Possible genes affecting fruit size in Vaccinium corymbosum
David Lloyd
Abstract
Fruit size is an economically important trait that, while extensively studied in other fruits,
has received relatively little attention in Vaccinium corymbosum, the highbush blueberry. The
purpose of this study was to identify and locate genes of potential importance to this trait in order
to enable breeders to produce plants with larger fruit. To this end, I BLASTed genes that have
been identified as affecting fruit size in other species against a set of scaffolds of the blueberry
genome. This project was conducted as part of an undergraduate course at Davidson College.
Introduction
Larger fruit has the obvious advantage of higher yields. However, the issue of size is not
limited to yield maximization, but also fulfills consumer preference for larger berries (Donahue
et al. 2000). In species of the genus Solanum (tomatoes and their relatives), increase in fruit size
quickly followed domestication. In fact, fruits from modern cultivars can reach masses up to one
thousand times that of their wild ancestors (Cong et al., 2008)
There are several possible mechanisms for production of larger fruit. In tomatoes, one
can observe a marked difference in anatomy between smaller and larger fruit. Larger cultivars
have a significantly increased number of locules (seed bearing compartments) compared to
smaller domestic and wild varieties that have only two (Cong et al., 2008). Though there is no
such variation in current blueberry cultivars, genes affecting floral organ variation in tomato may
still be worth further investigation in blueberry.
The majority of variation currently observable in blueberry is due to variations in cell
number. Johnson et al. (2011) conducted an investigation of the correlations among various
measurements of size. They found that fruit diameter is an accurate predictor of fruit mass with
a linear relationship, and that cell number is an accurate predictor of fruit diameter with a
logarithmic relationship. They also determined that average cell size did not demonstrate any
correlation with fruit diameter and therefore mass. For this reason, the genes presented in this
study which merit the most attention are those involved in cell cycle regulation and cell
proliferation during floral and fruit development.
In addition to cell cycle regulators, other genes have been shown to have an effect on
mature fruit mass. In tomato, a form of invertase, TIV1, has been shown to significantly
influence fruit size by regulation of sugar metabolism (Klann et al., 1996). In their study, Klann
et al. determined that fruit which stored more sucrose by virtue of nonfunctional TIV1 were
reduced in size by as much as 30%. This finding presents the possibility that fruit growth
patterns can be manipulated and potentially increased my manipulation of the timing of TIV1
activity.
As a wide range of processes can affect fruit size, there is no one pathway for the trait.
However, Anastasiou et al. (2007) indicated the most important pathway involved is cell
proliferation of fruit development (Figure 1).
Figure 1. The fruit
development pathway
described by Anastasiou.
(Reproduction of Figure 1
from Anasasiou et al.,
2007).
Methods
I selected genes from the literature based on their observed effects in Vaccinium or other
organisms. The genes chosen for analysis are shown in table 1.
Gene
fw2.2
ABP1
NtKIS1a
CYC1
FAS
fw1.1
fw1.2
fs1.1
fw1.3
fw7.1
fw7.2
fw2.3
fs8.1
fw3.1
fw9.1
fw4.1
TIVI
pBI121
fw11.3
lcn11.1
ADH2
TG10
Reference
(Cong et al., 2002; Frary et al., 2000; Grandillo et al., 1999; Johnson et al., 2011; Lippman et al.,
2001)
(Cong et al., 2008)
(Cong et al., 2008; Jasinski et al., 2002)
(Cong et al., 2008)
(Cong et al., 2008)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Grandillo et al., 1999)
(Klann et al., 1996)
(Klann et al., 1996)
(Lippman et al., 2001)
(Lippman et al., 2001)
(Nesbitt et al., 2002)
(Nesbitt et al., 2002)
TG11
TG91
TG167
ANT
ARF2
ARGOS
BB
ARL
AN3
(Nesbitt et al.,
(Nesbitt et al.,
(Nesbitt et al.,
(Anastasiou et
(Anastasiou et
(Anastasiou et
(Anastasiou et
(Anastasiou et
(Anastasiou et
2002)
2002)
2002)
al., 2007)
al., 2007)
al., 2007)
al., 2007)
al., 2007)
al., 2007)
Table 1. The genes in this table have been implicated by the cited authors as affecting variations
in fruit size in some fruiting plant.
I searched for each of the above genes in the NCBI database. I selected the sequence
from the organism most closely related to blueberry and saved those in FASTA format. In order
to BLAST the sequences, I used a command line process in the Mac OS X terminal (Figure 2).
The commands and steps given in the following figures will work on any Unix-based command
line interface.
A
B
C
Figure 2. Note that “dalloyd” and “davidlloyd” will be replaced by the username and computer
name of whomever is performing the task. The commands in (A) and (B) must be entered only
once per login. Step (A) creates the database in which to operate, and step (B) sets up the BLAST
commands. In (C) the actual blast command is entered. “tblastn” is used to blast a amino acid
sequence and can be replaced with “blastn” to perform a nucleotide blast. “CYC1prot.txt” is the
name of the plain text file containing the sequence to be blasted in FASTA format.
Figure 3: A sample result for the BLAST process, this figure shows the output for the query
performed in Figure 1.
I considered hits to be significant if the E value was less than 110-4 (Figure 3). I submitted
the scaffolds containing significant alignments to the SSR tool server at the blueberry genome
portal (vaccinium.org, year). This online tool finds SSRs on the submitted scaffolds and the
results are automatically compiled into a spreadsheet. I chose primers based first on their
proximity and inclusion of my gene of interest, and then on the number of repeats of their SSR
motif. A full list of my selected primer pairs for each gene can be found in Table 2. I submitted
this report to the breeders at Washington State University.
I and other members of the class also attempted to use a selection of tools from GenSas.
However, due to poor results we discontinued use of that technique after one try.
Results
Gene
Scaffold
Location
ABP1
1)
For Primer
Rev Primer
Repeats (ga) x20
327
108155
GTAACGAACGAACGAACGAAC
ACAACCTTGCTTCCTTGATGAT
PCR Product=206bp
start:95084
2)
For Primer
Rev Primer
Repeats (ga) x19
GGGAGGAATTGAATGATGGTAA
GGAACGACGAAGAACCTACCTT
PCR Product=145bp
start:89171
3)
For Primer
Rev Primer
Repeats (ga) x17
GATGGGGTTTGATGGATTCTAA
CACCACACCACCAAACAACTAC
PCR Product=161
start:31968
ADH2
1)
For Primer
Rev Primer
Repeats (ga) x31
1152
20288
AGCTCCACTAATCGGACTCAAG
GGCACTTTCGTTTTCTGTTTTC
PCR Product=284bp
2)
For Primer
Rev Primer
Repeats (ag) x23
GGTAGGTAGGTAGGAGGTAGGAGG
ACACGCTTTTAAGAATCACCGT
PCR Product=126bp
start:90446
3)
For Primer
Rev Primer
Repeats (tc) x10
GTCCCTGAAATCAAACAAGTCC
AGCGTAGAGAATCGAACCTGAG
PCR Product=221
start:1178
start: 25475
ANT
1)
For Primer
Rev Primer
Repeats (aaag) x4
162
33237
CCTAACGGTGTCAATTGGGTAT
GCGATCTCTATTGAAATCCTGG
PCR Product=166bp
start: 31644
2)
For Primer
Rev Primer
Repeats (tc) x12
TGTGGTAGGTACGTACTCGTGG
AAACATGCCCATAATGAAGTCC
PCR Product=271bp
start:107
3)
For Primer
Rev Primer
Repeats (ag) x10
CGGAATCTTTCGTTATTGCTTC
GTGGATGCAACTTAGGTGTCAA
PCR Product=279bp
start:38419
ARF2
1)
For Primer
Rev Primer
Repeats (aat) x13
51
50409
GATTTATGCACAACAAGGCTCA
GACAGAAGAGTTAGCCAAGGGA
PCR Product=237bp
start: 27931
2)
For Primer
Rev Primer
Repeats (ag) x11
GTTTGTGAACTCAGAGGCAGTG
TATGTGACCCCAGTACACAAGC
PCR Product=268bp
start:24413
3)
For Primer
TGAAAGAAATCAACACCGACAG
start:106486
Rev Primer
Repeats (ac) x10
GGTTTTCCCTGATGTCTGCTAC
PCR Product=197bp
BB
1)
For Primer
Rev Primer
Repeats (tg) x11
613
144727
CAGTTGTAGTTGGGGTTGGAAT
TGTTTGCACAGATGTAAAAGGG
PCR Product=149bp
start: 96291
2)
For Primer
Rev Primer
Repeats (ag) x11
AGTACGTATACACCCACCCCAG
AATGACACCCAAACGCTTTACT
PCR Product=157bp
start:131191
3)
For Primer
Rev Primer
Repeats (tc) x13
GTGACCAGTGCACATTTTTCAT
TTACTATCTGGGTGATGTGGGA
PCR Product=210bp
start:86304
CYC1
1)
For Primer
Rev Primer
Repeats (ta) x10
277
33237
ATAACCTTGTAATTGCCGGATG
ATCATAGGAATTGACACGACCC
PCR Product=200bp
start: 27802
2)
For Primer
Rev Primer
Repeats (tc) x16
TAGGTGTATGAACCCACGTGAA
CTTTATTTCCCAGCTTCCAATG
PCR Product=243bp
start:5780
3)
For Primer
Rev Primer
Repeats (tc) x10
TGGTTCCTGTTTTGATCTTCCT
AGTAGTGACAACCCAACCTCGT
PCR Product=264bp
start:72626
fw2.2
1)
For Primer
Rev Primer
Repeats (ag) x12
1222
11597
GTGTGTGTGTGTGTGTGTGTGA
ATGTGAACCCAAAGTTACCAGG
PCR Product=205bp
start: 16660
2)
For Primer
Rev Primer
Repeats (ct) x21
TAGGTGTATGAACCCACGTGAA
CTTTATTTCCCAGCTTCCAATG
PCR Product=287bp
start:23817
3)
For Primer
Rev Primer
Repeats (ct) x10
GTATGTCAACCACGGACAGTTG
TGGGACACCAAATATACGTTCA
PCR Product=272bp
start:5445
fw7.2
1)
For Primer
Rev Primer
Repeats (tc) x17
704
125210
GATGTTGGGTTGATCGTACAAA
CTTCCCAATACCAAAACCCTAA
PCR Product=289bp
start: 97640
2)
For Primer
Rev Primer
Repeats (ga) x10
GAGGGGGTGTGAATTTTGTCTA
CTATTTTCCCTCTCTCCTCCGT
PCR Product=265bp
start:78950
3)
For Primer
Rev Primer
Repeats (ct) x24
CCTATTTTTGGTGTTTTGTCCC
ATCCTCCAAAAGTGTACACCCA
PCR Product=271bp
start:78053
pBI121
1)
For Primer
Rev Primer
Repeats (tc) x21
14
326508
ATGTGTTGCCACACTATTGCAT
GGTTGTTGGTCTCTATCTTCCG
PCR Product=300bp
start: 290791
2)
For Primer
Rev Primer
Repeats (tc) x11
ACTTCAACCTTACCCCCTCATT
TCAGGAGGAATAACCCAAATGT
PCR Product=146bp
start:333807
3)
For Primer
Rev Primer
Repeats (ga) x11
TGTGTGGAGTGGAGAGAGAAGA
ACGGTAGCGAGACTACCCAATA
PCR Product=249bp
start:358798
TIV1
1)
For Primer
Rev Primer
Repeats (ga) x19
80
74980
GGTATCAACGAAAGCGTACCTC
CGTCCGACTTCTAATAANACACG
PCR Product=205bp
start: 103562
2)
For Primer
Rev Primer
Repeats (ga) x11
AAGTTGGGCTTTACGGGATATT
CAAGTGGAAAAACAAGTCCACA
PCR Product=126bp
start:59469
3)
For Primer
Rev Primer
Repeats (ga) x15
GCTGAAAACACCACCAGTAACA
TGCAAACGTAAACTTAAGCCCT
PCR Product=237bp
start:54986
Table 2. This table contains a list of the genes which produced significant alignments, their
locations on the given scaffolds, and the three primer pairs I selected for each.
GenSAS Results
To test the protein BLAST function on GenSAS. I tested auxin-binding protein 1 (ABP1) from
Zea mays. A tblastn query yielded a hit on scaffold 00327 with score=112 and E=2e-26. I then
uploaded the entire scaffold to GenSAS as a plain text file. I selected Protein blast from the tools
menu and created the task. I then received email confirmation that my task had started execution
my task started at 14:57. I received an email at 15:25 saying that my task had failed. I submitted
a detailed report of my experience with GenSAS to the developers, and it is my hope that the
problem will be solved for future researchers.
Discussion
The results of my experiments are promising. It is my hope that the primer pairs I have
provided will be useful to the breeders. My data will allow plant breeders to select individuals
that express the desired trait (large fruit size) and to track these genes through the progeny.
Hypothetically, one would subsequent generations which also expressed the large fruit
phenotype to share alleles of the genes I examined with their large-fruited forbearers. The
primer pairs that I have provided will allow the breeders to genotype progeny before they grow
to maturity that could greatly speed the breeding process. Immature plants that have the ideal
genotype for fruit size can be allowed to mature, reproduce and harvested.
There is a chance that the data that are presented here will enable the development of new
large-fruited cultivars. This would be a significant breakthrough for farmers who would be able
to increase their yields without planting more land. Of course, it could also mean more blueberry
mass per blueberry muffin, a possibility to which I doubt anyone would object.
Acknowledgements
Portions of the research for this paper were conducted jointly by the members of the spring 2013
section of BIO 343 taught by Dr. A. Malcolm Campbell, professor of biology at Davidson
College. Genotype sequence data was generated and provided by the lab of Dr. Allan Brown of
North Carolina State University. Dr. Jeannie Rowland of the USDA and Dr. Doreen Main of
Washington State University were also instrumental in the execution of this research. This
project was done in collaboration with Davidson College, NCSU, WSU, USDA and the David H.
Murdock Research Institute.
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