Journal of Crop Improvement
ISSN: 1542-7528 (Print) 1542-7536 (Online) Journal homepage: https://www.tandfonline.com/loi/wcim20
Validation of SNP marker linked to alc gene for
long shelf life of tomato
M.K. Osei, E. Danquah, A. Danquah, M. Massoudi, D. Maxwell, H. Adu-
Dapaah & E. Blay
To cite this article: M.K. Osei, E. Danquah, A. Danquah, M. Massoudi, D. Maxwell, H. Adu-
Dapaah & E. Blay (2019) Validation of SNP marker linked to alc gene for long shelf life of tomato,
Journal of Crop Improvement, 33:5, 669-682, DOI: 10.1080/15427528.2019.1657216
To link to this article:  https://doi.org/10.1080/15427528.2019.1657216
Published online: 27 Aug 2019.
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JOURNAL OF CROP IMPROVEMENT
2019, VOL. 33, NO. 5, 669–682
https://doi.org/10.1080/15427528.2019.1657216
Validation of SNP marker linked to alc gene for long shelf
life of tomato
M.K. Oseia,b, E. Danquahb, A. Danquahb, M. Massoudic, D. Maxwelld, H. Adu-
Dapaaha, and E. Blayb
aCSIR-Crops Research Institute, Kumasi, Ghana; bWest Africa Centre for Crop Improvement (WACCI),
University of Ghana, Accra, Ghana; cAg-Biotech Lab, Monterrey, CA, USA; dDepatment of Plant
Pathology, University of Wisconsin-Madison, USA
ABSTRACT ARTICLE HISTORY
Tomato (Solanum lycopersicum L.) fruits are naturally perishable Received 21 May 2019
and have short shelf life. Post-harvest losses can be devastating Accepted 14 August 2019
and can be associated with rapid ripening. The objective of this KEYWORDS
study was to identify a single nucleotide polymorphism (SNP) Alc gene; mutant; shelf life;
marker associated with alc gene conferring long shelf life in SNP marker; tomato
tomato. The tomato line CSIR/CRI-AT06 was crossed with Alc-
LA3134 to develop an F2 population. A total of 72 plants were
screened for the target loci. Genotypic data of the F2 popula-
tion were generated following the scoring system as [T:A] for
heterozygous individuals, [A:A] and [T:T] for plants homozy-
gous for the donor and recipient parents, respectively. An
assessment of the Sly10–14 SNP marker was completed by
comparing SNP score against fruit shelf life of the 72 F2 entries.
The pattern of segregation of SNP marker for the alc gene was
tested for goodness of fit to a 1:2:1 ratio using Chi-square test
(χ2) test. The results showed that the marker segregated in the
expected ratio. The analysis from 72 F2 population plants
indicated that SNP marker (Sly 10-14) had significant associa-
tion with shelf life at 1% significance level. From the analysis, it
can be established that a locus linked to Sly10-14 is significant.
The homozygous marker (A:A) has an effect of increasing shelf
life by 22 days and explains 87% of the observed variation. This
marker (Sly10-14) is linked to a gene that controls the shelf life
of tomato fruits. The effect of this gene is additive and it
increases the shelf life.
Introduction
The tomato fruits experience post-harvest losses, primarily because they are
naturally perishable and hence have poor shelf life. Even though tomato fruits
differ in their firmness and shelf life, the growing conditions, state of maturity and
postharvest handling also can affect the shelf life (Nasrin and Molla 2009). The
lack of storage facilities and precarious transportation often lead to losses. As a
result, farmers are compelled to sell their produce at lower prices, especially in
periods of availability (Issahaku 2012). In most situations, fresh market tomatoes
CONTACT M.K. Osei oranigh@hotmail.com
© 2019 Informa UK Limited, trading as Taylor & Francis Group
670 M. K. OSEI ET AL.
are harvested at physiological maturity (but unripe) and allowed to ripen artifi-
cially to overcome the post-harvest damage attributable to mechanical, biotic or
abiotic factors (Arah et al. 2015). This practice, however, does not meet the
consumer expectation for increasing shelf life of tomato (Rodriguez et al. 2010;
Casals et al. 2011; Yogendra and Ramanjini 2013; Pech et al. 2013). Hitherto,
enhancement of the genetic constitution is the most important factor to prolong
the shelf life of the crop. Currently, there is renewed interest in tomato ripening
mutants, which delay or impede ripening, thereby extending the shelf life
(Kopeliovitch et al. 1979; McGlasson et al. 1983; Osei et al. 2017).
Apart from many ripening mutants, like rin, nor, Nr and alc, S. pimpinel-
lifolium is also used as donor for extending the post-harvest life of tomato
(Costa et al. 2013). Rin is most common in fresh-market, whereas alleles of
nor are common in the processing industry. However, the alcobaca (alc)
allele of the nor gene is an extensively used mutant in tomato to extend shelf
life (Kopeliovitch et al. 1981; Mutschler et al. 1992; Ara´Ujo et al. 2002;
Dhatt, Singh, and Dhaliwal 2003; Dias et al. 2003; Faria et al. 2003; Garg,
Cheema, and Dhatt 2008; Casals et al. 2011; Yogendra and Ramanjini 2013).
Alc mutant tomatoes are common in Spain and Portugal and date back to the
1650s (Casals et al. 2011). The fruits of alc/alc genotype are characterized by
reduced carotenoid content, increased firmness and increased shelf life (Faria
et al. 2003). The effects are additive, and use of the allele in the heterozygous
condition can extend shelf-life and minimize fruit loss. At the molecular
level, thymine is replaced by adenine at position 317 in the wild type ALC
gene (Casals et al. 2011).
The use of molecular markers linked to genes for increased shelf life can
provide an efficient tool for plant breeding. This methodology accelerates transfer
of the genes into a cultivar throughmarker-assisted backcrossing compared to the
traditional methods, which are time consuming and laborious (Pratta et al. 2011).
Markers tightly linked to the genes of interest can distinguish the heterozygous
plants from the homozygous plants in segregating generations, thereby facilitating
the development of new long shelf life lines throughmarker-assisted backcrossing.
Thus, the molecular markers linked to the alc allele for prolonging shelf life can be
exploited in a marker-assisted selection (MAS) scheme. Hence, the present study
used an F2 population of tomato to validate the alc SNPmolecular marker, Sly10–
14, for use in breeding programs for long shelf life.
Materials and methods
Plant material
Parental lines CSIR/CRI-ATS06 (ALC/ALC) and Alc-LA3134 (alc/alc) of
Solanum lycopersicum with contrasting phenotypes with regards to firm-
ness/shelf life were obtained from CSIR-CRI, Kumasi, Ghana, and Tomato
JOURNAL OF CROP IMPROVEMENT 671
Genetics Resource Center, University of California-Davis, USA, respectively.
The lines were first crossed in April 2018 to produce F1 plants. The F2
generation was derived by selfing F1 plants in the greenhouse in August 2018.
SNP markers used for the parental polymorphism survey
In a previous study, 140 SolCAP SNPs were selected on the basis of their physical
distribution on each chromosome. These were tested on four Ghana breeding
lines and three long shelf life accessions (LA3134, LA1795, and LA3770) from
Tomato Genetic Center, University of California Davis. A subset of 22 SNP
markers that were polymorphic and located on different chromosomes was tested
for polymorphism between CSIR/CRI-ATS06 and Alc-LA3134 (Table 1).
Information on the primers is a trade secret of Ag-Biotech. These markers were
associated with chromosomes 2 to 12. Most of the markers were designed from
SolCAP SNPs (M. Massoudi, Personal communication, 2017). The Sly10–14 was
designed using the A/T SNP associated with the alc gene (Casals et al. 2011).
Molecular marker analysis of F2 population
Leaf sample collection, DNA extraction, and SNP marker analysis
A total of 88 F2 plants were grown in a greenhouse and were subjected to
screening using SNP alc marker, Sly10–14, together with other SNP markers
Table 1. List of single nucleotide polymorphism (SNP) markers used for parental polymorphism
evaluation.
No. SNP marker Chromosome no. Position (million nt) Locus
1. Sly02-9 2 54.031 Solyc02g092250
2. Sly03-8 3 66.529 Solyc03g115250
3. Sly04-7 4 45.179 Solyc04g050040
4. Sly05-5 5 9.768 Solyc05g015050
5. Sly05-9 5 60.740 Solyc05g050010
6. Sly06-1 6 1.672 Solyc06g007670
7. Sly06-6 6 33.047 Solyc06g050350
8. Sly06-7 6 41.280 Solyc06g065720
9. Sly07-1 7 1.820 Solyc07g007040
10. Sly08-1 8 0.035 Solyc08g005050
11. Sly08-8 8 53.294 Solyc08g065320
12. Sly08-9 8 56.001 Solyc08g067030
13. Sly09-1 9 0.048 Solyc09g005080
14. Sly10-1 10 0.476 Solyc10g005600
15. Sly10-alc 10 1.300 Solyc10g006880
16. Sly10-11 10 56.667 Solyc10g055410
17. Sly11-06 11 19.229 Solyc11g027880
18. Sly11-11 11 52.324 Solyc11g066300
19. Sly11-13 11 53.921 Solyc11g069050
20. Sly11-Rx4 11 53.876 Solyc11g069020
21. Sly12-09 12 63.959 Solyc12g056940
21. Sly12-10 12 67.286 Solyc12g098960
672 M. K. OSEI ET AL.
(Table 1). Fresh leaf tissues were harvested from the 88 individuals of the F2
population. They were placed in known positions of a 96 deep-well plate and
shipped to Ag-Biotech, Inc. via USDA-APHIS office in San Francisco, USA.
Samples of parents were also included in each plate as controls. Genomic
DNA (approximately 50 mg) of young leaf tissue from each plant was
extracted by using CTAB protocol (Saghai-Maroof et al., 1994; Doyle and
Doyle 1990). Approximately 50 ng/µL of DNA was prepared from each
sample for SNP genotyping. The SNP-based detection method was similar
to the Kompetitive Allele Specific PCR (KASP) genotyping platform (LGC
Genomics, Beverly, MA, USA). SNPs were assayed using KASP assay
(Semagn et al. 2014). All SNP assays were performed at Ag-Biotech, Inc.
Genotyping of F2 population
Genotypic data of the F2 population were generated following the scoring
system as [T:A] for heterozygous individuals carrying both alleles at the
target locus, [A:A] for plants homozygous for the allele of donor parent,
[T:T] and for plants homozygous for the allele of recipient parent.
Phenotyping of F2 population for fruit shelf life and statistical analyses of
the data
The F2 plants and their parents were grown in a greenhouse at the Kwadaso
research station of the CSIR-Crops Research Institute, Kumasi, Ghana during
August 2018. Random samples of five fruits per plant were harvested at breaker
stage and placed on shelves in a ventilated room at 26.7 oC – 29.1°C to determine
shelf life. The relative humidity ranged from 51% to 89%. Fruits were discarded at
24-h intervals at the first visual sign of deterioration, i.e. a slight wrinkling of the
fruit skin caused by fruit desiccation (Schuelter et al. 2003). The process of
discarding the fruits continued till the last fruit became unmarketable. To identify
the tomato lines with prolonged shelf life, the time (in days) taken by fruits from
matured color stage until the firmness is lost was recorded.
The segregation ratios of marker Sly10-14 associated with alc gene in the F2
progenies were subjected to Mendelian genetic models using the Chi-square (χ2)
test (Fisher 1922). SNP-marker data for the F2 individuals and their corresponding
phenotypic values were used for single-locus analysis through one-way ANOVA
using R statistical package to establish maker-phenotype association. Statistical
analyses were conducted using the linear model function “lm” in the R Core
package (R Core Team 2017). The simple marker-trait model described by
Edwards, Stuber, and Wendel (1987) was applied as:
yijk¼ μþMiþei
JOURNAL OF CROP IMPROVEMENT 673
where yi = the shelf-life rating for the i
th F2 progeny within marker (Mi). The
term ei represents the marker within genotype variance or error for the
model.
Results
Parental polymorphism survey
Of the 22 SNP markers used for parental polymorphism survey, only 19 were
polymorphic between the parents (CSIR/CRI-ATS06 and Alc-LA3134). All the 19
SNP markers, however, produced heterozygous F1s as expected, except SNP
markers Sly02-9 and Sly11-13. For Sly08-8 marker, the genotype of the donor
entry was heterozygous and the F1 was also heterozygous (Table 2).
SNP genotyping and phenotyping for shelf life for the F2 population
Phenotyping for long shelf life was carried out by considering the number of
days elapsed for the tomato fruit to deteriorate. Of the 88 F2 plants, six plants
died and 10 plants did not fruit. Table 3 contains overall assessment of Sly10-
14 (SNP score) versus shelf life of 72 F2 plants whose fruits were evaluated for
storage. Clearly, almost all the F2 lines with marker genotypes T:A and A:A
had corresponding long shelf life compared with T:T marker genotypes.
Table 2. Parental polymorphism survey between parents.
Marker/Entry Alc-LA3134 CSIR/CRI-ATS06 F1 generation Remarks
Sly02-9 A:A† T:T T:T PM¶
Sly03-8 A:A T:T T:A PM
Sly04-7 A:A T:T T:A PM
Sly05-5 A:A T:T T:A PM
Sly05-9 T:T‡ A:A T:A PM
Sly06-1 T:T A:A T:A PM
Sly06-6 T:T A:A T:A PM
Sly06-7 A:A T:T T:A PM
Sly07-1 T:T A:A T:A PM
Sly08-1 A:A T:T T:A PM
Sly08-8 T:A§ T:T T:A PM
Sly08-9 A:A T:T T:A PM
Sly09-1 T:T A:A T:A PM
Sly10-1 T:T A:A T:A PM
Sly10-alc A:A T:T T:A PM
Sly10-11 A:A T:T T:A PM
Sly11-13 T:T A:A T:T PM
Sly11-Rx4 A:A T:T T:A PM
Sly12-10 A:A T:T T:A PM
†A: A = Homozygous marker genotype.
‡T: T = Homozygous marker genotype.
§T: A = Heterozygous marker genotype.
PM = Polymorphic marker.
674 M. K. OSEI ET AL.
Table 3. Assessment of Sly10-14 (SNP score) against shelf life of F2 lines.
Shelf life
(days) Overall
Entry SNP score Shelf life (days) Overall assessment¶ Entry SNP score assessment
Parents F2 lines
CSIR/CRI-ATS06 T:T† 14 SSL F2 (E1)-45 T:T 21 SSL
Alc-LA3134 A:A‡ 47 LSL F2 (E1)-46 A:A 43 LSL
F2 lines F2 (E1)-47 A:A 55 LSL
F2 (E1)-1 T:A§ 39 LSL F2 (E1)-48 T:A 39 LSL
F2 (E1)-2 T:T 21 SSL F2 (E1)-49 T:T 18 SSL
F2 (E1)-3 A:A 41 LSL F2 (E1)-50 T:T 16 SSL
F2 (E1)-4 T:A 38 LSL F2 (E1)-51 T:A 40 LSL
F2 (E1)-5 T:A 40 LSL F2 (E1)-52 A:A 41 LSL
F2 (E1)-6 T:T 16 SSL F2 (E1)-53 T:A 28 SSL
F2 (E1)-7 T:T 12 SSL F2 (E1)-56 T:A 29 SSL
F2 (E1)-8 T:A 40 LSL F2 (E1)-57 T:T 18 SSL
F2 (E1)-9 T:A 40 LSL F2 (E1)-59 T:T 21 SSL
F2 (E1)-11 A:A 49 LSL F2 (E1)-60 T:T 16 SSL
F2 (E1)-13 T;A 39 LSL F2 (E1)-61 T:T 20 SSL
F2 (E1)-14 T:T 18 SSL F2 (E1)-62 T:T 16 SSL
F2 (E1)-16 A;A 52 LSL F2 (E1)-63 T:A 40 LSL
F2 (E1)-17 T:A 55 LSL F2 (E1)-65 T:A 38 LSL
F2 (E1)-18 T:T 14 SSL F2 (E1)-66 T:T 14 SSL
F2 (E1)-19 T:A 33 LSL F2 (E1)-67 T:A 39 LSL
F2 (E1)-20 T:A 37 LSL F2 (E1)-69 T:A 38 LSL
F2 (E1)-21 A:A 48 LSL F2 (E1)-70 T:A 38 LSL
F2 (E1)-23 T:T 16 SSL F2 (E1)-71 T:A 38 LSL
F2 (E1)-24 T:T 14 SSL F2 (E1)-72 T:A 38 LSL
F2 (E1)-25 T:A 52 LSL F2 (E1)-73 T:T 14 SSL
F2 (E1)-26 A:A 48 LSL F2 (E1)-74 T:A 40 LSL
F2 (E1)-27 A:A 44 LSL F2 (E1)-75 T:T 16 SSL
F2 (E1)-28 T:T 19 SSL F2 (E1)-76 A:A 44 LSL
F2 (E1)-30 T:T 21 SSL F2 (E1)-77 T:A 40 LSL
F2 (E1)-31 T:A 47 LSL F2 (E1)-78 A:A 51 LSL
F2 (E1)-32 T:A 33 LSL F2 (E1)-79 T:T 18 SSL
F2 (E1)-34 T:T 21 SSL F2 (E1)-80 T:T 17 SSL
F2 (E1)-35 T:A 36 LSL F2 (E1)-81 T:A 40 LSL
F2 (E1)-38 T:A 48 LSL F2 (E1)-83 T:T 16 SSL
F2 (E1)-39 T:T 19 SSL F2 (E1)-85 T:A 42 LSL
F2 (E1)-40 T:A 39 LSL F2 (E1)-86 T:A 42 LSL
F2 (E1)-41 T:T 18 SSL F2 (E1)-88 nil 40 LSL
F2 (E1)-42 A:A 40 LSL
F2 (E1)-43 A:A 40 LSL
F2 (E1)-44 A:A 44 LSL
†T:T = Homozygous marker genotype.
‡A:A = Homozygous marker genotype.
§T:A = Heterozygous marker genotype.
SSL = Short Shelf Life; LSL = Long Shelf Life.
Chi-square test of F2 population
The SNPmarker for the alc genewas tested for goodness of fit in a 1:2:1 ratio using
Chi-square test. The Chi-square value of 1.46 was found to be statistically non-
JOURNAL OF CROP IMPROVEMENT 675
Table 4. Chi-square test for testing goodness fit of F2 pop for Mendelian segregation ratios.
Expected χ2 =
Genotypic class Observed frequency (O) Frequency (E) O-E (O-E)2 ∑ [(O-E)2/E]
Recipient (CSIR/CRI-ATS06) 26 22.25 3.75 14.06 0.63
Recombinants 45 44.5 0.5 0.25 0.01
Donor (Alc-LA3134) 18 22.25 −4.25 18.06 0.82
Total 89 89 1.46
Table 5. One-way ANOVA for marker-trait association.
Df Sum Sq. Mean Sq. F value Pr (>F)
Genotype 2 9910 4955 246.5 <2e-16 ***
Residuals 68 1367 20
*** Significant at the 0.001 probability level.
significant between the expected and observed ratio for the alc SNP marker, for 2
degrees of freedom against the tabulated value. The results showed that themarker
segregated in the expected ratio (Table 4).
Tabulated χ2 value at 2 degrees of freedom: 5.991 at 5% probability level.
Marker association with alc gene for long shelf life
The phenotypic data collected from the screenhouse were combined with geno-
typic data to determine if the Sly10-14 marker was linked with long shelf life
(Table 3). The analysis from the 72 plants of the F2 population with the three SNP
marker alleles (A:A, T:A, T:T) indicated that SNP marker (Sly 10-14) had sig-
nificant association with shelf life at 1% level of significance (Table 5).
Mean comparisons among the three marker genotypes using LSD test at 5%
significance level are shown in Table 6. Significant differences existed among the
three genotypes, A:A (Alc-LA3134), T:T (CSIR/CRI-ATS06) and T:A. Long shelf
life of 46 dayswas recorded for theA:A genotype, whereas T:T genotype had a shelf
life of only 17 days. The heterozygous genotype could, however, be stored for 23
days longer than the T:T genotype and 6 days less than theA:A genotype (Table 6).
Table 7 contains multiple linear regression models to estimate additive and
dominance effects of the SNPmarker. From the adjusted R squared value based on
the number of observations, 87.5% of the variance can be explained by the SNP
marker Sly 10-14 for alc gene relative to shelf life. An estimated value of 31.5 days
represented the intercept (mid-parent value), whereas 14.16 and 7.96 days repre-
sented additive and dominance effect, respectively, indicating significance level of
1%. The heterozygous genotype, which gave 39.5 days (Table 6) of shelf life was
higher than the mid-parent value (intercept) of 31.5 days (Figure 1). Also, sub-
stitution with the A-allele in T:T genotype resulted in increased shelf life of about
23 days.
676 M. K. OSEI ET AL.
Table 6. Mean comparisons of shelf life (days) among the three marker genotypes.
Marker Genotype Shelf life Std. r LCL‡ UCL§ Min Max
A:A 45.7a† 4.81 14 43.32 48.11 40 55
A:T 39.5b 5.48 31 37.91 41.12 28 55
T:T 17.4c 2.56 26 15.63 19.14 12 21
†Treatments with the same letter are not significantly different at the 5% probability level (DF Error: 68;
Critical value of t: 1.995469).
‡LCL = lower control limit.
§UCL = upper control limit.
Table 7. Model summary and Coefficient
Table 7a Model summary
Model R R Square Adjusted R Square Standard error of estimate
1 0.9374 0.8788 0.8752 4.483
Table 7b Coefficient
Estimate Std. Error t value Pr (>|t|)
Intercept 31.5495 0.7431 42.457 < 2e-16 *** mid-parent value
add1 14.1648 0.7431 19.062 <2e-16 *** additive effect
dom1 7.9667 1.0957 7.271 4.6e-10 *** dominance effect
*** Significant at 0.001 probability level.
Discussion
Parental survey for polymorphism among the SNP markers
The use of molecular markers in tomato breeding programs has increased the
efficiency and accelerated the transfer of desirable genes among tomato
varieties and the introgression of novel genes from related wild species
(Karlik and Tombuloglu 2016; Osei et al. 2018). Screening of markers for
parental polymorphism among tomato cultivars forms the basis for tagging
of desired gene and subsequent marker-assisted introgression of the gene
through backcross breeding, involving foreground and background selection
(Osei et al. 2018). These parental polymorphic markers were identified on
target chromosomes so that they can be utilized in background selection
during marker-assisted transfer of the target gene in the genetic background
of recurrent parents.
Additive
towards
Mid-parent                TA [partial dominance]
TT AA
(17 days) 31.5 39.5 (46 days)
Figure 1. Flow chart of gene action.
JOURNAL OF CROP IMPROVEMENT 677
In this study, 22 SNPs were examined for polymorphism against the
parents. Nineteen of these markers were able to discriminate between geno-
types of the Alc-LA3134 and CSIR/CRI-ATS06; thus, endorsing the contrast-
ing nature of the parents and providing fundamental evidence about the
usefulness of the SNP markers in a backcross program. The allelic variations
observed within the genome of these parents were made possible with the
help of SNP markers. SNP markers are high-throughput sequence-based
markers. They have, across years, proved to be universals as well as the
most abundant forms of genetic variation among individuals of the same
species (Parida et al. 2012). The validation of a marker is the process of
designing an assay based on the discovered polymorphism and then geno-
typing a panel of diverse germplasm and segregating population
(Mammadov et al. 2014). Even though Sly08-8 showed polymorphism
between the two parents, the genotype marker for the Alc-LA3134 (X: Y)
was heterozygous instead of a homozygous; thus, it was not a worthwhile
polymorphic SNP marker. The parental polymorphic SNP markers identified
in the present study to be polymorphic between donor-recipient parent
combinations will be highly useful for deployment in background selection
aimed at introgression of alc gene into the genetic background of the CSIR/
CRI-ATS06.
Identification of SNP marker associated with the putative locus for shelf
life in the F2 population
The marker Sly10-14 revealed that the crosses between Alc-LA3134 and
CSIR/CRI-ATS06) were successful because this marker was able to detect
heterozygous marker genotypes in the progenies. These identified genotypes
are useful for an advanced breeding program as selections could be made of
lines to advance. The A/T SNP, which is shown to be a useful molecular
marker for breeding long shelf life tomatoes in this study, is in the NAC
domain of the NAC-NOR gene. This gene codes for an NAC domain
transcription fraction and the NAC domain functions in DNA binding as
well as in dimerization with other NAC proteins (Olsen et al. 2005). This
replacement of a T by an A in the NAC-NOR gene results in the replace of
thymine with adenine in the NAC domain. This mutated transcription factor
then results in the physiological changes in lower ethylene emission and
carotenoid levels as well as the down-regulation of several cell-wall modifying
genes (Kumar et al. 2018). These physiological changes are then associated
with increase in shelf life of the tomato fruit.
For effective use of the marker in selecting long shelf life genotypes with-
out false positives, Chi-square test was used. Non-significance of Chi-square
test values indicated that the marker followed expected Mendelian inheri-
tance pattern. This is in agreement with Pushpa et al. (2015), whose work
678 M. K. OSEI ET AL.
validated molecular markers linked to low glucosinolate quantitative trait loci
(QTL) and after a Chi-square test determined that there was no significance
of the test values. Chi-square test clearly showed that Sly10-14 marker
segregated in the expected ratio of 1:2:1 in the F2 population. According to
Sayed et al. (2002) and Xu et al. (1997), markers will generally segregate in a
Mendelian fashion, while occasionally distorted segregation ratios may be
encountered. The analysis further indicates that the trait under study exhibits
a polygenic segregation for long shelf life and poor shelf life phenotype. It is
noteworthy that, in this study, the genes behaved as expected.
In the present study, the phenotype of the F2 population derived from Alc
LA3134 and CSIR/CRI-ATS06 was determined. It was interesting to note that
entries that did not have the A-SNP allele produced poor shelf life, thus confirm-
ing the importance of introgressing the A-SNP allele of the alc gene into an
adapted tomato line. In other words, almost all the F2 individuals that had or
received the A-SNP allele showed increased shelf life compared with those with
TT-SNP alleles, which are associated with the maternal parent. This explains that
when an A-SNP allele of Alc-LA3134 is introduced into an adapted line with TT-
SNP alleles, shelf life is prolonged. This is in agreement with Pratta et al. (2011),
who also reported the presence of favorable alleles for fruit keeping quality in the
cultivated genotypes of tomato. To this effect, those lines in the F2 individuals that
had AA-SNP alleles showed prolonged shelf life. This is consistent with the report
of Kalenahalli and Gowda (2012), where the use of ripening mutants, such as
alcobaca (alc) tomato line, in crosses with other lines prolonged the shelf life. It is
therefore not surprising to see those lines in the F2 population with TA and/or AA
genotypes exhibiting high shelf life tomatoes.
Even though plants with TA or AA genotypes were reported in this study
to show longer shelf life than individuals with the AA marker genotypes, the
plants with AA genotypes had longer shelf life than those with TA marker
genotypes. This observation may be explained based on the fact that those F2
plants with the AA marker genotype had the same marker genotype as the
donor (Alc-LA3134) used in the crosses. Our results are consistent with those
of other reports (Sinha et al. 2014; Dias et al. 2003; Kopeliovitch et al. 1980),
where shelf life was increased when the alc gene was present.
Marker association with alc gene for long shelf life
To identify the association of polymorphic markers with the trait, single-marker
analysis was performed using R in the F2 population. An attempt wasmade to find
the putative SNP alleles associated with the shelf life traits, which could further
assist in marker-assisted backcross selection of genotypes. The analysis showed
that Sly10-14 marker had a strong link with the shelf life trait. This observation
strongly suggests the possibility of a significant putative genetic locus associated
with long shelf life on chromosome 10 near or at the location of the marker Sly10-
JOURNAL OF CROP IMPROVEMENT 679
14. The results of the present study validate the genomic location of the Sly10-14 to
be associated with long shelf life. This marker Sly10-14 supplied by Ag-Biotech is
novel in the identification of shelf life gene alc on the short armof chromosome 10.
Further attempt was made to estimate additive and dominance effects of
the SNP marker using multiple linear regression models. The adjusted R-
square value of 87.5% of the variance can be explained by the SNP marker Sly
10-14 for alc expressed in shelf life. With an increase of the TA genotype over
the mid-parent value (intercept), it can be proposed that partial dominance
of gene action is at play. Besides, substitution with the A-SNP allele in TT
genotype gave an increase of about 22 days in shelf life. This suggests that
any time an A-SNP allele is introduced or substituted in the TT genotype,
shelf life of that genotype would be improved by 3 weeks. It therefore
suggests gene action effect is additive but toward partial dominance.
According to Mutschler (1984), the altered ripening behavior and improved
storage quality in “Alcobaca” fruits are controlled by a single recessive gene “alc”.
Knowledge of gene action and relative amounts of additive and non-additive
genetic variance present in cross combinations will lead to successful results in any
future hybrid breeding. Moreover, insight into the nature of gene action involved
in the expression of various quantitative characters is essential to a plant breeder
for starting a judicious breeding program. An understanding of how heterozyg-
osity and homozygosity affect gene action and interaction will facilitate decisions
about whether the end product in breeding programs should be hybrids or inbred
lines. In this study, a preponderance of additive gene action was noticed and
according to Arunachalam (1984), reliance should be placed on mass selection
and progeny selection in self-pollinated species like tomatoes.
Conclusion
Considering the molecular analysis in Alc-LA3134 × CSIR/CRI-ATS06 (F2) of
tomato, 19 of the 22 SNP markers were found to be polymorphic. From the
analysis, it can be established that a locus linked to Sly10-14 marker was signifi-
cantly associated with shelf life (P-value = 0.0001). It had an effect of increasing
shelf life by 22 days (i.e. difference between TA_39 days and TT_17 days) and
explained 87% of the observed variation. This marker Sly10-14 was linked to a
gene that controls the shelf life of tomato fruits and this gene was additive in
nature and acted in a semi-dominant manner, increasing shelf life by 22 days.
This study described the validation of Sly10-14 SNP marker linked to shelf life
using F2 population. Hence, this marker will be useful for marker-assisted back-
crossing breeding in tomato to improve the shelf life of tomato fruits.
Additionally, this is the only known SNPmarker available for tagging the alc gene.
680 M. K. OSEI ET AL.
Acknowledgments
The authors thank the staff of Ag-Biotech for their enormous assistance regarding DNA
extraction and SNP marker analysis. This work was supported by Ag-Biotech Lab.,
Monterrey, CA, USA, and Syngenta.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Ag-Biotech Inn, Monterrey, CA, USA [Nil];Syngenta BV
[Nil].
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