Tree Genetics & Genomes (2010) 6:613–625
DOI 10.1007/s11295-010-0276-z
OPINION PAPER
The emerging role of genomic tools in mulberry (Morus)
genetic improvement
Kunjupillai Vijayan
Received: 7 September 2009 / Revised: 5 December 2009 / Accepted: 26 January 2010 / Published online: 23 February 2010
# Springer-Verlag 2010
Abstract Mulberry (Morus L) is one of the economically
important trees that have a long history of extensive
cultivation in Asia. Mulberry leaf is the sole food for the
silkworm Bombyx mori; hence, the sustainability of the
sericulture industry is dependent on the continuous supply
of nutritious mulberry leaf. Genetic improvement of
mulberry for leaf yield, leaf nutritional contents, and
adaptability has great socioeconomical importance because
the sericulture is the backbone of rutal economy in several
Asian countries. Much effort has been made on germplasm
collection, characterization, and varietal development
through conventional methods. However, the complexity
of important agronomic traits, the substantial lag time
between germination and sexual maturity, the absence of
inbred lines, and the outbreeding nature of mulberry delay
the genetic improvement via conventional breeding techni-
ques. In this postgenomic era, having the next generation
sequencing facilities in its fold, it is possible to integrate
modern genomic tools with conventional breeding tech-
niques to dissect the complex traits into their individual
components to gain better control over them. In mulberry,
such efforts are lacking. This paper, therefore, summarizes
the current position of genetic and genomics research in
mulberry and discusses the directions for future research,
utilizing the emerging technologies in molecular markers,
association genetics, quantitative trait locus mapping, tran-
scriptomics, metabolomics, marker-assisted selection breed-
ing, and transgenesis.
Communicated by: E. Dirlewanger.
K. Vijayan (*)
Institute of Plant and Microbial Biology, Academia Sinica,
Taipei, Taiwan 115, Republic of China
e-mail: kvijayan01@yahoo.com
Keywords Association mapping . Metabolomics .
Mulberry . Trancriptomics . Sericulture . QTL mapping
Introduction
Mulberry (Morus L; Moraceae) is a fast-growing deciduous
woody perennial tree belonging to the family Moraceae.
Mulberry has tremendous economic importance in most of
the developing countries in Asia, where sericulture is an
important industry. The silkworm Bombyx mori L. exclu-
sively feeds the leaf of mulberry, and hence, the sustain-
ability of sericulture heavily depends on the productivity of
high-quality mulberry leaves. Mulberry is also used as
animal fodder because it is highly nutritious, palatable, and
digestible (70–90%; Sanchez 2000a, b). Fruits of mulberry
are used for human consumption either as fresh fruit or in
the form of various confectionary products such as jam,
marmalade, frozen desserts, pulp, juice, paste, ice cream,
and wine (Soufleros et al. 2004; Yua et al. 2008). In spite of
this high commercial importance, very little effort has been
made to harness the benefits of the recent technological
advancements in plant genomics for mulberry genetic
improvement. Therefore, this article is intended to provide
an overview of the genomic tools that are suitable for
enhancing the productivity and adaptability in mulberry.
Origin, distribution, and cytology
Mulberry originated in the northern hemisphere, particular-
ly in the Himalayan foothills, and later it spread into the
tropics of the southern hemisphere (Hou 1994; Benavides et
al. 1994). Today, it is present in all regions between 50° N
lat. and 10° S lat. (Yokoyama 1962), from sea level to
614 Tree Genetics & Genomes (2010) 6:613–625

altitudes as high as 4,000 m (Machii et al. 1999; Tutin Table 1 Mulberry species recognized by Koidzumi (1917)
1996), which include Asia, Europe, North and South Sl. no. Species
America, and Africa. Although the taxonomy of mulberry
is yet to be resolved fully, according to the widely accepted 1 M. bombycis Koidz.
classification of Koidzumi (1917), the genus Morus is 2 M. alba L.
divided into two sections, the Dolichostylae (long style) 3 M. indica L.
and the Macromorus (short style), and each section is again 4 M. kagayamae Koidz.
divided into two groups namely Papillosae and Pubescen- 5 M. boninensis Koidz.
tae, based on the nature of stigmatic hairs. A total of 24 6 M. atropurpurea Roxb.
species and one subspecies are recognized in this classifi- 7 M. laevigata Wall.
cation (Table 1). Currently, however, more than 68 species 8 M. formosensis Hotta
are widely recognized (Datta 2000). Out of which, only a 9 M. mesozygia Stapf.
few species such as Morus alba, Morus indica, Morus 10 M. cathayana Hemsl.
bombycis, Morus latifolia, and Morus multicaulis are 11 M. microphylla Bickl.
cultivated for leaves to feed the silkworm while another 12 M. rabica Koidz.
species, Morus nigra, is cultivated for its fruits. Cytolog- 13 M. latifolia Poir.
ically, mulberry exhibits different ploidy levels. Most of the 14 M. acidosa Griff.
cultivating species are diploids (2x, 2n=28). But triploids 15 M. rotundiloba Koidz.
(3x, 3n=42; M. bombycis), tetraploids (4x, 4n=56; Morus 16 M. notabilis C. K. Schn.
laevigata, Morus cathayana, and Morus boninensis), 17 M. nigriformis Koidz.
hexaploids (6x, 6n=84; Morus serrata and Morus tiliaefo- 18 M. serrata Roxb.
lia), octoploids (8x, 8n=112; M. cathayana), and docosa- 19 M. nigra L.
ploids (22x, 22n=308; M. nigra) (Basavaiah et al. 1989) 20 M. rubra L.
and even haploids (M. notabilis) with 14 chromosomes are 21 M. celtidifolia Kunth
also available in nature (Maode et al. 1996).
22 M. tiliaefolia Makino
23 M. macroura Miq.
24 M. multicaulis Perr.
Phenotypic and genotypic variations

Owing to the great economic importance, large numbers of
mulberry germplasm accessions have been maintained in
several countries. For instance, China, India, Japan, Korea,
and Bulgaria have, respectively, more than 1,860, 1,120, 1,375,
615, and 140 germplasm accessions (FAO 2003; Machii et al.
1999; Pan 2000; Tzenov 2002; Tikader and Dandin 2006;
Tikader et al. 2009). Since information on phenotypic and
genotypic variability is vital for effective utilization of
germplasm accessions for long-term improvement of leaf
yield, leaf quality, adaptation, and resistance to pests and
diseases, efforts have continuously been made to characterize
and evaluate germplasm accessions. Evaluations based on
traits related to growth, development, leaf yield, leaf quality,
and adaptability to various agroclimatic conditions (Banerjee
et al. 2007; Bindroo et al. 1996; Machii et al. 1997, 2000;
Rajan et al. 1997; Suryanarayana et al. 2002; Tikader et al.
2003; Tikader and Kamble 2008a, 2009; Vijayan et al. 1999a,
b) and screening for tolerance to abiotic and biotic stresses
(Sujathamma and Dandin 1998; Susheelamma and Jolly
1986; Susheelamma et al. 1990; Yadav et al. 1993; Vijayan
et al. 2003, 2004a) revealed the presence of considerable
genetic variability among the germplasm accessions. Based
on the genetic distance estimated with different statistical
methods, the germplasm accessions were grouped into
different clusters, and utilizing this information, parental
selections were made for different breeding programs (Machii
et al. 1997, 2000; Rajan et al. 1997; Vijayan et al. 1999a, b;
Tikader et al. 2003; Tikader and Kamble 2008a, 2009).
Highly heritable morphological characters were documented
and utilized for the development and registration of varieties
(Rao 2003; Vijayan et al. 1997a, c). However, phenotypic
evaluation has several limitations as most of the agronomi-
cally important traits that are used for evaluations vary greatly
depending upon the developmental stages and environmental
conditions, and collection of data is time consuming,
laborious, and expensive and is influenced by human bias.
Therefore, molecular markers, which are highly polymorphic,
multiallelic, nonepistatic, neutral, and insensitive to environ-
ment influence, have been used for genetic evaluation of
mulberry germplasm resources. However, most of these
molecular markers that have been used for mulberry genetic
resource assessment are of anonymous types such as random
amplification of polymorphic DNA (RAPD; Bhattacharya and
Ranade 2001; Chatterjee et al. 2004; Lou et al. 1998;
Srivastava et al. 2004; Xiang et al. 1995; Zhao and Pan
2004), amplified fragment length polymorphism (AFLP;
Botton et al. 2005; Kafkas et al. 2008; Sharma et al. 2000;
Wang and Yu 2001), and intersimple sequence repeats (ISSR;
Tree Genetics & Genomes (2010) 6:613–625
Awasthi et al. 2004; Vijayan and Chatterjee 2003; Vijayan
2004; Vijayan et al. 2004b, c, 2005, 2006a, b; Zhao et al.
2006). Nonetheless, these studies further confirmed the high
genetic diversity among the accessions. Recently, more
reliable, robust, codominant, and better informative markers
such as simple sequence repeats (SSR) markers have been
developed in mulberry (Agarwal et al. 2004; Zhao et al.
2005). A list of the 16 SSR primers along with their
nucleotide sequences and related information is available in
Tikader et al. (2009). Thus, a number of proven marker
systems are now available for mulberry germplasm assess-
ment. Using these marker systems, effective assessments of
mulberry germplasm to identify genetically distant accessions
with desirable traits can be made to assist the breeders for
selection of parents. In the meantime, efforts can also be made
to develop single-nucleotide polymorphism (SNP) markers in
mulberry, as these are the abundant markers present in any
genome (Collins et al. 1997). Hence, SNP can make
genotyping more effective and faster than any other marker
systems. Owing to the current impetus on mulberry genomic
research, large numbers of expressed sequence tags (ESTs)
from mulberry have been started to be deposited in GenBank
(Lal et al. 2009; Zhao 2008; Sajeevan et al. unpublished).
Presently, nearly 1,366 ESTs from healthy and stress-
subjected plants of M. alba and M. indica are available.
Using these ESTs, new markers such as SNPs and SSRs can
be developed. Likewise, attempts can also be devoted to
develop SNPs from ISSR and RAPD markers that are
associated with valuable phenotypic traits through locus-
specific amplification and comparative resequencing from
multiple individuals (Rieder et al. 1998;). Recently, chloro-
plast genome of mulberry (M. indica cv. K2) has been
completely sequenced (Ravi et al. 2006). This 158,484-bp
circular DNA contains two identical inverted repeats of
25,678 bp each, separating a large single-copy region of
87,386 bp and a small single-copy region of 19,742 bp. The
sequence information on the cpDNA can be of much use to
develop suitable polymerase chain reaction (PCR) primers for
phylogenetic assessments, evolutionary investigations (Cruzen
et al. 1993), and bar coding of mulberry species (CBOL plant
working group 2009). Furthermore, information on the
complete sequence of chloroplast DNA has great potential
for manipulating the photosynthetic efficiency of mulberry
through genetic engineering because a large number genes
involved in the process of photosynthesis are located in the
chloroplast.
Trait improvement through conventional breeding
In mulberry, leaf is the primary product and, therefore, leaf
productivity was the principal trait targeted by most of the
breeding schemes. Leaf productivity is a multifactorial trait
615
that depends on a number of quantitative traits such as plant
height, number of branches, leaf retention capacity, nodal
length, leaf size and weight, total biomass, etc. (Bindroo et
al. 1990; Sahu et al. 1995; Tikader and Kamble 2008a,
2009; Vijayan et al. 1997b). The other important traits that
have been targeted by the mulberry breeders are the
adaptability, resistance to pests and diseases, tolerance to
abiotic stresses like drought, salinity, and cold, higher
vegetative propagation ability, better leaf quality, and better
coppicing ability. Coppicing ability is important in tropical
sericulture zones where silkworms are reared four to five
times per year, and to feed the silkworms with fresh leaves,
mulberry is pruned regularly for four to five times per year
to obtain juvenile shoots. Since mulberry is a cross-
pollinated perennial plant with high heterozygosity and
long juvenile period, traditional breeding methodologies
mostly relied on the production of F1 hybrids (Das 1984).
F1 hybrids are obtained either through controlled crossing
or by collecting open-pollinated seeds from selected female
parents. Intensive within-family selection and clonal prop-
agation were adopted to multiply superior hybrids. Once a
hybrid with desirable traits is identified, it is mass
multiplied for further assessment. Based on its performance
in multilocation trials, varieties suitable for a particular
agroclimatic zone is selected and released for filed
cultivations (Fig. 1). Mulberry varieties are multiplied
clonally (planting stem cuttings) to capture the additive
and nonadditive variations. Using these breeding methods,
leaf productivity has been increased significantly. Major
silk-producing countries like China and India have released
a number of mulberry cultivars for commercial exploitation
(Tikader et al. 2009; Vijayan 2009). In spite of the large
germplasm collections, breeders so far have exploited only
a few accessions mostly from M. alba and a few other
species closely related to it (Tikader and Kamble 2008b),
leaving huge genetic resources unutilized. This reduced
genetic base makes them susceptible to disease epidemics
and reduces the chance of their further improvement using
new combinations of genes (Tikader and Dandin 2007).
This limited use of germplasm resources is mainly due to
the paucity of information on the genetic control of most of
the agronomically important traits. Thus, it is difficult to
transfer desirable traits from the unadapted genotypes to the
elite lines. Introgression of characters from wild relatives to
the elite breeding line is, often, accompanied by undesirable
traits that are difficult to be removed because of their close
physical linkage with the desired traits, a phenomenon
called linkage drag. In order to get rid of these undesired
traits, back crossings are to be made for several generations.
Mulberry being a tree crop with long juvenile period, back
crossing for many generations is highly time consuming
and laborious and is almost prohibitive. Similarly, no inbred
lines or doubled haploid lines are available to study the
616
Germplasm evaluation 4-5 yrs
Promising cultivars/land races
Parental selection
Hybridization and seed collection
(Controlled crossing/Open pollination)
4-5 yrs
Seedling nurseries
Seedling assessment/
Progeny row Trials
(Growth and branching, leaf yield)
Primary Yield Trials with selections from PRT
(Growth, leaf yield, incidence of pests and diseases) 5-6 yrs
Final Yield Trails with selections
from PYTs 5-6 yrs
(Growth, leaf yield, propagation efficiency,
incidence of pests and diseases, bioassay)
Multi Location Trials with selections from FYT by
regional research stations under a breeding institute 5-6 yrs
Variety for that specific region
(Growth, leaf yield, adaptability, incidence of pests and diseases
under different cultural conditions of that zone)
All India Coordinated Trials with outstanding
selections from different breeding Institutes 5-6 yrs
(Growth, leaf yield, adaptability, incidence of pests
and diseases under different agro-climatic conditions)
Registration, mass multiplication and variety release 1-2 yrs
Fig. 1 Developmental stages of new mulberry varieties through
conventional breeding
genetic controls on important agronomic traits. Therefore,
except a few crossability, heritability, and combining ability
studies (Dandin et al. 1987; Dwivedi et al. 1989; Tikader
and Dandin 2001; Vijayan et al. 1997a, 2008), no effort was
made to understand the genetic control of major growth and
adaptability traits. The major challenges, thus, faced by the
mulberry breeders, such as (1) understanding the basis of
heterosis and prediction of hybrid performance, (2) identi-
fication of useful genetic factors in divergent populations or
lines, (3) introgression of desired traits with minimal
linkage drag, (4) understanding the genotype by environ-
ment interaction, remain unresolved. In order to resolve
these problems, proper understanding on (a) the number of
genetic factors (loci) influencing the expression of the traits,
(b) the chromosomal location of these loci, (c) the relative
size of the contribution of individual loci to trait expression,
(d) pleiotropic effects, (e) epistatic interactions among
genetic factors, and (f) variation of expression of individual
factors in different environments are required. This neces-
sitates the integration of recent advances in genomic
research with conventional breeding techniques to dissect
Tree Genetics & Genomes (2010) 6:613–625
the complexity of most of these traits to increase the
productivity and nutritional quality of the leaves and the
adaptability of the mulberry for sustaining the sericulture
industry.
Genomic research in mulberry
Thus, it is evident that growth-related traits play a
significant role in leaf productivity and adaptations in
mulberry. Understanding on these traits through genomic
approach is essential to tackle them effectively and
efficiently. Although much advancement has been made in
silkworm genomic research, the same could not be
achieved in mulberry. However, in many tree species like
Eucalyptus (Gratapaglia and Kirst 2008), Pinus (Pot et al.
2006), and Populus (Rae et al. 2006) both forward and
reverse genomic approaches were proven their worth in
elucidating genes involved in the expression of different
traits and chemical pathways. Generally, in forward
genomics, existing phenotypic traits are analyzed to
identify the underlying genetic variations whereas in
reverse genomic manipulation of specific gene through
transgenic technologies such as insertional mutagenesis,
overexpression of genes, and RNA interference (RNAi) and
miRNA approaches is used to study the gene–trait
relationship. Reverse genomic methods are, however,
mostly species specific, expensive, and transgenic or only
transiently disrupt gene functions. Forward genomic
approaches, thus, seem to be well suited for mulberry as
considerable phenotypic variation is present in the natural
populations for the important traits like leaf yield, leaf
quality, and adaptability. The commonly used technologies
in forward genomics are genotyping, genetic mapping,
discovery of quantitative trait loci (QTL), association
mapping, positional cloning and sequencing, functional
genomics, and the recently introduced metabolomics. The
following sections will deal with some of these tools and
their possible applications to resolve specific problems in
mulberry.
Genetic linkage mapping
Mulberry breeding thus far focused on improvement of leaf
yield and its component characters through selection on
phenotypes. This selection method, based on phenotype of
the principle character and its components, has achieved a
certain level of genetic gain as evidenced from the higher
leaf yield presently obtained from improved cultivars
(Tikader et al. 2009; Vijayan 2009). Further improvement
on the rate of genetic gain, particularly for the complex
traits such as growth rate, leaf size, delayed leaf senescence,
Tree Genetics & Genomes (2010) 6:613–625 617

propagation efficiency, resprouting, and survival, can be
achieved through indirect selection on trait-linked molecu-
lar markers. Furthermore, in mulberry, most of the
component traits of leaf yield were found under the control
of both additive and nonadditive gene actions (Misilamani
et al. 2000; Vijayan et al. 1997a, c, 2008). Hence, capturing
the nonadditive genetic variation through clonal propaga-
tion offers a potential method for considerably improving
the yield potential in mulberry. Therefore, molecular
markers tightly linked to traits of importance, identified
through genetic linkage mapping, would be a powerful tool
for accelerating the varietal developments in mulberry.
In most of the plants, genetic linkage maps are
constructed from segregating populations such as F2,
backcrosses, doubled haploids, recombinant inbred lines,
and near-isogenic lines. However, in outbreeding heterozy-
gous perennial crops like mulberry, such populations are
generally not available and are difficult to develop due to
the high genetic load and the long generation period.
Hence, linkage maps in tree crops are generally developed
from F1 progenies, either full sibs or half sibs, using
“pseudo-testcross strategy” (Grattapaglia and Sederoff
1994). The assumption behind this method is that in a
cross between two heterozygous parents the dominant
markers heterozygous in one parent and null in the other
will segregate in a 1:1 ratio in the progeny (Grattapaglia
and Sederoff 1994). Different software packages are
available for constructing linkage maps using information
from such crosses (Table 2). Although this method is
simple and is applicable for any type of maker systems, it
suffers from the limitation that maps are individual specific
and cannot be aligned or integrated. Therefore, linkage
maps for every elite breeding line have to be developed.
Nevertheless, it is possible to integrate the maps if multi-
allelic codominant markers such as microsatellite markers
are used. Nonetheless, linkage mapping through pseudo-
testcross strategy is well established in several tree plants
like Eucalyptus, Populus, etc. (Grattapaglia and Sederoff
1994; Thamarus et al. 2002; Zhang et al. 2004). However,
in mulberry, only a single map was reported (Venkateswarlu
et al. 2006), and it was constructed with RAPD, ISSR, and
SSR markers. The average map distances of 15.75 cM for
the female and 18.78 cM of the male parents clearly suggest
that the markers in this map are sparsely distributed. Thus,
the linkage information generated from this map has very
limited utility in identifying markers–trait association.
Therefore, attempts must be made urgently to develop
linkage maps with densely distributed polymorphic markers
such as AFLP, SSR, and SNPs. In this regard, it is
important to note that comparative genome analyses in
many crop plants have shown that gene content and orders
are often highly conserved at the map level across many
species. For instance, a comparative genomic analysis
revealed that all common markers in Eucalyptus grandis
and Eucalyptus globulus were collinear, and little evidence
was found for gross chromosomal rearrangements (Myburg
et al. 2003). Similarly, extensive synteny was found
between the linkage maps of maritime pine and loblolly
pine (Chagne et al. 2003) and between loblolly pine and
Douglas fir (Krutovsky et al. 2004). Results from these
studies suggest that comparative genome analysis between
even distantly related species can generate useful informa-
tion on genome evolution and location of homologous
chromosomal regions that harbor important traits. Such
approach can be used in mulberry to get more information
from the great advancement made on genetic linkage
analyses in other tree crops like Eucalyptus, Populus,
Ficus, apple, cacao, etc.
QTL discovery
Given the polygenic nature of most of the economically
important traits in mulberry, it is essential to map the QTLs

Table 2 Some of the useful softwares for linkage and QTL mappings

Software Availability Reference

MAPMAKER http://www.broad.mit.edu/ftp/distribution/software/ mapmaker3/ Lander et al. (1987)

http://www.genome.wi.mit.edu/genome_software
JOINMAP http://www.kyazma.nl/index.php/mc.JoinMap/ Stam (1993)
CARTHAGENE http://www.inra.fr/mia/T/CarthaGene/ de Givry et al. (2005)
MAPMAKER-QTL http://www-genome.wi.mit.edu/genome_software Lander and Botstein (1989)
MCQTL http://carlit.toulouse.inra.fr/MCQTL/ Jourjon et al. (2005)
MAPQTL http://www.mapqtl.nl Van Ooijen (2004)
PLABQTL https://www.uni-hohenheim.de/plantbreeding/software/ Utz and Melchinger 1995
QTLExpress http://qtl.cap.ed.ac.uk/ Seaton et al. (2002)
QTL cartographer http://statgen.ncsu.edu/qtlcart/cartographer.html Basten et al. (1998)
Qgene http://www.qgene.org/ Nelson (1997)
618 Tree Genetics & Genomes (2010) 6:613–625

for these traits. QTL mapping has been started to be viewed as
a powerful tool to dissect the complex traits into their
components (Grattapaglia et al. 2009). Recent reports on the
genetic architecture of QTLs in interspecific hybrids of trees
(Table 3) revealed that QTL mapping is effective for growth-
related traits in all the major tree plants (Grattapaglia et al.
1996). In many such studies, the major effect of QTLs
explained about 10–30% of phenotypic variations (Vaillancourt
et al. 1995; Byrne et al. 1997; Rae et al. 2008; Novaes et al.
2009). Encouraged by the results of these QTL mappings in
trees, mulberry breeders in India and China have taken up
QTL mapping in a big way. For instance, in India, the Central
Silk Board, Bangalore, with the financial support from the
Department of Biotechnology (DBT), Government of India,
has taken up research projects aiming to map QTLs for leaf
production, vegetative propagation water use efficiency,
epicuticular wax formation, and resistance to pest and diseases,
which are implemented at various research institutes.
The basic method of discovering a QTL is based on the
degree of covariation between markers and phenotypic
traits. Several different approaches like single marker
approach (Coffman et al. 2003), interval mapping (Lander
and Botstein 1989), and composite interval mapping (Zeng
1994) are used for identifying QTLs. A number of software
packages are also available for detecting QTLs and many
are freely available (Table 2). Regarding the QTLs in tree
species like mulberry, pseudo-testcross mapping strategy
detects only those QTLs for which one or both parents are
heterozygous and are not masked either by dominance
(Groover et al. 1994) or by the environment in which the
phenotyping is carried out (Bradshaw 1996). Lin and
Ritland (1996) are of the opinion that selective genotyping
might decrease the power of mapping multiple linked
QTLs. The number of independent measurement per line
and the number of lines used for analysis have great
influence on the outcome of QTL mapping (Hansen et al.

Table 3 A brief summary of the quantitative trait loci (QTL) mapping and association mappings (LD) in tree species

Plant Character studied Results References

Apple Fruit quality QTL mapping Kenis et al. (2008)

Apple Biotic stress QTL mapping Stoeckli et al. (2008)
Cacao Yield and growth LD mapping Marcano et al. (2009)
Coconut Wax components QTL mapping Riedel et al. (2009)
Eucalyptus Propagation QTL mapping Marques et al. (2005)
Eucalyptus Growth eQTL Krist et al. (2004)
Eucalyptus Biotic stress resistance QTL mapping Freeman et al. (2008a)
Eucalyptus Chemical compounds against herbivory QTL mapping Freeman et al. (2008b)
Eucalyptus Wood properties QTL mapping Freeman et al. (2009)
Douglas fir Growth QTL mapping Ukrainetz et al. (2008)
Pinus Biotic stress resistance LD mapping Neale (2007)
Pinus Growth QTL mapping Pot et al. (2006)
Pinus Growth LD mapping Yu et al. (2006)
Pinus Wood density QTL mapping Pot et al. (2006)
Pinus Wood density LD mapping Gonzalez-Martinez et al. (2007)
Pinus Carbon assimilation LD mapping Gonzalez-Martinez et al. (2008)
Pinus Abiotic stress tolerance LD mapping Gonzalez-Martinez et al. (2008)
Populus Bud phenology QTL mapping Rae et al. (2006)
Populus Bud phenology LD mapping Hall et al. (2007)
Populus Carbon assimilation QTL mapping Rae et al. (2007)
Populus Abiotic stress tolerance QTL mapping Street et al. (2006)
Populus Abiotic stress tolerance eQTL Street et al. (2006)
Populus Growth QTL mapping Rae et al. (2008)
Populus Adventitious root growth QTL mapping Zhang et al. (2009b)
Sweet cherry Fruit size QTL mapping Zhang et al. (2009a)
White spruce Biotic stress resistance LD mapping Namround et al. (2008)
White spruce Abiotic stress resistance LD mapping Namround et al. (2008)
White spruce Bud phenology LD mapping Namround et al. (2008)
Tree Genetics & Genomes (2010) 6:613–625
2008). Since most populations have a large number of
QTLs with small effects that can combine to cause
significant effect, it is important to increase replicated
measures from large collections of lines. Furthermore,
considering the caveat from Beavis (1998) that high
heterozygosity and experimental designs may lead to
overestimation of the magnitude of QTLs, adequate
precautions are essential while mapping QTLs in mulberry.
Another critical factor that can affect the success of QTL
mapping in mulberry is the phenotyping. Phenotyping
requires correct definition of traits, environmental measure-
ments, experimental design, and proper recording of data
and method of analysis.
Expression quantitative trait locus mapping
A QTL identified through the aforementioned approaches
may have genomic regions with several hundred genes
(Salvi and Tuberosa 2005). Thus, it is difficult to
determine which gene(s) is responsible for the variation
in the trait, for which extensive cloning and sequencing
are required (Mackay 2001). System biology approaches
such as transcriptomics has shown that modifications in
gene regulation cause wide phenotypic variations in
natural populations (Gilad et al. 2008; Hansen et al.
2008). Thus, mapping of QTLs that control the transcript
level of gene expression, called expression QTL (eQTL)
mapping or systems genetics, helps us understand the
relationship between the genome and the transcriptome.
Generally, two types of eQTLs are recognized, cis-QTLs
and trans-QTLs. cis-QTLs are due to polypmorphism in or
closer to the gene that codes for the mRNA product and,
thus, are likely to represent structural genes, while trans-
QTLs are due to polymorphism located elsewhere in the
genome but control the transcription of the interested gene
and, thus, are likely to be transcription regulator(s). The
major difference of eQTL mapping from traditional QTL
mapping is the ability to analyze thousands of traits at a
time to find the QTL responsible for the variations
(Chesler 2007). In order to achieve the full potential of
eQTL mapping, it is necessary to develop highly saturated
maps like those with SNP markers (Gilad et al. 2008).
Several software packages are freely available, with which
eQTLs can be identified from repository genomic data.
EQTL EXPLORER is one such soft ware available from
http://web.bioinformatics.ic.ac.uk/eqtlexplorer. The suc-
cess (Table 3) of eQTL mapping in Eucalyptus and
Populus (Krist et al. 2004) suggests that eQTL is much
suited to mulberry for identification of genes responsible
for adaptability, pathogen resistance, and early leaf
senescence and leaf nutritional qualities.
619
Metabolomic QTL mapping
The nutritional status of mulberry leaf has a high impact on
the cocoon quality as mature silkworms use more than 70%
of the leaf protein for synthesizing the silk proteins, fibroin,
and sericin. (Fukuda et al. 1959). Nutritional status of
mulberry leaf can be improved by integrating genetic and
metabolic approaches. Metabolic profiling facilitates un-
derstanding of plant metabolic networks (Fernie and
Schauer 2008). Pathway-based approaches have been found
useful in identifying the genetic determinants of crop
compositional quality. These approaches have identified
several mQTLs in tomato (Schauer et al. 2006, 2008),
Cucumis (Alba et al. 2005), and sesame (Laurentin et al.
2008). The most commonly used technologies for meta-
bolic profiling are mass spectrometry and nuclear magnetic
resonance (NMR). Different types of mass spectrometry are
available. Of which, gas chromatography–mass spectrom-
etry, gas chromatography–time-of-flight mass spectrometry,
liquid chromatography–mass spectrometry, capillary elec-
trophoresis–mass spectrometry, and Fourier transform–ion
cyclotron resonance–mass spectrometry are most widely
used. NMR uses magnetic nuclei of atoms after application
of a constant magnetic field. NMR has the advantage of
being used for assessing living cells; it provides subcellular
information, and it is easier to drive atomic information for
flux modeling from NMR than from mass-spectrometry-
based approaches (Fernie and Schauer 2008). Considering
the great potentials of mQTL mapping, it can be adopted
for improving the leaf quality in mulberry to enhance the
silk productivity per unit area, which will have a great
impact on the sustainability of sericulture.
Association mapping
Association mapping or linkage disequilibrium (LD)
mapping is an emerging technique in plants, though it
was well established in human. Association mapping differs
from traditional linkage mapping in many aspects (Table 4);
it exploits the historical and evolutionary recombination
events rather than the limited recombinations available in
biparental mapping populations (Nordborg and Tavaré
2002). LD mapping uses the genetic diversity present in
natural populations to identify molecular markers that are
tightly linked to complex phenotypic traits on the basis of
significant allele frequency differences between individuals
with the phenotype of interest (“cases”) and a set of
unrelated control individuals (Pritchard et al. 2000). Based
on the nature of the study, LD mapping can be categorized
into candidate gene association mapping and genome-wide
association mapping (Zhu et al. 2008). Candidate gene
association mapping relates polymorphisms in selected
620 Tree Genetics & Genomes (2010) 6:613–625

Table 4 Major differences between biparental linkage mapping and association mapping

Linkage mapping Association mapping

Family-based Population-based
Pedigree data are essential Pedigree data are not essential
Recombination from only 2–3 generations is available Recombination from many generation is available
Parental lines must be genotypically and phenotypically No parents
divergent and must be compatible
Mapping population is to be developed Natural populations are used
Information on a single genetic background is available Information from a large genetic back ground is available
High-resolution mapping requires large population Natural population is large
Alleles are screened only in pairs in diploids Large number of alleles may be present in a single locus
Less prone to false positive Highly prone to false positive due to population
structure and admixture of populations

candidate genes with phenotypic variation for specific traits
whereas genome-wide association mapping or gene scan
surveys genetic variation in the whole genome to locate
genes or narrow regions that have significant statistical
connections to various complex traits (Zhu et al. 2008).
Many computer softwares can be used for visualizing
linkage disequilibrium (Table 5). Association mapping has
already proven its efficiency in many tree plants (Table 3).
Detection of gene–trait associations for important growth
traits like phenology, disease resistance, drought resistance,
and wood properties in those trees suggest the suitability of
association mapping in mulberry, though it has not been
attempted in mulberry.
Marker-assisted selection breeding
Variety development in mulberry based on phenotypic
characters needs large-scale multienvironmental testing
(Fig. 1). In addition, mulberry needs at least 3–4 years to
become well established to be able to express important
agronomic traits properly. For appropriate statistical
analysis, data have to be recorded in all the seasons for a
minimum of 3 years. Thus, field testing of a large number
of progenies of mulberry requires huge space, time, labor,
and money. Therefore, assistance from environmentally
insensitive, selectively neutral, and less expensive and
more time-saving methods like molecular marker-assisted
selection (MAS) techniques can be used for accelerating
mulberry breeding as it is easier, faster, nondestructive,
and cheaper than the phenotypic assessment, especially for
complex traits which are expensive to assess (Koebner and
Summers 2003). The most important requirement for
MAS is molecular markers that are tightly linked to traits
of interest (Collard et al. 2005). If there is a marker linked
tightly to a particular phenotype, then one can select the
phenotype indirectly based on the presence or absence of
the marker on a gel or on autoradiogram, depending on the

Table 5 Commonly available

softwares for visualization of Software Available from
linkage disequilibrium
GOLD http://www.sph.umich.edu/csg/abecasis/GOLD
GRR http://www.sph.umich.edu/csg/abecasis/GRR/
GOLDsurfer http://www.umbio.com
HAPLOPAINTER http://haplopainter.sourceforge.net/html/ManualIndex.htm
HAPLOT http://info.med.yale.edu/genetics/kkidd/programs.html
HaploView http://broad.mit.edu/mpg/haploview/index.php
HaploVisual http://www.cs.helsinki.fi/u/prastas/haplovisual/
JLIN http://www.genepi.com.au/projects/jlin
LDheatmap http://stat-db.stat.sfu.ca:8080/statgen/research/LDheatmap
LDMAP http://cedar.genetics.soton.ac.uk/pub/PROGRAMS/LDMAP
Marker http://www.gmap.net/marker
PowerMarker http://statgen.ncsu.edu/powermarker/index.html
SNPanalyzer http://www.istech.info/istech/board/login_form.jsp
TASSEL http://www.maizegenetics.net
Tree Genetics & Genomes (2010) 6:613–625
marker system (Hospital 2009), because the banding
patterns indirectly reveal the presence or absence of a
specific chromosomal segment which carries the desired
alleles (Varshney et al. 2004). Although tightly linked
markers for phenotypic traits through genetic mapping
have not been identified in mulberry, using multiple
regression analysis on germplasm accessions, several
molecular markers associated with many important agro-
nomic and biochemical characters have been identified
(Vijayan and Chatterjee 2003; Vijayan et al. 2006a, 2009;
Kar et al. 2007). After appropriate validations, these
markers can initially be employed for MAS breeding in
mulberry.
Genetic transformation system
Reverse genomic tools such as insertional mutagenesis,
overexpression of genes, and RNAi are increasingly
being used for dissection of complex traits in trees.
Insertional mutants with suppressed or activated target
genes are used for identifying phenotypes (Busov et al.
2005). Overexpression of dominant genes in transgenic
plants leads to visible phenotypic changes and it has been
shown in trees (Grattapaglia et al. 2009). RNAi is another
powerful tool that has been developed recently for
validating gene function in the context of plant develop-
ments. The essence of RNAi is the delivery of double-
stranded RNA into the plant to induce a sequence-specific
RNA degradation mechanism that effectively silences
targeted gene (Waterhouse and Helliwell 2003). For
elucidating genes involved in many biochemical path-
ways, this methods appear to be very promising (Capell
and Christou 2004). In order to apply these technologies
in mulberry, an efficient transformation system is a
prerequisite. Bhatnagar et al. (2002, 2003) have developed
an efficient Agrobacterium-mediated transformation sys-
tem for the mulberry. Using this transformation system, a
glycinin gene AlaBlb, an oryzacystatin gene OC (Wang et
al. 2003), and a barley HVA1 gene (Lal et al. 2008) were
successfully incorporated into the mulberry genome. Thus,
an efficient genetic transformation system in mulberry is
ready for the breeders to take advantage of. Overexpres-
sion of genes and reverse genetics are also promising
options to study the trait–gene relationships in mulberry.
However, transgenesis of a highly cross-pollinated plant
like mulberry should be done with great caution. In this
regard, it is important to note that chloroplast genetic
engineering has been viewed as a safer method of
transgenic plant development as, unlike nuclear genes,
transgenes in the chloroplast DNA have little chances of
dissemination through cross-pollination.
621
Conclusion and perspectives
Given the astonishing speed with which the current
genomic research is progressing due to the revolutionary
technological advancements in various fields of biology
especially in DNA sequencing and bioinformatics, it is easy
to envisage that studies of trait–gene associations will
become as simple as doing a PCR amplification reaction in
a well-equipped laboratory. Indeed, it is true that the
genomic research in mulberry has not been developed as
much as that in silkworms. Therefore, mulberry breeders
could not get much benefit from the tremendous achieve-
ments made in plant genomic research. Nonetheless,
considering the developed marker technology, large genetic
resources, and trained manpower, it is envisaged that
deployment of the emerging genomic tools discussed in
this article would be applied soon for resolving the major
bottlenecks in mulberry breeding such as germplasm
evaluations, parental selections, progeny analyses, intro-
gression of traits from unadapted genotypes and species to
elite breeding lines. The efficient transformation system
available for mulberry is another tool that can be used for
improving mulberry via genetic engineering. Side-by-side
efforts can also be made for developing adequate number of
mapping populations from well-planned crosses, evolving
efficient phenotyping methodologies, discovering and val-
idating SNP markers, and developing more ESTs. These
resources are essential for the successful application of the
emerging genomic tools in mulberry to enhance its
productivity and adaptability for sustaining a vibrant
sericulture industry in Asia.
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