Mapping QTL for cold-tolerance trait in

a GIFT-derived tilapia line by

ddRAD-seq Chun Hui Ai
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 Aquaculture 556 (2022) 738273

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Mapping QTL for cold-tolerance trait in a GIFT-derived tilapia line by

ddRAD-seq
Chun Hui Ai a, 1, Bi Jun Li a, 1, Jun Hong Xia a, b, *
a
College of Life Sciences, State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic
Animals, Sun Yat-Sen University, Guangzhou 510275, PR China
b
Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming 525000, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Tilapia is an economically important fish in the world. High mortality caused by low temperature is a serious
ddRAD-seq problem to tilapia farming during overwintering. In this study, a genome-wide significant QTL interval, located
Cold tolerance on chrLG18, controlling cold-tolerance trait was identified in a full-sib family of GIFT originated tilapia line using
QTL
ddRAD-seq-based QTL mapping. The QTL explained 21.5% of phenotypic variances. Six pairs of microsatellite
Microsatellite marker
markers were successfully developed within this interval. For the microsatellite marker SSR613719, which had
two genotypes in the population, ‘lm’ and ‘ll’, the number of individuals with the ‘ll’ genotype was significantly
higher than the number of individuals with the ‘lm’ genotype in the cold sensitive population (p < 0.05). This
independent test indicated the reliability of the detected QTLs. The expression of candidate genes (pak1ip1,
loc100696456 and loc100697261) in the QTL interval was significantly affected under cold treatment as indi­
cated by qRT-PCR test. This study provides important information for marker-assisted selection of cold tolerant
tilapia lines.

1. Introduction decrease and the content of polyunsaturated fatty acids to increase,

Water temperature is one of the most important physicochemical
factors in the aquatic environment. Fish live in water and are poikilio­
thermic animals (Brett, 1971). Water temperature has a direct impact on
the development, growth, reproduction, metabolism, behavior of fish,
which in turn has a huge impact on their entire life history (Ney, 1993).
Fish have to endure daily and seasonal changes in water temperature. If
the climatic conditions of the habitat change in such a way that the
water temperature exceeds the tolerance range of fish, this can have
extremely detrimental consequences for fish and can even lead to death
(Lopez-Olmeda and Sanchez-Vazquez, 2011).
Tilapia is an important aquaculture fish worldwide for its fast growth
rate and high adaptability (Li et al., 2002). Tilapia is highly affected by
environmental temperature. Low temperature results in poor growth
and high mortality during overwintering period (Chervinski and Lahav,
1962). A number of studies have been carried out on low temperature
stress in tilapia. Previous studies have found that low temperatures
cause the proportion of saturated fatty acids in the liver of tilapia to
although saturated and monounsaturated fatty acids are always the
main energy contributors to the fish (Corrêa et al., 2017; Hsieh et al.,
2007b). Cholesterol and triglyceride levels in the blood are stimulated
by low temperature conditions (Ma et al., 2015), and serum glucose and
cholesterol levels were significantly reduced after 120 h of cold treat­
ment (He et al., 2015). Under short-term cold stress, the tilapia liver
indicated increased levels of superoxide dismutase, glutathione oxidase,
catalase and glutathione. Fish first use saturated fatty acids for energy
production under low temperature stress. As the stress time increases,
fish begin to produce polyunsaturated fatty acids to meet their energy
requirements, but the resulting peroxides may cause damage to the fish
(He et al., 2015). At 15 ◦ C, the ion concentration in tilapia serum
remained unchanged, glucose content tended to increase and then
decrease. Triglyceride, cholesterol and LDL-cholesterol content
increased significantly, while HDL-cholesterol content remained stable.
The activities for aspartate aminotransferase, alanine aminotransferase
and lactate dehydrogenase increased significantly, while the activities
for superoxide dismutase and catalase tended to increase and then

* Corresponding author at: College of Life Sciences, State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key
Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou 510275, PR China
E-mail address: xiajunh3@mail.sysu.edu.cn (J.H. Xia).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.aquaculture.2022.738273
Received 24 February 2022; Received in revised form 10 April 2022; Accepted 16 April 2022
Available online 22 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
C.H. Ai et al.
decrease (Shi et al., 2015). Cold stress may lead to dysregulation of free
radical and lipid metabolism, reduce superoxide dismutase activity,
macrophage rupture and phagocytic activity, and increase malondial­
dehyde content in tilapia head-kidney, which adversely affect their
physiological adaptation and eventually lead to death (Qiang et al.,
2018).
Marker-assisted selection (MAS) allows selection at an early stage
and overcomes the difficulty in identifying traits that are not obvious,
thus significantly improving the accuracy of selection and shortening
the breeding cycle (Lande and Thompson, 1990). Using a set of markers
that are closely linked to traits, MAS can be more effectively applied to
the selection of cold tolerant lines in tilapia. Mapping quantitative trait
loci (QTL) and finding the linked markers for cold-tolerant traits are the
key steps in the improvement of the cold stress tolerance in tilapia. A lot
of QTL intervals associated with, e.g., growth (Liu et al., 2014), disease
resistance (Barría et al., 2021), sex (Zhu et al., 2022), salt tolerance (Gu
et al., 2018), hypoxia tolerance (Li et al., 2017), ammonia nitrogen
tolerance (Zhu et al., 2021) and body color (Li et al., 2019) have been
identified in tilapia. QTL mapping for cold tolerance in two unrelated F2
families of interspecific tilapia hybrids (Oreochromis mossambi­
cus×Oreochromis aureus) conducted using tens of microsatellites sug­
gests a QTL, near UNH879 for cold tolerance (Cnaani et al., 2003). A
genome scan of a cross between a (Oreochromis niloticus×Sarotherodon
galilaeus) male and a (O. mossambicus×O. aureus) female supports the
existence of a QTL for cold tolerance on ChrLG23 using microsatellite
and AFLP marker data (Moen et al., 2004). Due to low density of markers
(e.g., 10 markers on LG23) mapped in populations, QTL for cold toler­
ance is broadly spanned the region of the chromosome. Moreover, QTL
for cold tolerance in Nile tilapia-originated lines is unclear, which may
hinder the selection of cold tolerant lines using MAS techniques.
Double-digest restriction-site associated DNA sequencing (ddRAD-
seq) is an inexpensive method for de novo SNP discovery, genotyping
and QTL mapping of important traits (Peterson et al., 2012). This
technique is already being applied in QTL mapping of many species (Ba
et al., 2017; Laila et al., 2019; Nyinondi et al., 2020; Tan et al., 2019).
In southern China, cold snap during over-wintering causes huge
economic losses (Wang et al., 2021), and the cold temperatures had a
negative impact on tilapia production (Ma et al., 2015; Shi et al., 2015).
QTL mapping for cold tolerant trait and then carrying out selection of
cold tolerant tilapia lines using MAS is plausible solutions to these
problems. In this study, we conducted QTL mapping to identify QTL
intervals for cold tolerant trait in a full-sib family (N = ~ 600) generated
using a pair of genetically improved farmed tilapia (GIFT)-derived ti­
lapias using ddRAD-seq. The expression of candidate genes under low
temperature stress was explored. Our finding provides a basis for con­
ducting molecular marker-assisted selection and functional analysis of
cold tolerance in tilapia.
2. Methods and Materials
2.1. Ethics statement
All experiments in this project were approved by the Animal Care
and Use Committee of the School of Life Sciences, Sun Yat-sen
University.
2.2. Family construction and management of experimental fish
In this study, a full-sib family (N = ~ 600) was generated from a pair
of GIFT tilapias in Wulonggang Aquaculture company, Ltd. (Guangzhou,
Guangdong, China). The family was raised in the pond at the hatchery of
the company, and then was transferred to a circulating freshwater cul­
ture system at the college of Life Sciences, Sun Yat-sen University at 60
days post-hatch (dph).
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Aquaculture 556 (2022) 738273
2.3. Cold stress treatment and collection of trait data for QTL mapping
The cold stress experiment was conducted in an indoor circulating
water culture system at the College of Life Sciences, Sun Yat-sen Uni­
versity. The starting temperature of the aquatic water was 26 ◦ C. The
cooling process was carried out at a rate of 1 ◦ C/h using a chiller until
the temperature reached to 15 ◦ C. The cooling rate was then slowed
down at a rate of 0.5 ◦ C/h, until the water temperature was close to
around 8.5 ◦ C. The temperature was then kept stable by using water
chiller. Unconsciousness is an unresponsive state. Tilapia may appear
unconsciousness under cold challenge depending on its cold tolerance.
However, an unconscious animal is not necessarily a dead one. The
response status of the test fish was continually observed. Once a fish
showed unconsciousness, the time point (Tn) for each fish (the nth fish)
was recorded immediately. The first unconscious fish was observed after
half hours of challenge at around 8.5 ◦ C. The survival time (min) was
then calculated using a formula: Tn-T1 and was treated as trait value for
cold tolerance analysis in this study. The fin sample for each unconscious
fish were cut and stored in absolute Ethanol for subsequent DNA
extraction.
2.4. Cold stress and sample collection for expression analysis
The offspring of the full-sib family that cultured in a tank for 7 days
were randomly divided into experimental and control groups. The water
temperature in the experimental group was decreased to 15 ◦ C with a
water chiller and the temperature in the control group was maintained
at 26 ◦ C. Gill, heart, liver, intestine, muscle and brain tissues from both
groups were collected at 12 h post treatment for RNA extraction.
2.5. DNA extraction, ddRAD-seq and SNP genotyping
Genomic DNA was extracted from fin tissue using AMPureXP beads
(Beckman Coulter, Fullerton, CA, USA) according to the manufacturer’s
protocol, and the RNA was removed by RNase A, then the quality of
genomic DNA was evaluated with 0.8% agarose gel electrophoresis and
quantified with Qubit Fluorometer (ver3.0; Invitrogen).
The ddRAD-seq genotyping technique was used to genotype 96 in­
dividuals including the first 48 samples showing the most sensitive to
cold stress and the last 48 samples showing the most tolerant during cold
stress treatment in the family. Two hundred nanograms of genomic DNA
was taken from each individual. The ddRAD-seq library was constructed
using a technique as described in the protocol (Peterson et al., 2012).
The library and genomic DNA from two parents was submitted to the
company Novogene (Beijing, China) for Illumina HiSeq PE150
sequencing.
The clean data for 96 offspring and 2 parents were mapped to the
Nile tilapia reference genome (oreNil2.fa) with the program bowtie
(version 2.0)(Langmead and Salzberg, 2012). SNP calling was carried
out using the mpileup pipeline that implemented in the program sam­
tools and bcftools (with the parameter setting as Dugf). The output data
was first filtered with a total minimum depth of 200 and a minimum
quality of 10. Then, for offspring, SNPs with the read depth < 10, ge­
notype quality <10 and missing data rate > 2% were removed. For the
parent data, SNPs with the read depth > 20 and read quality >10 were
remained. The resulted SNP datasets were merged with their parent SNP
data to remove the furious genotyping data.
2.6. QTL analysis
QTL analysis was performed using the software MapQTL 6.0 (Van
Ooijen, 2009), based on the SNPs obtained from ddRAD-seq and the
values of cold tolerance traits in tilapia. The genotyping data with phase
data were exported from JoinMap software (Van Ooijen, 2006). Per­
mutation test with 50,000 times was conducted to determine the
genome-wide and chromosome significant logarithm of odds (LOD)
C.H. Ai et al.
thresholds according to the interval at the relative cumulative value of
0.95. Interval mapping analysis was first performed to identify cofactors
within QTL regions using the automated cofactor selection analysis and
followed by composite interval mapping to finally identify QTL above
the genome-wide threshold levels.
2.7. Validation of the QTL using microsatellite markers
To confirm the genome-wide QTLs, six novel microsatellite markers
were successfully developed in the QTL region of chrLG18 using the
online tandem repeat finder (http://tandem.bu.edu/trf/trf.html) and
the program Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-bl
ast/). PCR amplifications were performed in a 20 μL volume using a
2× PCR Master Mix and 40 ng genomic DNA and 0.5 μM forward and
reverse primers (Dongsheng, China). The reactions were conducted in a
Bio-Rad cycler (Bio-Rad) using the following program: one cycle of 3
min at 94 ◦ C and 35 cycles of 30 s at 94 ◦ C, 30 s at 60 ◦ C, and 20 s at
72 ◦ C, followed by a final extension of 10 min at 72 ◦ C. PCR products
were resolved by running electrophoresis of 6% polyacrylamide gels and
silver staining. The genotyping for each microsatellite was manually
called. All microsatellite markers were genotyped for the 96 individuals
from two extreme trait groups of the QTL mapping family.
2.8. RNA isolation and qRT-PCR analysis for candidate genes
Total RNA was isolated using TRIzol reagent (Invitrogen, UK) ac­
cording to the manufacturer’s protocol. The candidate genes near and on
the QTL peak region (chrLG18: 18.93 Mb ~ 20.42 Mb) were explored.
Ef1α gene was used as an internal reference gene. qRT-PCR was per­
formed on the Roche Light Cycler 480 Real-time PCR System. Each
sample was performed with three replicates. Complementary DNA
(cDNA) products were diluted 1:10 before used as qPCR templates. The
total volume of the reaction system was 10 μL, containing 5 μL 2× SYBR
Green mix, 0.2 μL forward primer, 0.2 μL reverse primer, and 4.6 μL of
diluted cDNA. The qPCR program includes 3 min of 95 ◦ C pre-
incubation, followed by 40 cycles of amplification at 95 ◦ C for 15 s,
60 ◦ C for 15 s, and 72 ◦ C for 20 s. The melting curve procedures include
95 ◦ C for 15 s, 65 ◦ C for 15 s, and slowly increase to 95 ◦ C. The result was
analyzed according to the ΔΔCt method. The expression differences of
candidate genes between groups were investigated using Student’s t-
test.
2.9. Statistical analysis for microsatellite marker data
The trait differences among different microsatellite genotypes were
compared by t-test that was implemented in Microsoft Excel. The two-
tailed probability for frequency distribution differences of a microsat­
ellite genotype between extreme trait groups was calculated using an
online calculator of Fisher’s exact test (http://www.danielsoper.com/s
tatcalc3/).
3. Result
3.1. Cold-tolerance trait data for the full-sib family
Since a peak with a large number of tilapias died appeared during
acute cold stress experiment, the collecting of the trait data for all in­
dividuals was impossible. Therefore only individuals with accurately
recorded survival time were applied in the QTL mapping and following
verification. The mean of body weight for the sampled individuals
(extreme traits) of the full-sib family (n = 192) was 12.44 ± 3.97 g. The
offspring began to show unconscious at around 8.5 ◦ C, but with differ­
ential responses depending on its cold tolerance. The survival time for
the first fish showing unconscious was defined as 0 min. The survival
time for the last fish was recorded as 227 min.
The 96 individuals with extreme traits were used for QTL mapping.
3
Aquaculture 556 (2022) 738273
The first 48 individuals showing the most sensitiveness to cold stress
have a mean survival time of 40.34 min (SD = 24.03 min) and the last 48
individuals showing the most tolerance to cold stress have a mean sur­
vival time of 215.75 min (SD = 7.00 min) (Fig. 1a).
The 192 individuals including 96 extremely cold tolerant individuals
and 96 extremely cold sensitive individuals were applied for QTL vali­
dation using microsatellite markers. The survival time data for the 96
extremely cold sensitive individuals was 81.54 min (SD = 47.57 min)
and the survival time data for the 96 extremely cold tolerant individuals
was 203.75 min (SD = 13.86 min) (Fig. 1b).
3.2. SNP genotyping for the mapping family by ddRAD-seq
The 96 individuals for QTL mapping were genotyped using ddRAD-
seq technique. The two parents were genotyped using whole genome
resequencing technique. The samtools pipelines identified 81,279 vari­
ants. A total of 2,151,523 SNPs were identified between the parents. We
further filtered the variants and finally got 1160 high quality SNP data
for the mapping family (Supplementary Table 1).
QTL analysis was performed using SNP data in combination with
survival time data for the mapping family. One significant QTLs at
genome wide level were detected on chrLG18 (p = 0.05, Fig. 2). The SNP
marker chrLG18_19,316,719 showed the most significant association
with cold-tolerant trait (p < 0.05) with the explained percentage of
variance of 21.5% and an LOD value of 5.6 (the genome wide signifi­
cance threshold = 4.5). In addition, the SNP marker chrLG8-
24_16,262,132 on chrLG8 showed a LOD value of 3.9 and the
explained percentage of variance of 14.5% (the chromosome wide sig­
nificance threshold = 3.1). The SNP chrLG4_4,189,125 on chrLG4 has
the explained percentage of variance of 9.9% with an LOD value of 3.2.
Information of peak SNP markers significantly associated with cold-
tolerance trait is summarized in Table 1.
3.3. Validation and fine mapping of the QTL on chrLG18
To verify the reliability of the QTL that identified by ddRAD-seq, six
novel microsatellite markers with polymorphisms between parents were
successfully developed between 11 Mb and 20 Mb of chrLG18 (Sup­
plementary Table 2).
Genotyping of the 96 individuals in the cold tolerant group and the
96 individuals in the cold sensitive group showed that three of the mi­
crosatellite markers were significantly associated with cold tolerant
traits. The microsatellite marker SSR613719 was the most associated
markers with the cold tolerant trait. There are two genotypes for the
marker SRR613719, ‘lm’ and ‘ll’, detected in the population. The ge­
notype ‘ll’ has a higher percentage (56.3%) in cold sensitive group,
while genotype ‘lm’ has a higher percentage (63.2%) in the cold tolerant
group (Chi-square test with Yates’ correction, p = 0.010979, Fig.3). The
microsatellite marker analysis confirmed the reliability of the QTL that
identified by ddRAD-seq.
By combining the microsatellite markers with the SNP markers, a
composite interval mapping analysis was carried out on the linkage
group chrLG18. The microsatellite marker SSR1136961 showed the
highest LOD value of 5.67, and the explained percentage of variance of
24%. Based on the fine mapping results, the QTL controlling for cold
tolerance was localized between 18.93 Mb and 20.42 Mb on chrLG18 in
the GIFT tilapia (Fig.4).
3.4. Characterization of candidate genes in the QTL intervals
It is hypothesized that genes located within this QTL interval may
play a role in cold tolerance in tilapia. We identified 43 genes in the QTL
interval. The gene names and their annotation information are shown in
Supplementary Table 3.
Heart, gill, liver, intestine, muscle and brain tissues were collected
from the test GIFT tilapia at 12 h post cold treatment (15 ◦ C) and from
C.H. Ai et al. Aquaculture 556 (2022) 738273

Fig. 1. The distribution of cold tolerant trait data for the full-sib family of GIFT tilapia. a. The survival time of tilapia individuals (N = 96) used for ddRAD-seq. b. The
survival time of tilapia individuals (N = 192) used for microsatellite marker validation of QTL.

Fig. 2. QTL analysis for cold tolerant trait in the full-sib family using SNP data. The genome-wide threshold value for LOD was 4.5 (p = 0.05). One major genome-
wide significant QTL interval was identified with LOD value of 5.6 and a percentage of explained variance of 21.5%.

under cold treatment (p < 0.05). In contrast, the gene expression for
Table 1
loc100697261 in heart, gill, gut and brain was significantly down-
The expression level for 9 genes in response to cold stress in different tissues.
regulated under cold treatment (p < 0.05). In addition, loc100696456
Gene name Heart Gill Liver Intestine Muscle Brain was significantly down-regulated in heart, muscle, gills and brain (p <
ndufv2 *↑ 0.05). These genes may play an important role in tilapia under cold
mtcl1 *↓ stress.
LOC100696456 *↓ *↓ **↓ **↓
paklipl *↑ *↑ *↑
LOC102083181 *↓ **↓
4. Discussion
LOC100697261 **↓ **↓ **↓ **↓
cc2d1b *↓ *↓ 4.1. QTL mapping on temperature tolerance traits in fishes
napg *↑
rab12 *↑
Temperature tolerance is one of the most important traits in fish.
* p < 0.05 ** p < 0.01 ↑ upregulation under cold stress ↓ downregulation under However, genetically improving temperature tolerance in fish is
cold stress. generally difficult with traditional methods since the difficulties in
measurements and low heritability with the traits. The heritability of
the control group at 12 h (25 ◦ C). Primer pairs were successfully cold tolerance in juvenile Nile tilapia is estimated to be 0.08 ± 0.17 for
developed for 14 of the candidate genes located in the QTL interval. We cooling degree hours (CDH) and 0.09 ± 0.19 for temperature at death
found 9 of the genes were significantly regulated by temperature using (TAD), estimated with an animal model (Charo-Karisa et al., 2005).
qRT-PCR (Table 2). There was also a study concluded that the heritability was not likely to
The gene pak1ip1, loc100696456 (cathepsin Z) and loc100697261 be more than 0.15 in their laboratory population for killifish (Heteran­
(acetyl-coenzyme A synthetase, cytoplasmic) showed significant expres­ dria formosa) and did not detect a significant response to selection (Baer
sion changes in three and more tissues under treatment (Fig.5). The and Travis, 2000). Therefore MAS-based methods with linked markers
pak1ip1 gene was significantly upregulated in heart, gills and intestine may be alternative methods for selection of cold tolerant lines in fish.

4
C.H. Ai et al. Aquaculture 556 (2022) 738273

explained percentage of variance of 21.5% and a chromosome-level

Fig. 3. The significant relationship between the genotypic data of SRR613719
and the cold tolerant data in the full-sib family. There are two genotypes for the
microsatellite marker SRR613719, ‘lm’ and ‘ll’. The genotype ‘ll’ has a higher
percentage in the cold sensitive group, while the genotype ‘lm’ has a higher
percentage in the cold tolerant group (Chi-square test with Yates correction, p
= 0.010979).
significant QTL on chrLG8 showed the explained percentage of vari­
ance of 14.5%. Compared to the low density of markers in former studies
on hybrid tilapia with slower growth rate, much higher marker density
was applied and novel QTLs and more precise QTL positions were
detected in this study. More importantly, these QTLs were detected in a
major cultured tilapia line with fast growth trait. These may suggest it is
possible to select a not only cold tolerant, but also fast growing lines
from GIFT tilapia using MAS techniques.
The measurement of stress tolerant traits is often a key step in QTL
mapping. The accuracy of QTL mapping is influenced by several factors,
including the genetic characteristics of the QTL, experimental design,
environmental effects, marker density and information, genotyping and
the accuracy of trait measurements (Broman, 2001; Doerge, 2002). In
tilapia, the size, age and genetic background may affect cold tolerance
(Atwood et al., 2003; Cnaani et al., 2000; Nitzan et al., 2016). In this
study, the acute cold treatment was conducted to the juvenile tilapia
population. The QTL data may reflect the genetic architectures that
associated with the acute cold response of juvenile tilapia. Future
evaluation under long-term low-temperature stress may give us an
overall understanding of the genetic mechanisms of tilapia under cold
stress. In addition, the QTL identified in this tilapia line needs to be
verified in multiple populations.
4.2. Candidate Genes related to cold tolerance

In this study, 9 candidate genes, e.g., pak1ip1, loc100696456

(cathepsin Z) and loc100697261 (acetyl-coenzyme A synthetase, cyto­
plasmic) showed significant expression changes under 12 h cold stress.
The loc100697261 gene encodes an acetyl coenzyme A synthase. GO
functional annotation of the gene indicates that it plays a role in the
biosynthesis of propionate (GO:0019542), acetyl coenzyme biosynthesis
of acetate (GO:0051790) and short-chain fatty acid biosynthesis
(GO:0006629). The fluidity of membranes may be related to the low
temperature tolerance (Holmstrup et al., 2014). The increased propor­
tion of unsaturated fatty acids at low temperature may help to maintain
membrane fluidity (Shivaji and Prakash, 2010). The lipid composition of
Fig. 4. Fine mapping of the QTL on chrLG18 with SNP and microsatellite tilapia was found to correlate with its low temperature stress response in
marker data in GIFT tilapia. The X-axis indicated the physical location (Mb) of a few previous studies (He et al., 2015; Hsieh et al., 2007a; Ma et al.,
SNPs and microsatellite markers in chrLG18. The Y-axis indicated the 2015). This cytosolic process incorporates carbons from acetyl coen­
LOD values. zyme A into the growing fatty acid chain during de novo synthesis of
fatty acids (Kanoh and Lindsay, 1972). In addition, during starvation,
cells must typically shift from growth to survival mode and alter meta­
Table 2
bolism towards functions important for viability. Instead of shipping
Information of peak SNP markers significantly associated with cold-tolerance
trait.
acetyl units out to the cytosol, there is now a greater requirement for
acetyl-CoA to be oxidized in the mitochondria for ATP synthesis. Most
Marker Chr Pos LOD value Expl (%)
organisms, as well as particular tissue microenvironments in vivo
chrLG18_19,316,719 chrLG18 19,316,719 5.6 21.5 experience challenges in the nutrient environment that might limit
chrLG8_24_16,262,132 chrLG8_24 16,262,132 3.9 14.5 acetyl-CoA biosynthesis or availability (e.g., carbon starvation or hyp­
chrLG4_4,189,125 chrLG4 4,189,125 3.2 9.9
oxia) (Shi and Tu, 2015). These may suggest the gene play roles in
Chr: Chromosome. Pos: position. LOD: logarithm of odds. Expl: Percentage of response to low nutrient environment, such as in cold stress, which may
phenotypic variance explained. cause fasting to fish.
Cathepsins are a group of lysosomal proteases that have a key role in
The genetic bases of temperature tolerance traits have been studied cellular protein turnover and play important roles in stress response.
in some economic fish, such as turbot (Scophthalmus maximus) (Ma et al., Cathepsin B links oxidative stress to the activation of NLRP3 inflam­
2021), common carp (Cyprinus carpio L.) (Sun and Liang, 2004), rainbow masome (Bai et al., 2018). Loss of Cathepsin C function also induced ER
trout (Oncorhynchus mykiss)(Perry et al., 2001), Arctic charr (Salvelinus stress-mediated JNK phosphorylation accompanied by the translocation
alpinus)(Quinn et al., 2011). In tilapia, QTL for cold tolerance was re­ of mitochondrial cyt c followed by apoptotic cell death in colorectal
ported for several interspecific tilapia hybrids (O. mossambicus x carcinoma cells (Khaket et al., 2018). Cathepsin K is regulated by shear
O. aureus) using tens of microsatellites (Cnaani et al., 2003) or a cross stress (Platt et al., 2007). Differential gene expression analyses indicated
generated using a (O. niloticus×S. galilaeus) male and a that the cathepsin L (CTSL) gene was significantly upregulated (with
(O. mossambicus×O. aureus) female using microsatellite and AFLP FDR < 0.05 and log2 fold change value >1). Given that the cathepsin L
marker genotypic data (Moen et al., 2004). These studies reported QTL protein was related to invasion of the novel coronavirus (SARS-CoV-2),
for cold tolerance in hybrid tilapia. In this study, QTL analysis for cold mild cold stress might alter the susceptibility to coronavirus disease-19
tolerance in a GIFT tilapia line was performed using 1160 SNP markers. in humans (Yasukochi et al., 2021). In addition, PAK1IP1 is a nucleolar
We reported one genome-wide significant QTL on chrLG18 with the stress induced nucleolar protein with nuclearoplasmic localization,

5
C.H. Ai et al.
Fig. 5. The expression level of the gene pak1ip1, loc100696456 and loc100697261 in five tissues of GIFT tilapia. Under cold treatment, the expression of gene pak1ip1
was up-regulated in heart, gill and intestine; the expression of gene loc100697261 was down-regulated in heart, gill, intestine and brain; the expression of
loc100696456 was down-regulated in heart, muscle, gill and brain.
encodes a nucleolar factor involved in ribosomal stress response (Ross
et al., 2013). These genes may be important candidates for further
studies on cold regulation in tilapia.
5. Conclusion
In this study, we identified a genome-wide significant QTL interval
on chrLG18 associated with cold tolerance in the GIFT tilapia by using
ddRAD-seq. Six microsatellite markers were successfully developed near
or on the QTL regions of chrLG18 and was significantly associated to
cold-tolerance trait. Nine candidate genes (e.g., pak1ip1, loc100696456
and loc100697261) in this QTL were found differentially expressed
exposed to cold stress. This research supplies a basis for marker-assisted
selection of super lines with a high level of tolerance against cold stress
in the tilapia, and lays a foundation for the mining of regulatory genes
under cold stress in tilapia.
Funding
This work was supported by Key-Area Research and Development
Program of Guangdong Province (no.2021B0202020001), Independent
Research and Development Projects of Maoming Laboratory
(2021ZZ007), National Natural Science Foundation of China
(No.32072970).
Availability of data and materials
All the read data were available at the DDBJ database (BioProject
Accession: PRJDB12823).
Authors’ contributions
Jun Hong Xia: Conceptualization, Writing- Reviewing and Editing,
Supervision. Bi Jun Li: Methodology, Software, Visualization, Investi­
gation. Chun Hui Ai: Data curation, Writing- Original draft preparation,
Validation.
Ethics approval and consent to participate
All experiments in this study were approved by the Animal Care and
Use Committee of the School of Life Science at Sun Yat-Sen University
and were performed according to the regulations and guidelines estab­
lished by this committee.
6
Aquaculture 556 (2022) 738273
Consent for publication
All authors have approved the manuscript and given their consent for
publication.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We thank all anonymous reviewers for their comments on the
manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2022.738273.
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Another random document with
no related content on Scribd:
artist, the decorator, the paper mills’ agent, and, last of all, the printer and the
binder. This was not the way the old-time printers had planned their books.
With all their mechanical limitations, they had followed architectural lines kept
consistent and harmonious because controlled by a single mind, while the
finished volume of the eighteen-nineties was a composite production of many
minds, with no architectural plan. No wonder that the volumes manufactured,
even in the most famous Presses, failed to compare with those produced in
Venice by Jenson and Aldus four centuries earlier!
When I succeeded John Wilson as head of the University Press in 1895, I
determined to carry out the resolution I had formed four years earlier, while
sitting in on the Eugene Field conference, of following the example of the
early master-printers so far as this could be done amidst modern conditions.
Some of my publisher friends were partially convinced by my contention that
if the printer properly fulfilled his function he must know how to express his
clients’ mental conception of the physical attributes of prospective volumes in
terms of type, paper, presswork, and binding better than they could do it
themselves. The Kelmscott publications, which appeared at this time, were of
great value in emphasizing my contention, for William Morris placed printing
back among the fine arts after it had lapsed into a trade.
I had no idea, when I presented my plan, of persuading my friends to
produce typographical monuments. No demand has ever existed for volumes
of this type adequate to the excessive cost involved by the perfection of
materials, the accuracy of editorial detail, the supreme excellence of
typography and presswork, and the glory of the binding. Sweynheim and
Pannartz, Gutenberg’s successors, were ruined by their experiments in Greek;
the Aldine Press in Venice was saved only by the intervention of Jean Grolier;
Henri Étienne was ruined by his famous Thesaurus, and Christophe Plantin
would have been bankrupted by his Polyglot Bible had he not retrieved his
fortunes by later and meaner publications. Nor was I unmindful of similar
examples that might have been cited from more modern efforts, made by
ambitious publishers and printers.
What I wanted to do was to build low-cost volumes upon the same
principles as de luxe editions, eliminating the expensive materials but retaining
the harmony and consistency that come from designing the book from an
architectural standpoint. It adds little to the expense to select a type that
properly expresses the thought which the author wishes to convey; or to have
the presses touch the letters into the paper in such a way as to become a part
of it, without that heavy impression which makes the reverse side appear like
an example of Braille; or to find a paper (even made by machine!) soft to the
feel and grateful to the eye, on which the page is placed with well-considered
margins; or to use illustrations or decorations, if warranted at all, in such a way
as to assist the imagination of the reader rather than to divert him from the
text; to plan a title page which, like the door to a house, invites the reader to
open it and proceed, its type lines carefully balanced with the blank; or to bind
(even in cloth!) with trig squares and with design or lettering in keeping with
the printing inside.
By degrees the publishers began to realize that this could be done, and
when once established, the idea of treating the making of books as a
manufacturing problem instead of as a series of contracts with different
concerns, no one of which knew what the others were doing, found favor. The
authors also preferred it, for their literary children now went forth to the
world in more becoming dress. Thus serving in the capacity of book architect
and typographical advisor, instead of merely as a contrasting printer, these
years have been lived in a veritable Kingdom of Books, in company with
interesting people,—authors and artists as well as publishers,—in a delightfully
intimate way because I have been permitted to be a part of the great
adventure.

During these years I have seen dramatic changes. Wages were somewhat
advanced between 1891 and the outbreak of the World War, but even at this
latter date the cost of manufacturing books was less than half of what it is now.
This is the great problem which publishers have to face today. When the cost
of everything doubled after the World War, the public accepted the necessity
of paying twice the price for a theater ticket as a matter of course; but when
the retail price of books was advanced in proportion to the cost of
manufacture, there was a great outcry among buyers that authors, publishers,
and booksellers were opportunists, demanding an unwarranted profit. As a
matter of fact, the novel which used to sell at $1.35 per copy should now sell
at $2.50 if the increased costs were properly apportioned. The publisher today
is forced to decline many promising first novels because the small margin of
profit demands a comparatively large first edition.
 Unless a publisher can sell 5,000 copies as a minimum it is impossible for
him to make any profit upon a novel. Taking this as a basis, and a novel as
containing 320 pages, suppose we see how the $2.00 retail price distributes
itself. The cost of manufacture, including the typesetting, electrotype plates,
cover design, jacket, brass dies, presswork, paper, and binding, amounts to 42
cents per copy (in England, about 37 cents). The publisher’s cost of running
his office, which he calls “overhead,” is 36 cents per copy. The minimum
royalty received by an author is 10 per cent. of the retail price, which would
give him 20 cents. This makes a total cost of 98 cents a copy, without
advertising. But a book must be advertised.
Every fifty dollars spent in advertising on a five thousand edition adds a
cent to the publisher’s cost. The free copies distributed for press reviews
represent no trifling item. A thousand dollars is not a large amount to be spent
for advertising, and this means 20 cents a copy on a 5000 edition, making a
total cost of $1.18 per copy and reducing the publisher’s profit to 2 cents, since
he sells a two-dollar book to the retail bookseller for $1.20. The bookseller
figures that his cost of doing business is one-third the amount of his sales, or,
on a two-dollar book, 67 cents. This then shows a net profit to the retail
bookseller of 13 cents, to the publisher of 2 cents, and to the author of 20
cents a copy.
Beyond this, there is an additional expense to both bookseller and
publisher which the buyer of books is likely to overlook. It is impossible to
know just when the demand for a book will cease, and this means that the
publisher and the bookseller are frequently left with copies on hand which
have to be disposed of at a price below cost. This is an expense that has to be
included in the book business just as much as in handling fruit, flowers, or
other perishable goods.
When a publisher is able to figure on a large demand for the first edition,
he can cut down the cost of manufacture materially; but, on the other hand,
this is at least partially offset by the fact that authors whose books warrant
large first editions demand considerably more than 10 per cent. royalty, and
the advertising item on a big seller runs into large figures.

I wish I might say that I had seen a dramatic change in the methods
employed in the retail bookstores! There still exists, with a few notable
exceptions, the same lack of realization that familiarity with the goods one has
to sell is as necessary in merchandizing books as with any other commodity.
Salesmen in many otherwise well-organized retail bookstores are still painfully
ignorant of their proper functions and indifferent to the legitimate
requirements of their prospective customers.
Some years ago, when one of my novels was having its run, I happened to
be in New York at a time when a friend was sailing for Europe. He had
announced his intention of purchasing a copy of my book to read on the
steamer, and I asked him to permit me to send it to him with the author’s
compliments. Lest any reader be astonished to learn that an author ever buys a
copy of his own book, let me record the fact that except for the twelve which
form a part of his contract with the publisher, he pays cash for every copy he
gives away. Mark Twain dedicated the first edition of The Jumping Frog to “John
Smith.” In the second edition he omitted the dedication, explaining that in
dedicating the volume as he did, he had felt sure that at least all the John
Smiths would buy books. To his consternation he found that they all expected
complimentary copies, and he was hoist by his own petard!
With the idea of carrying out my promise to my friend, I stepped into one
of the largest bookstores in New York, and approached a clerk, asking him for
the book by title. My pride was somewhat hurt to find that even the name was
entirely unfamiliar to him. He ran over various volumes upon the counter, and
then turned to me, saying, “We don’t carry that book, but we have several
others here which I am sure you would like better.”
“Undoubtedly you have,” I agreed with him; “but that is beside the point. I
am the author of the book I asked for, and I wish to secure a copy to give to a
friend. I am surprised that a store like this does not carry it.”
Leaning nonchalantly on a large, circular pile of books near him, the clerk
took upon himself the education of the author.
“It would require a store much larger than this to carry every book that is
published, wouldn’t it?” he asked cheerfully. “Of course each author naturally
thinks his book should have the place of honor on the bookstalls, but we have
to be governed by the demand.”
It was humiliating to learn the real reason why this house failed to carry
my book. I had to say something to explain my presumption even in assuming
that I might find it there, so in my confusion I stammered,
 “But I understood from the publishers that the book was selling very
well.”
“Oh, yes,” the clerk replied indulgently; “they have to say that to their
authors to keep them satisfied!”
With the matter thus definitely settled, nothing remained but to make my
escape as gracefully as circumstances would permit. As I started to leave, the
clerk resumed his standing position, and my eye happened to rest on the pile
of perhaps two hundred books upon which he had been half-reclining. The
jacket was strikingly familiar. Turning to the clerk I said severely,
“Would you mind glancing at that pile of books from which you have just
risen?”
“Oh!” he exclaimed, smiling and handing me a copy, “that is the very book
we were looking for, isn’t it?”
It seemed my opportunity to become the educator, and I seized it.
“Young man,” I said, “if you would discontinue the practice of letting my
books support you, and sell a few copies so that they might support me, it
would be a whole lot better for both of us.”
“Ha, ha!” he laughed, graciously pleased with my sally; “that’s a good line,
isn’t it? I really must read your book!”

The old-time publisher is passing, and the author is largely to blame. I have
seen the close association—in many cases the profound friendship—between
author and publisher broken by the commercialism fostered by some literary
agents and completed by competitive bids made by one publishing house to
beguile a popular author away from another. There was a time when a writer
was proud to be classified as a “Macmillan,” or a “Harper” author. He felt
himself a part of the publisher’s organization, and had no hesitation in taking
his literary problems to the editorial advisor of the house whose imprint
appeared upon the title pages of his volumes. A celebrated Boston authoress
once found herself absolutely at a standstill on a partially completed novel. She
confided her dilemma to her publisher, who immediately sent one of his
editorial staff to the rescue. They spent two weeks working together over the
manuscript, solved the problems, and the novel, when published, was the most
successful of the season.
Several publishers have acknowledged to me that in offering unusually
high royalties to authors they have no expectation of breaking even, but that to
have a popular title upon their list increases the sales of their entire line. The
publisher from whom the popular writer is filched has usually done his share
in helping him attain his popularity. The royalty he pays is a fair division of the
profits. He cannot, in justice to his other authors, pay him a further premium.
Ethics, perhaps, has no place in business, but the relation between author
and publisher seems to me to be beyond a business covenant. A publisher may
deliberately add an author to his list at a loss in order to accomplish a specific
purpose, but this practice cannot be continued indefinitely. A far-sighted
author will consider the matter seriously before he becomes an opportunist.

In England this questionable practice has been of much slower growth.

The House of Murray, in London, is one of those still conducted on the old-
time basis. John Murray IV, the present head of the business, has no interest in
any author who comes to him for any reason other than a desire to have the
Murray imprint upon his book. It is more than a business. The publishing
offices at 50a, Albemarle Street adjoin and open out of the Murray home. In
the library is still shown the fireplace where John Murray III burned Byron’s
Memoirs, after purchasing them at an enormous price, because he deemed that
their publication would do injury to the reputation of the writer and of the
House itself.
John Murray II was one of the publishers of Scott’s Marmion. In those days
it was customary for publishers to share their contracts. Constable had
purchased from Scott for £1,000 the copyright of Marmion without having
seen a single line, and the honorarium was paid the author before the poem was
completed or the manuscript delivered. Constable, however, promptly
disposed of a one-fourth interest to Mr. Miller of Albemarle Street, and
another one fourth to John Murray, then of Fleet Street.
By 1829 Scott had succeeded in getting into his own hands nearly all his
copyrights, one of the outstanding items being this one-quarter interest in
Marmion held by Mr. Murray. Longmans and Constable had tried in vain to
purchase it. When, however, Scott himself approached Murray through
Lockhart, the following letter from Mr. Murray was the result:
So highly do I estimate the honour of being even in so small a degree the publisher
of the author of the poem that no pecuniary consideration whatever can induce me to
part with it. But there is a consideration of another kind that would make it painful
to me if I were to retain it a moment longer. I mean the knowledge of its being required
by the author, into whose hands it was spontaneously resigned at the same instant that
I read the request.
There has always been a vast difference in authors in the attitude they
assume toward the transformation of their manuscripts into printed books.
Most of them leave every detail to their publishers, but a few take a deep and
intelligent personal interest. Bernard Shaw is to be included in the latter group.
A leading Boston publisher once telephoned me that an unknown English
author had submitted a manuscript for publication, but that it was too
socialistic in its nature to be acceptable. Then the publisher added that the
author had asked, in case this house did not care to publish the volume, that
arrangements be made to have the book printed in this country in order to
secure American copyright.
“We don’t care to have anything to do with it,” was the statement; “but I
thought perhaps you might like to manufacture the book.”
“Who is the author?” I inquired.
“It’s a man named Shaw.”
“What is the rest of his name?”
“Wait a minute and I’ll find out.”
Leaving the telephone for a moment, the publisher returned and said,
“His name is G. Bernard Shaw. Did you ever hear of him?”
“Yes,” I replied; “I met him last summer in London through Cobden-
Sanderson, and I should be glad to undertake the manufacture of the book for
Mr. Shaw.”
“All right,” came the answer. “Have your boy call for the manuscript.”
This manuscript was Man and Superman.
 From that day and for many years, Shaw and I carried on a desultory
correspondence, his letters proving most original and diverting. On one
occasion he took me severely to task for having used two sizes of type upon a
title page. He wrote four pages to prove what poor taste and workmanship this
represented, and then ended the letter with these words, “But, after all, any
other printer would have used sixteen instead of two, so I bless you for your
restraint!”
We had another lengthy discussion on the use of apostrophes in printing.
“I have made no attempt to deal with the apostrophes you introduce,” he
wrote; “but my own usage is carefully considered and the inconsistencies are
only apparent. For instance, Ive, youve, lets, thats, are quite unmistakable, but Ill,
hell, shell, for I’ll, he’ll, she’ll, are impossible without a phonetic alphabet to
distinguish between long and short e. In such cases I retain the apostrophe, in
all others I discard it. Now you may ask me why I discard it. Solely because it
spoils the printing. If you print a Bible you can make a handsome job of it
because there are no apostrophes or inverted commas to break up the
letterpress with holes and dots. Until people are forced to have some
consideration for a book as something to look at as well as something to read,
we shall never get rid of these senseless disfigurements that have destroyed all
the old sense of beauty in printing.”
“Ninety-nine per cent. of the secret of good printing,” Shaw continued, “is
not to have patches of white or trickling rivers of it trailing down a page, like
rain-drops on a window. Horrible! White is the enemy of the printer. Black,
rich, fat, even black, without gray patches, is, or should be, his pride. Leads and
quads and displays of different kinds of type should be reserved for insurance
prospectuses and advertisements of lost dogs.…”
His enthusiasm for William Morris’ leaf ornaments is not shared by all
booklovers. Glance at any of the Kelmscott volumes, and you will find these
glorified oak leaves scattered over the type page in absolutely unrelated
fashion,—a greater blemish, to some eyes, than occasional variation in spacing.
Shaw writes:
If you look at one of the books printed by William Morris, the greatest printer of
the XIX century, and one of the greatest printers of all the centuries, you will see that
he occasionally puts in a little leaf ornament, or something of the kind. The idiots in
America who tried to imitate Morris, not understanding this, peppered such things all
over their “art” books, and generally managed to stick in an extra large quad before
each to show how little they understood about the business. Morris doesn’t do this in
his own books. He rewrites the sentence so as to make it justify, without bringing one
gap underneath another in the line above. But in printing other people’s books, which
he had no right to alter, he sometimes found it impossible to avoid this. Then, sooner
than spoil the rich, even color of his block of letterpress by a big white hole, he filled it
up with a leaf.
Do not dismiss this as not being “business.” I assure you, I have a book which
Morris gave me, a single copy, by selling which I could cover the entire cost of printing
my books, and its value is due solely to its having been manufactured in the way I
advocate; there’s absolutely no other secret about it; and there is no reason why you
should not make yourself famous through all the ages by turning out editions of
standard works on these lines whilst other printers are exhausting themselves in dirty
felt end papers, sham Kelmscott capitals, leaf ornaments in quad sauce, and then
wondering why nobody in Europe will pay twopence for them, whilst Kelmscott books
and Doves Press books of Morris’ friends, Emery Walker and Cobden-Sanderson,
fetch fancy prices before the ink is thoroughly dry.… After this I shall have to get you
to print all my future books, so please have this treatise printed in letters of gold and
preserved for future reference
CHAPTER III

Friends through Type

 III
FRIENDS THROUGH TYPE

In 1903 I again visited Italy to continue my study of the art of printing in

the old monasteries and libraries, sailing on the S. S. Canopic from Boston to
Naples. Among the passengers on board I met Horace Fletcher, returning to
his home in Venice. At that time his volume Menticulture was having a
tremendous run. I had enjoyed reading the book, and in its author I discovered
a unique and charming personality; in fact, I have never met so perfect an
expression of practical optimism. His humor was infectious, his philosophy
appealing, his quiet persistency irresistible.
To many people the name of Horace Fletcher has become associated with
the Gladstonian doctrine of excessive chewing, but this falls far short of the
whole truth. His scheme was the broadest imaginable, and thorough
mastication was only the hub into which the other spokes of the wheel of his
philosophy of life were to be fitted. The scheme was nothing less than a
cultivation of progressive human efficiency. Believing that absolute health is
the real basis of human happiness and advancement, and that health depends
upon an intelligent treatment of food in the mouth together with knowledge
of how best to furnish the fuel that is actually required to run the human
engine, Horace Fletcher sought for and found perfect guides among the
natural human instincts and physiologic facilities, and demonstrated that his
theories were facts.

During the years that followed I served as his typographic mentor. He was
eager to try weird and ingenious experiments to bring out the various points of
his theories through unique typographical arrangement (see opp. page). It
required all my skill and diplomacy to convince him that type possessed rigid
limitations, and that to gain his emphasis he must adopt less complicated
methods. From this association we became the closest of friends, and
presuming upon this relation I used to banter him upon being so casual. His
copy was never ready when the compositors needed it; he was always late in
returning his proofs. The manufacture of a Fletcher book was a hectic
experience, yet no one ever seemed to take exceptions. This was characteristic
of the man. He moved and acted upon suddenly formed impulses, never
planning ahead yet always securing exactly what he wanted, and those
inconvenienced the most always seemed to enjoy it.

A Page of Horace Fletcher Manuscript

“I believe,” he used to say, “in hitching one’s wagon to a star, but I always
keep my bag packed and close at hand ready to change stars at a moment’s
notice. It is only by doing this that you can give things a chance to happen to
you.”
 Among the volumes Fletcher had with him on board ship was one he had
purchased in Italy, printed in a type I did not recognize but which greatly
attracted me by its beauty. The book bore the imprint: Parma: Co’tipi Bodoniani.
Some weeks later, in a small, second-hand bookstore in Florence, I happened
upon a volume printed in the same type, which I purchased and took at once
to my friend, Doctor Guido Biagi, at the Laurenziana Library.
“The work of Giambattista Bodoni is not familiar to you?” he inquired in
surprise. “It is he who revived in Italy the glory of the Aldi. He and Firmin
Didot in Paris were the fathers of modern type design at the beginning of the
nineteenth century.”
“Is this type still in use?” I inquired.
“No,” Biagi answered. “When Bodoni died there was no one worthy to
continue its use, so his matrices and punches are kept intact, exactly as he left
them. They are on exhibition in the library at Parma, just as the old Plantin
relics are preserved in the museum at Antwerp.”
 GIAMBATTISTA BODONI, 1740–1813
From Engraving at the Bibliothèque Nationale, Paris

I immediately took steps through our Ambassador at Rome to gain

permission from the Italian Government to recut this face for use in America.
After considerable difficulty and delay this permission was granted, with a
proviso that I should not allow any of the type made from my proposed
matrices to get into the hands of Italian printers, as this would detract from
the prestige of the city of Parma. It was a condition to which I was quite
willing to subscribe! Within a year I have received a prospectus from a revived
Bodoni Press at Montagnola di Lugano, Switzerland, announcing that the
exclusive use of the original types of Giambattista Bodoni has been given them
by the Italian Government. This would seem to indicate that the early
governmental objections have disappeared.
While searching around to secure the fullest set of patterns, I stumbled
upon the fact that Bodoni and Didot had based their types upon the same
model, and that Didot had made use of his font particularly in the wonderful
editions published in Paris at the very beginning of the nineteenth century. I
then hurried to Paris to see whether these matrices were in existence. There,
after a search through the foundries, I discovered the original punches, long
discarded, in the foundry of Peignot, to whom I gave an order to cast the
different sizes of type, which I had shipped to America.
This was the first type based on this model ever to come into this country.
The Bodoni face has since been recut by typefounders as well as for the
typesetting machines, and is today one of the most popular faces in common
use. Personally I prefer the Bodoni letter to that of Didot (see opp. page). The
Frenchman succumbed to the elegance of his period, and by lightening the
thin lines robbed the design of the virility that Bodoni retained. I am not in
sympathy with the excessive height of the ascending letters, which frequently
extend beyond the capitals; but when one considers how radical a departure
from precedent this type was, he must admire the skill and courage of the
designers. William Morris cared little for it,—“The sweltering hideousness of
the Bodoni letter,” he exclaimed; “the most illegible type that was ever cut,
with its preposterous thicks and thins”; while Theodore L. De Vinne, in his
Practice of Typography, writes:
The beauty of the Bodoni letters consists in their regularity, in their clearness, and
in their conformity to the taste of the race, nation, and age in which the work was first
written, and finally in the grace of the characters, independent of time or place.
When authorities differ to such a wide extent, the student of type design
must draw his own conclusions!
 The Bodoni Letter (bottom) compared with the Didot Letter (top)

Fletcher’s idea of an appointment was something to be kept if or when

convenient, yet he never seemed to offend any one. He did nothing he did not
wish to do, and his methods of extricating himself from unwelcome
responsibilities always amused rather than annoyed. “If you don’t want to do a
thing very badly,” he confided to me on one such occasion, “do it very badly.”
 HORACE FLETCHER IN 1915

On board the Canopic Fletcher was surrounded by an admiring and

interested group. General Leonard Wood was on his way to study colonial
government abroad before taking up his first administration as Governor of
the Philippines. On his staff was General Hugh Lennox Scott, who later
succeeded General Wood as Chief of Staff of the United States Army. The
conversations and discussions in the smokeroom each evening after dinner
were illuminating and fascinating. General Wood had but recently completed
his work as Governor of Cuba, and he talked freely of his experiences there,
while General Scott was full of reminiscences of his extraordinary adventures
with the Indians. He later played an important part in bringing peace to the
Philippines.
 It was at one of these four-cornered sessions in the smokeroom that we
first learned of Fletcher’s ambition to revolutionize the world in its methods of
eating. That he would actually accomplish this no one of us believed, but the
fact remains. The smokeroom steward was serving the coffee, inquiring of
each one how many lumps of sugar he required. Fletcher, to our amazement,
called for five! It was a grand-stand play in a way, but he secured his audience
as completely as do the tambourines and the singing of the Salvation Army.
“Why are you surprised?” he demanded with seeming innocence. “I am
simply taking a coffee liqueur, in which there is less sugar now than there is in
your chartreuse or benedictine. But I am mixing it with the saliva, which is
more than you are doing. The sugar, as you take it, becomes acid in the
stomach and retards digestion; by my method, it is changed into grape sugar,
which is easily assimilated.”
“To insalivate one’s liquor,” he explained to us, “gives one the most
exquisite pleasure imaginable, but it is a terrific test of quality. It brings out the
richness of flavor, which is lost when one gulps the wine down. Did you ever
notice the way a tea-taster sips his tea?”
As he talked he exposed the ignorance of the entire group on physiological
matters to an embarrassing extent, clinching his remarks by asking General
Wood the question,
“Would you engage as chauffeur for your automobile a man who knew as
little about his motor as you know about your own human engine?”
No one ever loved a practical joke better than Horace Fletcher. I was a
guest at a dinner he once gave at the Graduates’ Club in New Haven. Among
the others present were President Hadley of Yale, John Hays Hammond,
Walter Camp, and Professor Lounsbury. There was considerable curiosity and
some speculation concerning what would constitute a Fletcher dinner. At the
proper time we were shown into a private room, where the table was set with
the severest simplicity. Instead of china, white crockery was used, and the chief
table decorations were three large crockery pitchers filled with ice water. At
each plate was a crockery saucer, containing a shredded-wheat biscuit. It was
amusing to glance around and note the expressions of dismay upon the faces
of the guests. Their worst apprehensions were being confirmed! Just as we
were well seated, the headwaiter came to the door and announced that by
mistake we had been shown into the wrong room, whereupon Fletcher, with
an inimitable twinkle in his eye, led the way into another private dining room,
where we sat down to one of the most sumptuous repasts I have ever enjoyed.
Today, twenty years after his campaign, it is almost forgotten that the
American breakfast was at that time a heavy meal. Horace Fletcher
revolutionized the practice of eating, and interjected the word fletcherize into
the English language. As a disciple of Fletcher Sir Thomas Barlow, physician-
in-chief to King Edward VII, persuaded royalty to set the style by cutting
down the formal dinner from three hours to an hour and a half, with a
corresponding relief to the digestive apparatus of the guests. In Belgium,
during the World War, working with Herbert Hoover, Fletcher taught the
impoverished people how to sustain themselves upon meager rations. Among
his admirers and devoted friends were such profound thinkers as William
James who, in response to a letter from him, wrote, “Your excessive reaction
to the stimulus of my grateful approval makes you remind me of those rich
soils which, when you tickle them with a straw, smile with a harvest”; and
Henry James, who closes a letter: “Come and bring with you plenary
absolution to the thankless subject who yet dares light the lamp of gratitude to
you at each day’s end of his life.”

My acquaintance with Henry James came through my close association

with the late Sir Sidney Lee, the Shakesperian authority, and Horace Fletcher.
“Don’t be surprised if he is brusque or uncivil,” Sir Sidney whispered to
me just before I met him at dinner; “one can never tell how he is going to act.”
As a matter of fact, I found Henry James a most genial and enjoyable
dinner companion, and never, during the few later occasions when I had the
pleasure of being with him, did he display those characteristics of ill humor
and brusqueness which have been attributed to him. It may not be generally
known that all his life—until he met Horace Fletcher—he suffered torments
from chronic indigestion, or that it was in Fletcherism that he found his first
relief. In a typically involved Jamesian letter to his brother William he writes
(February, 1909):
It is impossible save in a long talk to make you understand how the blessed
Fletcherism—so extra blessed—lulled me, charmed me, beguiled me, from the first
into the convenience of not having to drag myself out into eternal walking. One must