Published October 6, 2016
o r i g i n a l r es e a r c h
AGHmatrix: R Package to Construct Relationship
Matrices for Autotetraploid and Diploid Species:
A Blueberry Example
Rodrigo R. Amadeu, Catherine Cellon, James W. Olmstead, Antonio A. F. Garcia,
Marcio F. R. Resende Jr., and Patricio R. Muoz*
Abstract
Progress in the rate of improvement in autopolyploid species has
been limited compared with diploids, mainly because software
and methods to apply advanced prediction and selection meth-
odologies in autopolyploids are lacking. The objectives of this
research were to (i) develop an R package for autopolyploids to
construct the relationship matrix derived from pedigree informa-
tion that accounts for autopolyploidy and double reduction and
(ii) use the package to estimate the level and effect of double
reduction in an autotetraploid blueberry breeding population with
extensive pedigree information. The package is unique, as it can
create A-matrices for different levels of ploidy and double reduc-
tion, which can then be used by breeders to fit mixed models in
the context of predicting breeding values (BVs). Using the data
from this blueberry population, we found for all the traits that tet-
rasomic inheritance creates a better fit than disomic inheritance.
In one of the five traits studied, the level of double reduction was
different from zero, decreasing the estimated heritability, but it did
not affect the prediction of BVs. We also discovered that different
depths of pedigree would have significant implications on the
estimation of double reduction using this approach. This freely
available R package is available for autopolyploid breeders to
estimate the level of double reduction present in their populations
and the impact in the estimation of genetic parameters as well as
to use advanced methods of prediction and selection.
Published in Plant Genome
Volume 9. doi: 10.3835/plantgenome2016.01.0009
Crop Science Society of America
5585 Guilford Rd., Madison, WI 53711 USA
This is an open access article distributed under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
the pl ant genome november 2016 vol . 9, no . 3 1
Core Ideas
We developed an R package for autopolyploids to
construct the relationship matrix.
We estimated the level and effect of double reduction
in blueberry.
The package is unique as it can create A-matrices for
different levels of ploidy.
The package can create A-matrices for different levels
of double reduction
S
everal important agronomic and horticultural
crops such as alfalfa (Medicago sativa L.), potato
(Solanum tuberosum L.), and highbush blueberry (Vac-
cinium corymbosum L.) are autotetraploids, with four
homologous chromosomes present in each linkage group.
As more than two homologous chromosomes exist in
autotetraplopids, during meiosis chromosome pairing can
occur between a pair of randomly chosen chromosomes
(bivalents) or between more than two homologous chro-
mosomes (multivalents) (Fisher, 1947). This polysomic
R.R. Amadeu and A.A.F. Garcia, Dep. of Genetics, Luiz de Queiroz
College of Agriculture, Univ. of Sao Paulo, Av. Padua Dias 11, P.O.
Box 87, 13400-970, Piracicaba, SP, Brazil; C. Cellon and J.W. Ol-
mstead, Horticultural Sciences Dep., Institute of Food and Agricultural
Sciences, Univ. of Florida, P.O. Box 110690, Gainesville, FL 32611,
USA; M.F.R. Resende Jr., RAPiD Genomics LLC, 756 2nd Avenue,
Gainesville, FL, 32601, USA; P.R. Muoz Agronomy Dep., Institute of
Food and Agricultural Sciences, Univ. of Florida, P.O. Box 110965,
Gainesville, FL 32611, USA. R.R. Amadeu and C. Cellon contributed
equally to the manuscript. Received 25 Jan. 2016. Accepted 10
June 2016. *Corresponding author (p.munoz@ufl.edu). Assigned to
Associate Editor Jesse Poland.
Abbreviations: AIC, Akaike information criteria; BLUP, best linear
unbiased prediction; BV, breeding value; REML, restricted maximum
likelihood.
of 10
inheritance results in different segregation ratios for a
given locus compared with diploid species, as up to four
copies of an allele lead to the possibility of higher orders
of allele interaction (see details in Gallais, 2003). Thus,
considering a biallelic marker, there are five possible states
for a given A allele (referred hereafter as dosage): nulli-
plex, simplex, duplex, triplex, and quadruplex represent-
ing aaaa, Aaaa, AAaa, AAAa, and AAAA, respectively.
Moreover, the phenomenon known as double reduction,
where sister chromatids segregate into the same gamete,
creates difficulties when studying quantitative or popula-
tion genetics in autotetraploid species. Despite the fact
that double reduction is not well studied, its occurrence
has practical implications in plant breeding because it can
result in increased inbreeding even when two unrelated
individuals are crossed (Mather, 1935; Mather, 1936; Gal-
lais, 2003; Kerr et al., 2012). These challenges, and the fact
that these differences are often ignored in breeding strate-
gies, have been identified as the main reasons why the rate
of genetic improvement has been limited and sometimes
null in autotetraploid species as compared with their dip-
loid counterparts (Brummer, 1999; Katepa-Mupondwa et
al., 2002; Jansky, 2009). As an example, the genetic gain
for yield in alfalfa since 1940 has been between 0 and
0.30% yr1 on average, while 2% has been estimated in
annual diploid crops (Brummer, 1999).
Breeding autotetraploid species also typically take
longer than diploid species, and the development of
an improved cultivar can take between 10 and 20 yr
(Lyrene, 2008; Bradshaw and Bonierbale, 2010). One
reason is that it takes longer for autotetraploids to elimi-
nate deleterious alleles because of the greater number
of possible states (i.e., Aaaa, AAaa, and AAAa) (Gallais,
2003). Even though progress has been made in these spe-
cies, materialized in the many cultivars released, current
improvement strategies (e.g., phenotypic recurrent selec-
tion) and analysis methods are rudimentary and known
to be inefficient (Slater et al., 2013).
One of such autotetraploid species is blueberry,
including highbush, southern highbush (V. corymbosum
interspecific hybrids), and lowbush (V. angustifolium L.).
Blueberries are an economically important small fruit
crop for the United States. In 2014, 0.25 Tg (543.5 million
pounds) of blueberries were harvested for fresh produc-
tion and valued at $824.9 million (USDA, 2015). United
States production has increased an average of 20% every 2
yr since 2008, while acreage has increased by nearly 75%
between 2005 and 2012. This increase is based in part on
the reputation of blueberry fruit and products as health
foods because of the high amounts of antioxidants such
as anthocyanins. Antioxidants have been correlated with
beneficial health effects in numerous biological systems
including immunology, ophthalmology, cardiology, neu-
rology, metabolism, and inflammation (Cassidy et al.,
2013; Fan et al., 2012; Heim et al., 2012; Krikorian et al.,
2010; Li et al., 2013; Liu et al., 2012; McAnulty et al., 2011;
Shukitt-Hale, 2012; Tipton et al., 2013; Yousef et al., 2013).
2 of 10 the pl ant genome
The blueberry breeding program at the University of
Florida develops southern highbush blueberry cultivars
(2x = 4n = 48). These hybrid cultivars are derived mainly
from crosses between northern highbush tetraploid cul-
tivars (V. corymbosum) and diploid species (V. darrowii),
with lesser contributions from various other Vaccinium
species such as V. arboreum, V. elliottii, and V. virgatum
(Brevis et al., 2008; Lyrene 1997, 2008; Olmstead et al.,
2013). Many traits under selection in this species are
polygenic (Lyrene 1993) and the few inheritance studies
of tetraploid V. corymbosum and V. corymbosum V.
darrowii crosses have shown inheritance patterns that
follow expected autotetraploid segregation ratios (Draper
and Scott, 1971; Krebs and Hancock, 1989; Qu and Han-
cock, 1995). Additionally, the degree of double reduc-
tion present in blueberry is not yet known, and previous
reports were not conclusive (Krebs and Hancock, 1989).
The use of quantitative genetic methods for breed-
ing of blueberries or any other autotetraploid plant is not
as developed as diploid species. In conventional diploid
breeding, the use of linear mixed models, popularly
known as best linear unbiased prediction (BLUP), is a
method used to estimate variance components through
restricted maximum likelihood (REML) and predict
BVs. This methodology was developed by Henderson
in the 1950s (Van Vleck, 1998) and was first applied to
livestock breeding (VanRaden, 2008). In the past two
decades, BLUP has gained popularity in plant breeding,
first with perennial and forestry crops and more recently
with annual crops (Piepho et al., 2008). In this period,
the applicability of BLUP has been tested and adopted
in breeding diploid crops (Bernardo, 1994, 1996; Crossa
et al., 2010; Resende et al., 2012; Massman et al., 2013).
However, its use in many polyploidy species, such as
blueberries, has not been explored yet.
One of the main characteristics in the use of REML
and BLUP in breeding is the use of a numerator matrix
(A) containing an estimate of the pairwise relationship
among individuals. This information is used to model
the variance covariance between the populations. Kerr
et al. (2012) developed an extension of the numerator
relationship matrix based on pedigree values (A-matrix)
for autopolyploids. This calculation includes the ploidy
level and w coefficient (proportion of the alleles which
are identical by descent, double reduction). Using this
new tetraploid A-matrix, Slater et al. (2013) found that
higher genetic gains could be obtained for quantitative
traits in autotetraploid potato when compared with the
traditional phenotypic selection. However, there is cur-
rently no software available to construct such matrices so
that autotetraploid breeders can test and implement these
new and more efficient selection methods.
The objective of this study was two-fold: (i) to develop
an R package for construction of an autotetraploid rela-
tionship matrix that considers autopolyploidy and double
reduction derived from pedigree information and (ii)
to estimate the level of double reduction present in the
University of Florida blueberry breeding germplasm and
november 2016 vol . 9, no . 3
to study its effects on model fitting and genetic param-
eter estimations. This information will allow the future
implementation of BLUP and REML methods to estimate
BVs in the breeding program, study of genetic diversity,
inbreeding, definition of heterotic groups, etc.
Materials and Methods
Blueberry Breeding Population and Pedigree
The southern highbush blueberry population used in
this study was generated from the breeding program at
the University of Florida. This population was developed
through 124 controlled crosses of 148 selected parents
made in February 2011. Seeds were extracted from the
resulting mature fruit, cold stratified for 5 mo, and
planted in November 2011 as a family in 2-L pots in a
greenhouse. One hundred seedlings from each family
were transplanted to trays in January 2012 and planted
in May 2012 in a high-density (~20,000 plants per 0.2
ha) nursery at the Plant Science Research and Education
Unit in Citra, Florida. In May 2013, five to 32 plants were
selected from each family, and the remaining plants were
removed from the planting, leaving 1996 plants.
The pedigree of the University of Florida blueberry
breeding program was assembled using internal pedi-
gree records, the NCGRCorvallis Vaccinium Catalog
(National Center for Genome ResourcesCorvallis, 2012),
and the Brooks and Olmo Register of Fruit and Nut Vari-
eties (Brooks and Olmo, 1952, 1997). The file was ordered
chronologically beginning with the founding parents in
1908. The pedigree file contains 7755 lines with anno-
tated pedigree since 1908.
Phenotyping
Phenotypic data collected for selected seedlings in
2014 and 2015 included plant yield, fruit weight, fruit
diameter, fruit firmness, and fruit stem scar diameter.
Yield was measured using a 1-to-5 rating scale, where
1 equaled none to very few berries on the plant and 5
equaled a yield comparable with commercial cultivars
of the same age plant. Yield ratings were recorded over a
2-wk period before the blueberries were fully mature.
Fruit traits were determined using five randomly
sampled representative berries from each genotype.
Because of the diversity of harvest timing among the
individual selections, the berries were collected over a
6-wk period from the beginning of April to the middle
of May in each year. The total sample of berries was hand
harvested from each genotype when they were fully
mature and had no insect or visual damage. After har-
vest, the berries were kept in a cooler and stored at 4C
overnight prior to analysis. Weight (g) was measured on
each individual berry using an analytical scale (CP2202S,
Sartorius Corp.). The same five berries were oriented
equatorially for fruit size diameter (mm) and firmness
(g mm1 compression force) measurement using a Firm-
Tech II (BioWorks Inc.). The minimum and maximum
force thresholds were set at 50 g and 350 g, respectively.
a m adeu et al .: aghm atrix : r pack age to construct rel ationshi p m atri ces 3
Subsequently, each of the berries was placed with the
stem scar positioned upward on a tray in a light box
with a digital SLR (Pentax K-x, Ricoh Imaging) camera
positioned 50 cm above the berries. The camera was set
at a shutter speed of 1/50, aperture F 5.6, and ISO 200. A
ruler was placed in each image as a size reference. Subse-
quently, the images were uploaded into FIJI (Schindelin
et al., 2012) and the scale was set using the ruler. Scar
diameter (mm) was measured and recorded for each
fruit. The average weight (g), diameter (mm), firmness
(g mm1), and scar diameter (mm) of the five berries was
calculated and used for subsequent analyses.
Analysis and Model Comparison
Variance components were estimated with REML and
BVs with BLUP using ASReml version 3.0 (Gilmour et al.,
2009) with the following univariate linear mixed models:
y = 1n + Xb + Z1c + Z2r + Z3a + Z 4ba + e
where y is a vector of the trait being analyzed (i.e., yield,
weight, firmness, diameter, or scar), 1 is a vector of ones,
is the overall mean, b is the fixed effect of year, c is the
random effect of column nested within year ~N(0, 2c), r
is the random effect of row nested within year ~N(0, 2r),
a is the random effect of genotype ~N(0, A2g), ba is
the random effect of the interaction of year genotype
~N(0, 2ba), e is the random residual effect ~N(0, I2e), X,
and Z1Z 4 are incidence matrices for year, column, row,
genotype and year genotype interaction, respectively.
The unbiased heritability was calculated as h2 = 2g/(2g
+ 2c + 2r + 2bg + 2e). The A matrix was constructed
with the AGHmatrix R package as described below.
Matrix Construction with AGHmatrix
To build the pedigree-based relationship matrices, we
constructed an R package named AGHmatrix. The pack-
age is freely available for download at https://github.com/
prmunoz/AGHmatrix. To build the relationship matrix
derived from the pedigree using the AGHmatrix pack-
age, the pedigree information needs to be formatted in
three columns (individual, Parent 1, and Parent 2) and
read as a table in R (using read.table or read.csv func-
tions). The pedigree file should be sorted from older to
newer generations, thus the oldest generation (founders)
without pedigree information should appear at the top of
the file, while the current generation being phenotyped
is at the bottom of the file. Before proceeding to build
the relationship matrix, the AGHmatrix verifies that the
above order is correct, and if not, it will flag and permu-
tate the order of misplaced individuals. More details of
the packages functions can be found in the manual at
https://github.com/prmunoz/AGHmatrix/blob/master/
vignettes/tutorial.pdf.
The autotetraploid pedigree-based relationship
matrix Aw was calculated as presented in Slater et al.
(2013). The algorithm first computes the matrix K
of 10
considering the proportion of parental gametes that are
identity by descent as a result of double reduction w. Thus
for every individual ki with parents s and d,
If s and d of ai are unknown, they are assumed unre-
lated, and
kij = kji = 0; for j = 1:(i 1)
kii = (1 + w)/4
If only s is known, d is assumed nonrelated, and
kij = kji = 0.5(kjs); for j = 1:(i 1)
kii = [5 + 7w + 4k ss(1 w)]/24
If s and j are known, then
kij = kji = 0.5(kjs + kjd); for j = 1:(i 1)
kii = [1 + 2w + (1 w)k ss + (1 w)kdd + 3k sd]/6
Finally, the matrix Aw is given by Aw = 4K. Six Aw matri-
ces were constructed from the pedigree assuming vary-
ing levels of double reduction (w): 0, 0.05, 0.10, 0.15, 0.20,
and 0.25. Additionally, we also built the traditional dip-
loid pedigree-based relationship matrix (A-matrix) using
the AGHmatrix R package, which is calculated according
to the recursive method presented in Mrode (2014) and
described by Henderson (1976). The AGHmatrix R pack-
age can also build the genomic-base relationship matrix,
G-matrix, for diploids using either the method proposed
by VanRaden (2008) or from Powell et al. (2010). To cal-
culate the G-matrix, molecular data needs to be in table
format where n is the number of individuals (in rows)
and m is the number of loci (in columns). The loci infor-
mation is set to 1, 0, 1, for the genotypes aa, Aa, and
AA, respectively. Although the G-matrix capability is
present in the package, the genomic matrix was not built
or used in this study.
The autotetraploid matrices with different levels of
double reduction were used in the linear mixed model
described above. These autotetraploid models were com-
pared against the diploid counterpart using the Akaike
information criteria (AIC). As the level of double reduc-
tion w is unknown, we inferred its level based on the
model that maximized the likelihood of the data given
the parameters of the linear mixed model.
Results and Discussion
Using AGHmatrix to Determine Trait-Specific
Levels of Double Reduction
A unique challenge to plant breeders dealing with auto-
tetraploid species has been the determination of the
degree of double reduction and its impact on heritabil-
ity and BV estimates. This challenge has been exacer-
bated with the lack of molecular markers and genetics
4 of 10 the pl ant genome
resources in most autotetraploid species. Therefore, the
AGHmatrix package presented in this study may be the
solution for many autotetraploid breeders to obtain an
estimation of double reduction in their populations.
Using blueberry as an example, we demonstrated the
method to achieve such goals. We first built seven rela-
tionship matrices (A-matrices) base on the pedigree: one
assuming a diploid system and the other six assuming
an autotetraploid system with different levels of double
reduction ranging from 0 to 0.25. Each of these matrices
was fit in the same mixed model equation using the same
phenotypic data for each of the traits considered. We then
obtained the AIC value from the REML. The AIC dif-
ference between the disomic and tetrasomic model (Fig.
1) was used to indicate the model that best fit the data.
Yield, weight, and diameter had similar negative slopes,
each decreasing more dramatically with increasing dou-
ble reduction. The pattern of scar was similar but with a
marginal decrease from double reduction varying from
0 to 0.25. Firmness was the only trait that had the best fit
when double reduction was larger than zero; the fitness of
the model was maximized when double reduction ranged
from 0.15 to 0.20. The tetraploid model assuming w = 0
was the best fit for four of the five traits. The absence of
double reduction for the majority of traits is similar to
the findings of Krebs and Hancock (1989) when study-
ing blueberry based on segregation of a limited number
of isoenzyme loci in V. corymbosum. The most plausible
explanation for the null double reduction comes from the
physical barrier imposed by the size of the chromosomes.
Blueberries have small chromosomes (1.52.5 m), which
could limit multivalent formation, an essential step for
double reduction to occur (Krebs and Hancock, 1989;
Hall and Galletta, 1971). Also, cytogenetic studies of
meiotic pairing in V. corymbosum found the majority
of pairing to be bivalent with few quadrivalent forma-
tions (Jelenkovic and Harrington, 1971; Jelenkovic and
Hough, 1970; Vorsa and Novy, 1995; Qu et al., 1998). Qu
et al. (1998) found that >90% of the chromosomes formed
bivalents in a highbush cultivar and a wild tetraploid
selection. Jelenkovic and Hough (1970) found similar
results when studying three V. corymbosum cultivars,
where up to four quadrivalents were found in a pollen
cell, but most cells had only one. Additionally, preferen-
tial pairing lowers the expected frequency of multivalent
formation between homologous chromosomes (Sybenga,
1996). Preferential pairing has been reported in V. corym-
bosum and V. corymbosum V. darrowii hybrids (Draper
and Scott, 1971; Vorsa and Novy, 1995).
While the above explanations are feasible for the
zero double reduction found for four of the five traits,
they do not explain the high double reduction estimated
for firmness. However, a similar study in autotetra-
ploid potato found that the model best estimating the
proportion of double reduction varied between 0 and
0.25 depending on the trait considered (Slater et al.,
2013). Double reduction is expected to differ from trait
to trait because it is a position-dependent phenomenon
november 2016 vol . 9, no . 3
Fig. 1. The Akaike information criterion (AIC) difference between the diploid model and the tetraploid models assuming double
reduction values from 0 to 0.25. The AIC values were obtained from ASReml 3.0.

fluctuating between chromosomes based on the fre- Table 1. The heritability of five breeding traits calcu-
quency of multivalent formation and within chromo- lated assuming disomic and tetrasomic inheritance.
somes depending on where the measured loci reside Tetrasomic inheritance was estimated assuming no
(Wu et al., 2001). Bourke et al. (2015) found a parallel double reduction for all traits except firmness where
increase between the rate of double reduction and the 0.15 was used.
distance from the centromere using a high-density set
Disomic inheritance Tetrasomic inheritance
of markers in autotetraploid potato. The rate of double
reduction increases toward telomeres because there is a Trait Heritability Standard error Heritability Standard error
greater probability of a crossover to occur between the Yield 0.46 0.02 0.45 0.02
centromere and the loci (Bourke et al., 2015; Luo et al., Weight 0.58 0.02 0.58 0.02
2006; Welch, 1962; Wu et al., 2001). The high propor- Firmness 0.40 0.03 0.35 0.02
tion of double reduction for firmness suggests that genes Fruit diameter 0.26 0.03 0.26 0.03
controlling this trait may reside toward the distal end Scar diameter 0.54 0.02 0.54 0.02
of a chromosome. Thus, in the same line, the genes con- Tetrasomic inheritance was calculated assuming double reduction of 0.15.
trolling yield, weight, diameter, and scar may be located
closer to the centromere, be quantitative in nature and
dispersed over the whole genome, or located on chromo- showed a better fit with the tetrasomic model consider-
somes that do not exhibit much multivalent formation. ing higher levels of double reduction, thus the heritability
for the tetrasomic model was estimated using w = 0.15,
Impact of Increasing Double Reduction Levels which was the level of double reduction that fit the data
on Heritability and Relationship Estimates best (Table 1).
Heritability estimations were not significantly different The heritabilities estimates for firmness had the larg-
when assuming tetrasomic inheritance with zero double est difference between the two models, decreasing from
reduction (w = 0) instead of disomic inheritance for all 0.40 to 0.35 as a result of the larger proportion of double
traits (Table 1). However, firmness was the only trait that reduction used in the estimation. As seen in Fig. 2, there

a m adeu et al .: aghm atrix : r pack age to construct rel ationshi p m atri ces 5 of 10
Fig. 2. The estimated narrow-sense heritability for firmness assuming disomic inheritance and tetrasomic inheritance with increasing
coefficients of double reduction.

is a small difference between the disomic-estimated heri- Table 2. Average of the relationship coefficient be-
tability for firmness and the tetrasomic-estimated herita- tween two individuals assuming various levels of
bility with no double reduction. The difference becomes double reduction.
more pronounced, and the narrow-sense heritability for
Double First Third
the trait decreases as the proportion of double reduction Reduction Min. quartile Median Mean quartile Max.
increases. This trend was seen in all the traits measured
0 0 0.067 0.106 0.115 0.148 1.000
(data not shown). Comparing disomic and tetrasomic
0.05 0 0.071 0.113 0.123 0.158 1.050
inheritance with w = 0.10 in autotetraploid potatoes,
0.10 0 0.076 0.120 0.130 0.168 1.100
Slater et al. (2013) observed no consistent trend and
0.15 0 0.080 0.126 0.138 0.177 1.150
found the difference in heritability ranged from 0.01 to
0.20 0 0.084 0.133 0.145 0.186 1.200
0.13 depending on the trait being analyzed.
0.25 0 0.088 0.139 0.151 0.194 1.250
The downward trend in heritability observed
for blueberry fruit firmness can be explained by the
increased inbreeding caused by increasing the levels of by comparing the kinship coefficients obtained for w =
double reduction. As double reduction increased, the 0 and those obtained for w = 0.25 (Table 2). The effects
kinship coefficient also increased (Table 2). The maxi- of double reduction are cumulative with larger kinship
mum coefficient was determined by the amount of dou- coefficients more susceptible to its effects. In the first
ble reduction, whereas the minimum kinship coefficient quartile, the difference between the kinship coefficients
was unaltered by the level of double reduction. A large estimated assuming w = 0 and the coefficient estimated
number of the individuals in the pedigree had unknown assuming w = 0.25 was 0.021, while this difference almost
ancestry, which negatively skewed the distribution. doubles (0.046) in the third quartile.
Unknown ancestry was a result of missing information, The BLUP analysis uses these kinship coefficients
unspecified clones, and wild selections. These individu- and estimations of heritability to predict BVs. Thus, inac-
als were assumed to be unrelated and thus unaffected curate estimations of double reduction could lead to bias
by double reduction. As double reduction increased the predictions. Higher relationships should suffer a larger
kinship coefficient increased as well. This can be seen impact as these are more affected by double reduction.

6 of 10 the pl ant genome november 2016 vol . 9, no . 3

Fig. 3. Correlation of breeding values for firmness estimated assuming disomic and tetrasomic inheritance with w = 0.15.
Nevertheless, there was a strong correlation (r = 0.999)
between the diploid and tetraploid (w = 0.15) estimated
BVs for firmness (Fig. 3). This strong correlation was
seen for all traits when analyzed with tetraploid models
assuming w = 0 (data not shown). Similarly, Slater et al.
(2013) found a strong correlation (>0.95) for eight of the
nine traits when comparing the diploid estimated BVs
with the autotetraploid estimated BVs. The depth of the
pedigree could help balance and compensate errone-
ous estimations, resulting in small changes to the BVs
over various levels of double reduction. In this case, the
pedigree covered the whole breeding program beginning
with its origin in 1908.
Effect of Pedigree Depth on Double
Reduction Estimate
In the example data set we have used, there is an extensive
pedigree available beginning with known founder clones
dating back to 1908 (Coville, 1937). However, the detail of
pedigree information available for blueberry may not be
available for other autotetraploid plant breeders. To study
the consequences of using truncated or incomplete pedi-
gree information while studying double reduction using
these methods, we truncated the pedigree to 5, 10, 20, and
30 yr (back to 2009, 2004, 1994, and 1984, respectively).
Different levels of double reduction maximize the
fitting of the data when using different pedigree depths
a m adeu et al .: aghm atrix : r pack age to construct rel ationshi p m atri ces 7
(Fig. 4). By using only 5 yr of data (back to 2009) the level
of double reduction is estimated to be the maximum
coefficient tested in this study, 0.25, for four out of five
traits, and 0.20 for the last trait. In this case, all the traits
had the worst fit assuming no double reduction. When
10 yr of pedigree information was used, three traits fol-
lowed the same pattern as when using 5 yr of pedigree
information, no double reduction was estimated for one
trait, while the fit for the final trait was maximized at a
double reduction level of 0.05. As more pedigree infor-
mation was added, the slopes continued to change, but
even when 30 yr of pedigree information were used,
it was not enough to observe the estimation of double
reduction seen when all the pedigree information was
used. Thus, shallow pedigrees underestimate the actual
kinship coefficient between two individuals. For example
Boyce (1983) found that shallow pedigrees miscalculate
the level of inbreeding when analyzing 10 Standardbred
stallions with 30 generations worth of pedigree informa-
tion. In blueberries, there has been a steady increase in
inbreeding with each consecutive generation as a result
of recurrent selection (Brevis et al., 2008; Hancock and
Siefker, 1982; Ehlenfeldt, 1994). Relationships are further
intensified by the narrow gene pool created in the found-
ing event. Based on pedigree information, Ehlenfeldt
(1994) concluded that three cultivars, Brooks, Sooy, and
Rubel, have contributed the largest portion of genes in
of 10
Fig. 4. In order from left to right, pedigree depths of 5, 10, 20, and 30 yr and their influence on the estimated amount of double
reduction for each trait.
79 highbush cultivars (23.5, 12.5, and 28.6%, respec-
tively) and V. darrowi contributions can be traced back
to germplasm sources Florida 4B, Florida 4A, Florida
9A, and clone D (Brevis et al., 2008). The limited pedi-
gree information cannot accurately account for these
intertwined relationships. Thus, the higher proportions
of double reduction compensated for the lack of informa-
tion by increasing the assumed relationships between
the individuals and thus better fitted the model. Atkin
et al. (2009) and Mehrabani-Yeganeh et al. (1999) found
the accuracy of estimating additive variance components
and BVs improved when using more pedigree informa-
tion. Atkin et al. (2009) discovered that the variances
were estimated with the same precision once five genera-
tions were used, suggesting older generations do not con-
tribute much information to the model. This is mainly
because the coefficients of relationships with the cur-
rent population become smaller as generation distance
increases. However, using shallow pedigrees can lead the
erroneous conclusions in the estimation of relationship
8 of 10 the pl ant genome
coefficients and thus incorrect determination of the level
of double reduction for each trait. This will also impact
the estimated heritability, inbreeding, and BVs.
Conclusion
We have developed an R package, AGHmatrix, that can
be used to quickly develop relationship matrices for
autotetraploid crop species. To illustrate the utility of
this package, we used the pedigree and phenotypic data
from a blueberry breeding program. The majority of
traits (yield, weight, diameter, and scar) had the best fit
assuming tetrasomic inheritance with no double reduc-
tion. The only exception was firmness, which had the
best fit when double reduction was 0.15. Changes in the
estimation of heritability occurred with larger propor-
tions of double reduction. However, there was no sig-
nificant improvement in BV estimates for the blueberry
traits assessed when comparing tetrasomic inheritance
accounting for the correct levels of double reduction with
november 2016 vol . 9, no . 3
a simple disomic inheritance model. The effects of double
reduction are cumulative, and shallow pedigree depths
can underestimate the true kinship coefficient and, as a
result, report inflated levels of double reduction, which
can affect the estimation of the genetic parameters. As
double reduction is trait dependent, this package should
allow autotetraploid breeders to estimate the levels of
double reduction and how it affects genetic parameters
and ultimately breeding progress.
Acknowledgments
This material is based on work that is supported by the National Institute of
Food and Agriculture, USDA under award no. 2014-67013-22418 to PRM,
JWO, and JBE and by the Science without Borders/CAPES scholarship
(proc. No. 88888.020298/2013-00) program from Brazil awarded to RRA.
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