Genome-wide analysis of dry (tamar) date palm fruit color
Shameem Younuskunju1,5, Yasmin a.
Mohamoud1, Lisa Sara Mathew4, Klaus
F. X. Mayer5,6, Karsten Suhre3 and
Joel a. Malek1,2*
1Genomics Laboratory, Weill Cornell Medicine-Qatar,
Doha, 24144, Qatar.
2Department of Genetic Medicine, Weill Cornell
Medicine-Qatar, Doha, 24144, Qatar.
3Department of Physiology, Weill Cornell
Medicine-Qatar, Doha, 24144, Qatar.
4Clinical Genomics Laboratory, Sidra Medicine, P.O Box
26999, Doha, Qatar.
5Shool of Life Sciences, Technical University of
Munich, 85354, Munich, Germany.
6Plant Genome and Systems Biology, Helmholtz Center
Munich, 85764, Munich, Germany
*Corresponding Author:
Joel A. Malek
e-mail:
jom2042@qatar-med.cornell.edu
tel: +974-4492-8420
Abstract
Date palm (Phoenix dactylifera) fruit are an economically and culturally
significant crop in the Middle East and North Africa. There are hundreds
of different commercial cultivars producing dates with distinctive
shapes, colors, and sizes. Genetic studies of some Date palm traits have
been performed, including for date palm sex-determination, sugar content
and fresh fruit colour. In this study, we used genome sequences and
image data of 199 dry date fruit (Tamar) samples collected from 14
countries to identify genetic loci associated with the color of this
fruit stage. Here, we find loci across multiple linkage groups (LG)
associated with dry fruit color phenotype. We recover the previously
identified VIR genotype associated with fresh fruit yellow or red color
and new associations with the lightness and darkness of dry fruit. This
study will add resolution to our understanding of the date palm fruit
color phenotype especially at the most commercially important tamar
stage.
Introduction
Date palm (Phoenix dactylifera ) is one of the oldest and most
economically important fruit crops in the Middle East and North Africa
(Chao & Krueger, 2007; Weiss, Zohary, & Hopf, 2012). While there are
thousands of cultivars or varieties, likely, only a few hundred are
commercially important (Zaid & Arias-Jimenez, 1999). These cultivars
produce fruit (Dates) that have distinctive shapes, colors, and sizes of
fruit. Fruit development and ripening involve many complex biological
processes, and color changes of the fruit are closely associated with
the ripening stage (Abbas & Ibrahim, 1998). Dates have five different
development stages: Hababauk, Kimri, Khalal, Rutab, and Tamar
(Al-Mssallem et al., 2013; Siddiq & Greiby, 2013). Hababauk and Kimri
are the first two development stages, and fruit skin color is
whitish-green. In the Khalal stage, dates partially ripen and gain
maximum size and weight. During this stage, fruit color changes from
green to yellow or red depending on the cultivar. Dates fully mature in
the Rutab stage and the color begins its change to brown. Tamar is the
final stage of ripening, during which fruit water content is reduced to
less than 25%, sugar content increases to 70 to 80 % and the color
turns dark brown. During fruit ripening process amino acids act as
building blocks for the synthesis of key intermediates and end products
(Seymour, Taylor, & Tucker, 2012) and the enrichment of both free amino
acids and color or flavor-conferring phenylpropanoids was observed in
the early ripening stage of dates as in other fruits (Diboun et al.,
2015).
Dates are classified as climacteric fruit like other commercial fruit
such as apples, bananas and peach where ethylene functions as a key
regulator in fruit ripening (Abbas & Ibrahim, 1998; Al-Qurashi & Awad,
2011; Serrano, Pretel, Botella, & Amoros, 2001). Fruit ripening is
associated with an increase in respiration rate, burst of ethylene
production, oxidation process, sugar accumulation, chlorophyll break
down, pigment synthesis and other processes (Barreveld, 1993; Osorio,
Scossa, & Fernie, 2013; Stepanova & Alonso, 2005). Anthocyanin is a
class of secondary metabolite synthesized in higher plants and plays a
crucial role in pigmentation (red, pin Later k, purple & blue) in fruit
and vegetables (Kong et al., 2003; Kayesh E et al.,2013). In anthocyanin
biosynthesis, the R2R3 MYB transcription factors act as a regulator (Xie
et al., 2020). Previous studies from Hazzouri et al. (K. M.
Hazzouri et al., 2015; Khaled M. Hazzouri et al., 2019) revealed that a
retrotransposon insertion (named Ibn Majid ) and a start codon mutation
in an R2R3 MYB transcription factor encoded by the ortholog of the oil
palm VIRESCENS gene (VIR gene ), are likely the key genetic changes
leading to the red and yellow color phenotype in dates at the Khalal
stage (fresh fruit). Studies by Awad (2007) and Shareef (2020) showed
that the color transition form from yellow to brown ( Khalal-to-Rutab)
is associated with an increase in endogenous Abscisic Acid (ABA)
concentration (Awad, 2007; Shareef & Al-Khayri, 2020). A recent study
by Saar Elbar et al. provided evidence of gradual increase of
pulp ABA during the ripening stage (Khalal-to-Rutab-to-Tamar) (Elbar et
al., 2022). They also showed that the color transition from yellow to
brown (Khalal-to-Rutab) was preceded by an arrest of xylem-mediated
water transport into water.
Studies of the genetic basis of Date palm’ traits have significantly
increased due to the crop’s economic importance. Genome-wide association
studies (GWAS) are a powerful method for mapping the association between
genetic variation and phenotype (Cantor, Lange, & Sinsheimer, 2010;
Korte & Farlow, 2013). Previous GWAS in date palm identified
significant associations for fruit sugar content (Khaled M. Hazzouri et
al., 2019; Malek et al., 2020), confirmation of previous findings of the
sex determination region on LG12 (Khaled M. Hazzouri et al., 2019; Lisa
S. Mathew et al., 2014) and fresh fruit color (Khaled M. Hazzouri et
al., 2019). A goal of GWAS is to identify the association between the
variance of the phenotype of interest and genomic region or loci at
genome-wide significance. Challenges in conducting GWAS in date palm
exist including the use of clonal propagation, rare existence of outbred
populations and geographical population structure. The population
structure and cryptic relatedness have the potential to confound the
GWAS results and can lead to false discoveries (Chen et al., 2016;
Horton et al., 2012; Vilhjálmsson & Nordborg, 2013). Along these lines,
multiple studies (Chaluvadi, Khanam, Aly, & Bennetzen, 2014; Flowers et
al., 2019; Zehdi-Azouzi et al., 2015), including our own (L. S. Mathew
et al., 2015) have confirmed two major subpopulations (western and
Eastern populations) in the date palm population. Indeed, our recent
study reported that at least three and possibly four novel
subpopulations contribute to the current date palm population (Mohamoud
et al., 2019) making population structure an important consideration in
any date palm genome-wide association study. Different algorithms have
been developed to correct for population structure and increase the
computational efficiency and statistical power in GWAS (H. M. Kang et
al., 2008; Q. Wang, Tian, Pan, Buckler, & Zhang, 2014; Y. Zhang et al.,
2018). Fixed and random model Circulating Probability Unification
(FarmCPU ) is a statistically powerful and computationally efficient
GWAS method to control spurious associations (Liu, Huang, Fan, Buckler,
& Zhang, 2016; J. Wang & Zhang, 2021). The iterative usage of the
fixed effect and random effect models in the FarmCPU method incorporates
population structure and kinship matrix as covariates and eliminates the
false positive and false negative association results. We therefore
hoped to implement these methods in the challenge of GWAS in the highly
structured date palm population.
In this study, we conducted GWAS using 199 date palm samples to identify
the significantly associated genetic loci and possible candidate genes
with the color variation of Tamar stage fruit (dry fruit). The samples
used in this study are extensively diverse in their country of origin
and variety, collected from 14 countries.
Materials and Methods