RNA Extraction and Phenotypic Data Collection of Mangifera indica Cultivars for SNP Discovery and Gene Expression

Jose Judy Stervil (1), Barbara Hyacinthe (1), Dora Pilar Maul (1), Barbara Freeman (2) David Kuhn(2).
(1) School of Science, St. Thomas University, 16401 NW 37th Avenue, Miami Gardens, FL 33054, (2) USDA-ARS-SHRS, 13601 Old Cutler Road, Miami, FL 33158 .
I.Introduction

III. Results

Mango (Mangifera indica) is a highly consumed tropical crop because of its unique
1. RNA extraction and quantification of Mango
flavor, color, and shape. It belongs to the Anacardiaceae family of the flowering plants
and is native to South and Southeast Asia. There are four genetically diverse groups of
mango: Indian, Floridian, Southeast Asian, and West Indies (Schnell et al., 2006)
Due to the great demands of commercially favored mangoes, the United States
Department of Agriculture-Agricultural Research Service - Subtropical Horticulture
Research Laboratory (USDA-ARS-SHRL) in Miami, FL has established several
research projects among which are Mango SNP discovery and gene expression in
developing mango fruit. Both projects involve RNA extraction steps. After RNA
Sequencing, sequences can be used to detect Single-Nucleotide Polymorphism (SNPs),
which can be used as markers to create a genetic map in order to determine the location
of the mutations in the chromosomes. Whether a gene is expressed can also be
identified using the extracted RNA combined with other techniques. Mango phenotypic
data can be compared to the genetic map in order to link phenotypic traits to genetic
Figure 1. The samples were
Figure 2. Chloroform was used to
markers.
pulverized using liquid nitrogen
separate the RNA from the rest of
In this experiment, RNA was isolated from four stages of developing fruit, which
the organic compounds. The RNA
during the extraction of RNA.
remained in the top layer.
includes exocarp, mesocarp, seed coat and seed. In addition, phenotypic data of various
mango cultivars harvested from the station were collected for a period of 4 weeks.
Objectives of the present experiment:
Extract RNA to be used for SNP discovery and gene expression
2. Collection of Mango Phenotypic Data
Collect the phenotypic data for several cultivars of mangoes from the USDA-ARS
station

II. Materials and Methods

V. Future Steps
Location

RINe

E1

4.3

28S/18S
Conc. [ng/l] Sample Description
(height)
0.7
34.0
29_1-2seedMauri

A1

4.1

0.4

2.89

TA47_4-3scoatTAtkins

B1

4.0

0.3

155

TA48_4-3seedTAtkins

C1

6.4

1.9

1670

27_1-2exocarpMauri

D1

7.2

1.5

650

28_1-2mesocarpMauri

F1

7.4

0.8

112

30_1-3exocarpMauri

G1

6.3

1.0

142

31_1-3mesocarpMauri

H1
A2

5.3
5.0

0.7
1.8

6.85
315

32_1-3seedMauri
33_2-2exocarpMauri

B2

8.3

1.3

174

34_2-2mesocarpMauri

C2
D2
A0

4.4
3.0

1.3
0.9

122
30.9
33.3

Pot1_PI545851
Pot2_PI545851
ladder

Liquid Nitrogen Method of RNA Extraction

Day 1: Samples were collected and stored at -80oC. Samples were pulverized using
liquid nitrogen and added to 65oC RNA extraction buffer (Bailey et al., 2005) in
Table 1. Gel image, concentration, and purity readings from
centrifuge tubes. They were homogenized for 1 min and placed in water bath. 15 ml of
e stands for RNA
Tapestation
results
for
mango
and
lychee
RNA.
RIN
chloroform was added to each sample, homogenized and centrifuged for 30 min at 4oC
Integrity Number and has a value between 1 and 10, where 10
(8000 rpm). Top layer was transferred to new tubes with one-third volume of 8M
represent the highest quality RNA sample.
Lithium Chloride and stored overnight in ice water bath 4oC refrigerator.
Day 2: The tubes were centrifuged at 8000rpm for 1 hour at 4oC. Samples were
B2: 34_2-2mesocarpMauri
B1: TA48_4-3seedTAtkins
Figure 3. Measurements Figure 4. From left to right, Mango was cut and the
cleaned with the Qiagen RNeasy kit following manufacturers instructions. Water,
of the fruit were
brix was taken using a Digital Refractometer.
Dnase 1 and 10x Dnase buffer were added to tubes and incubated at 37 oC for 30 min.
recorded.
180ul of phenol chloroform was added, vortex and centrifuged for 10 min. at 4oC. Top
layer was then transferred to new 1.5ml cross-linked tubes and 20 ul of 3M NaOAc and
600 ul 100% EtOH were added. Tubes were vortex and RNA precipitated overnight at Cultivar
Fruit Weight (g)
Shape
Brix
(%)
80oC.
Day 3: Tubes were centrifuged at 14000g at 4oC for 30 min. and supernatant was
Table 2. Electropherogram of RNA samples using the TapeStation.
discarded and tubes were left open to dry. Pellet was rinsed with 500ul of 80% EtOH
Aroemanis
277.79
1
16.05
and re-centrifuged. All liquid was removed and placed in dry bath for 3-5min. at 50oC.
It was then re-suspended in 50ul of Rnase-free H2O; incubate for 10 min. before
Becky
560.99
5
10.12
quantification.
Quantification of RNA
Turpentine
324.54
2.25
13.6
Nanodrop 2000: The nanodrop was calibrated with 1ul of Rnase-free water. Samples
Table 3. Average fruit weight, shape and brix obtained from twelve
were vortexed and centrifuged. 0.6ul of each sample was measured using the small
replicate fruits from three of the mango cultivars.
sample volume setting. Calibration was tested periodically by measuring blank samples
with water.
TapeStation: TapeStation can read between 2-500ng/uL. The majority of RNA
extractions required a 1:20 dilution. Samples were prepared by mixing 4ul of R6K
Table 4. Quantification result of RNA extracted from mango and lychee
sample buffer with 1ul of RNA, denatured at 72oC for 3 minutes, placed on ice for 2
samples using the Nanodrop 2000. Most of the RNA extracted were high
minutes, and loaded onto the TapeStation. Samples were measured using an R6K
in concentration and pure.
IV. Conclusions:
ScreenTape.
Field Data Collection of Mango
1. RNA was successfully extracted from several cultivars of mango and lychee to be used for SNP discovery and gene expression.
A total of 47 cultivars of mangoes were collected from different locations at the
2. The quantification results of the Nanodrop and the TapeStation determined that most of the samples were high in purity and concentration
USDA-ARS station. Measurements of the fruit, stone and seed were taken, such as
with no degradation of the isolated RNA.
weight, length, width and thickness. Colorimetric, shape, anthracnose, brix and fiber
3. Phenotypic data for 47 cultivars of mangoes from the USDA-ARS station were collected.
data were also obtained for the fruit. The seeds were classified as monoembryonic or
polyembryonic.

RNA Sequencing from the RNA

extraction will be used to detect
Single-Nucleotide Polymorphism
(SNPs), which will be used to
create a genetic map.
Mango phenotypic data will be
compared to the genetic map to
link phenotypic traits to genetic
markers

VI. Acknowledgements
The authors want to acknowledge
Ashley Johnson, Leah Schwartz,
Herma Pierre, Tomas AyalaSilva. A special thanks to Carlos
Vazquez for his support. Partial
funding for this project came the
USDA-HIS-funded FCCAgE
Agricultural Education grant and
from the U.S. Department of
Education - STEM TRAC
grant(P03C110190), MDC
School of Science-STU School
of Science, Technology and
Engineering management.

VII. References
Bailey et al., 2005. Gene
expression in leaves of Theobroma
cacao in response to mechanical
wounding, ethylene, and/or methyl
jasmonate. Plant Science 168,
1247-1258.
Kuhn et al., 2012. Identification
and mapping of conserved ortholog
set (COS) II sequences of cacao
and their conversion to SNP
markers for marker-assisted
selection in Theobroma cacao and
comparative genomics studies.
Tree Genetics & Genomes 8, 97
111.
Schnell et al., 2006. Mango
Genetic Diversity Analysis and
Pedigree Interferences for Florida
Cultivars Using Microsatellite
Markers. J. Amer. Soc. Hort. Sci.
131(2), 214-224.