	0.65	0.65	0.65	0.65	0.65
MgCl_² (50mg/mL)	0.91	0.78	0.78	0.78	0.78	0.65	0.78	0.78	0.91	0.78	0.91	0.78	0.78	0.78
H_²O	0.86	0.99	0.99	0.99	0.99	4.41	0.99	0.99	0.86	0.99	0.86	0.99	0.99	0.99
Annealing temperature (ºC)	64-54	66-56	66-56	66-58	68-58	60-68	64-54	64-54	60-50	66-56	64-54	66-56	66-56	66-56

Table 3 Rate of DNA extraction success (proportion of samples that presented bands in the agarose gel), average DNA amount (± standard deviation), and rate of microsatellite amplification success (average, minimum and maximum number of loci that were successfully amplified) from 10 samples for each of the five tissue types of the palm Euterpe edulis.

Tissue type	DNA extraction success %	Amount of DNA (ng/ μL)	Amplification success (%)							Amplification success% [minimum, maximum]
			EE3	EE23	EE25	EE45	EE47	EE52	EE54
Leaf	100	140 ± 53.2	100	100	100	100	100	100	100	100 [100,100]
Stem	100	94 ± 19.6	90	90	100	100	100	100	80	95 [80,100]
Root	40	6 ± 6.2	50	0	50	40	20	80	10	40 [20,80]
Endocarp (not dispersed)	100	108 ± 63.7	100	100	100	100	100	100	100	98.75 [90,100]
Endocarp (dispersed seed)	80	53 ± 28.2	50	60	50	70	70	40	50	56.25 [40,70]
Embryo	50	82.5 ± 63.1	70	70	80	90	70	80	80	75 [60,90]

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Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

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[1] 3. Corresponding author: marilia_souza2@hotmail.com