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Applications of Molecular Markers in Plant Genome Analysis and Breeding, 2015: 119-142
ISBN: 978-81-308-0560-3 Editor: Ksenija Taški-Ajduković
5. Development and utilization of DNA
markers for genetic improvement of bast
fibre crops
Pratik Satya1 and Mridul Chakraborti2 1Senior Scientist, Crop Improvement Division, Central Research Institute for Jute and Allied Fibres,
Barrackpore, Kolkata-700120, India; 2Scientist, ICAR-National Research Centre for Orchids, Darjeeling-734101, West Bengal, India.
Abstract. DNA markers serve as helping hands in plant breeding
for achieving greater efficiency in selection. Unlike food crops,
genomic resources in bast fibre crops have received little attention
with sporadic research attempts, resulting in gaps in basic
information on DNA marker development and application. SSR and
AFLP markers have been the choice in bast fibre crops for genetic
map construction, while genetic diversity and evolution have been
assessed through traditional (RFLP, RAPD, SSR, AFLP) and novel
marker systems (IRAP, SRAP, SCoT, POG, PALG). Availability of
genomic and transcriptomic resources have helped in large scale
SSR and SNP marker discovery and high density map construction
in some species like flax and jute, but good quality maps are not
available in other bast fibre crops. Due to low genetic
polymorphism, complex genomic structure and unique reproductive
behaviour, the development and application of DNA markers in bast
fibre crops are really challenging. With the aid of next generation
sequencing based marker discovery, many more developments are
expected in coming years in this field of research.
Correspondence/Reprint request: Dr. Pratik Satya, Senior Scientist, Crop Improvement Division, Central
Research Institute for Jute and Allied Fibres, Barrackpore, Kolkata-700120, India. E-mail: [email protected]
Pratik Satya & Mridul Chakraborti 120
1. Introduction
The technology of utilizing allelic or non-allelic DNA fragments for
genetic analysis and selection has revolutionized the field of crop science.
Use of molecular markers and molecular breeding are now common
approaches in crop breeding and genetics. Molecular markers have enabled
us to precisely identify genetic location of complex traits and to practice
selection assisted by marker genotype, trace the evolutionary history of
crops, characterize and categorize large genetic resources within a short
period and identify gene sequences controlling the phenotype (Satya, 2007).
However, as it is true with phenotype based genetic analysis and selection,
the prime attention to develop and utilize molecular markers focussed on few
major crop species, such as rice, wheat, maize, soybean and cotton.
Researches on application of molecular markers on other crops, particularly
of less economic importance are scarce.
The bast fibre crops are nature’s unique gift to mankind, fulfilling the
need of clothes, bags, ropes, papers with several additional secondary uses.
Considering economic importance, the bast fibres crops come just next to
cotton. These natural fibre crops are our best allies for reducing environment
pollution. The bast fibre based products are best alternatives for reduction of
synthetic polypropylene/polyethylene fibre materials that cause severe
pollution. In addition, these crops having high biomass add high carbon
credit by assimilation of carbon-di-oxide from polluted air and purify air by
adding substantial amount of oxygen (Maiti et al., 2011). Each bast fibre has
unique domains of application, which are based on the structure and quality
of fibre. The ramie and the flax, being finer fibre are in high demand in the
textile industry for fine quality, durability, strength and lustre, while the
coarser fibres like jute and kenaf dominate the non-textile fibre industry for
manufacturing of sacks, hessian, bags, ropes, twines and carpet backings
(Maiti et al., 2011). Development of improved cultivars with high fibre
yield, fine fibre quality and high biomass are primary targets of bast fibre
crop breeding programmes. Most of these traits are quantitative being
influenced by pre- and post-harvest environmental modifications. Direct
selection for these traits are often not possible in segregating generations, as
direct measurement of fibre yield and quality requires harvesting of the plant
before it attains reproductive stage. Indirect selection is the only plausible
solution in such cases, where DNA markers can be of immense help to
improve selection efficiency.
While the research in major crops have already moved from fragment
based markers to sequence based markers utilizing the benefits of next
generation sequencing (NGS) technologies, the minor crops could not
DNA marker research in bast fibre crops 121
harness the benefits of DNA marker technologies of the past twenty years.
Apart from cotton, other fibre crops have received little research efforts and
funding for development of molecular markers and their utilization. Despite
availability of several varieties, the genetic gain in these crops is limited.
Marker database and genomic resources are building up slowly in these crops
from sporadic research efforts. Still a lot remains to be done to reap tangible
benefits from molecular markers in these crops, which offers great challenges
for the researchers. This review will focus on four major bast fibre crops, jute
(Corchorus capsularis L. and C. olitorius L.), flax (Linum usitatissimum L.),
kenaf or mesta (Hibiscus cannabinus L. and H. sabdariffa L.) and ramie
(Boehmeria nivea L. Gaud.), with a general but necessary introduction on the
evolution of molecular markers crucial for new developments of DNA marker
applications in fibre crops.
2. Evolution of molecular markers
A marker is a unique character of an individual or any descriptor that
distinguishes it from others. A molecular marker is a specific genomic
region identified in a genotype, which is unique or at least different from
sequence of same region in another genotype. The difference may either
arise from change in base sequence, deletion of part or whole of the
sequence in one genotype, sequence inversion or from repetition of the
sequence. Molecular markers have come a long way from the first
generation hybridization based markers like restriction fragment length
polymorphism (RFLP) (Botstein et al., 1980) to the new generation
sequence based markers like SNP, InDel and DArT markers. RFLP markers
were developed by genomic DNA digestion with restriction endonucleases
(EcoRI, HindIII, EcoRV, DraI etc.) followed by hybridization with
previously designed probes and were more utilized in human genetic
analysis. But the application of RFLP was not very handy for plant breeding
due to low polymorphism, complex protocol and low number of markers
generated. In the nineties, development of PCR based markers greatly
increased speed and accuracy of marker based analyses in biological science.
A few hour of PCR cycle eliminated need of laborious and time consuming
hybridization protocols, reducing experimental time from weeks to days.
PCR also reduced the requirement of template DNA from microgram to
nanogram, which further broadened the applicability of DNA markers in
anthropological and forensic studies. Use of random and semi-arbitrary
primers increased the number of marker loci, enhanced polymorphism and
reproducibility of results. The arbitrary primer based marker systems were
designed on a common principle of amplifying random genomic locations
Pratik Satya & Mridul Chakraborti 122
followed by comparison of the patterns of amplification among genotypes.
Since primers are binding in random genetic regions, amplicons of same size
may not be allelic. These marker systems are thus multi-locus, generating
presence-absence type polymorphism. Such markers are more advantageous
in differentiating genotypes, but have limited scope for genetic mapping,
where locus specificity is an important prerequisite.
2.1. Microsatellite sequence based markers
Microsatellites are small tandem repeats of 1–6 nucleotides, often called
as SSR or VNTR. Specifically, the term variable number of tandem repeats
(VNTR) includes both microsatellites and minisatellites (repeat length 6–500
nucleotides). The markers developed based on primers designed on flanking
sequence of microsatellite repeat regions are more commonly known as
simple sequence repeat (SSR) markers (Li et al., 2002). SSRs are second
most abundant marker system after SNPs, as large portion of genomic DNA
contains small repetitive regions. Development of SSR markers requires
cloning and characterization of microsatellite rich regions in the genome and
designing of primers on the basis of sequence information, thus marker
development and validation are cost intensive and time consuming.
Techniques like PCR isolation of microsatellite arrays (PIMA), enrichment
of genomic libraries with microsatellite specific sequences, oligonucleotide
repeat primer extension etc. are used for cloning and sequencing of flanking
regions of microsatellites (Satya, 2007). However, once a microsatellite
database is developed, it can be simply used to develop other PCR based
markers, producing locus specific, universal and codominant alleles, which
have numerous applications in comparative and phylogenetic studies as well
as genetic analysis of breeding populations. These markers target non-coding
DNA sequences for amplification, which are evolutionary neutral. Once a set
of microsatellite markers are developed for a species, it can be tested in
related species for cross-species amplification and validation (Peakall et al.,
1998). The rich database of cotton microsatellite is particularly valuable for
fibre crops, as many fibre crops like jute, kenaf, Urena and Sida belong to
the same Malvaceae family.
Other microsatellite sequence based marker systems are principally
based on either amplification of microsatellite regions by designing primers
for the conserved flanking regions of microsatellites revealing the variation
in repeat length and number or by amplifying internal regions of two
microsatellites by using microsatellite specific primers. Inter simple
sequence repeats (ISSR), single primer amplification reaction (SPAR),
random amplification of microsatellite polymorphism (RAMPO) and
DNA marker research in bast fibre crops 123
microsatellite primed PCR (MP-PCR) are techniques where primers are
designed on the basis of microsatellite repeat sequences (Satya, 2007).
2.2. Next generation sequencing (NGS)
A major problem faced by the geneticists with the first and second
generation molecular markers is the limitation of marker number during
construction of a high density map. Although thousands of microsatellites
have been discovered in major crop species, development of high density
genetic map is a real challenge from segregating populations due to low
marker polymorphism among the parental lines. More than 4000 SSRs have
been screened for parental polymorphism during several map construction
efforts in cotton, but only few hundred SSR markers could be mapped. The
problem is more acute for species with high genome size, such as polyploids,
where sufficient markers are required to have a moderate coverage of the
genetic map.
More number of markers can be obtained if single nucleotide
polymorphism (SNPs) is used as a marker system. SNPs are single base pair
changes in the genome, which are more abundant and polymorphic than any
other marker system. However, discovery of SNPs following sequence
comparison in a ‘clone by clone’ approach or from whole genome sequence
data is time consuming and costly. The advent of next generation sequencing
(NGS) technologies (Elshire et al., 2011) and powerful computational
pipelines has reduced the cost of genome sequencing by many folds allowing
discovery, sequencing and genotyping of thousands of markers at one step.
This powerful tool has opened up new avenues in species where reference
sequence is not available (Ray and Satya, 2014). The NGS based genomics
and transcriptomics techniques are now being used more and more as
compared to other existing functional genomics tools like differential
display, cDNA-AFLP and micro-array technology for SNP discovery and
quantifying gene expression. Techniques like RAD-seq and Genotyping-
by‐Sequencing (GBS) are already being utilized for SNP based genetic
mapping in fibre like cotton and jute. In coming years, these technologies are
going to be pivotal for molecular breeding of fibre crops.
3. DNA marker development in bast fibre crops and their
applications in genetics and breeding
The bast fibre crops, including jute, flax, ramie, and kenaf are the source
of textile and non-textile fibres. A common feature of these crops is that
cultivation of these crops is driven by the demand of the industry; this
Pratik Satya & Mridul Chakraborti 124
industrial nature is a crucial differentiation of these crops from food and feed
crops.
3.1. Jute (Corchorus capsularis and C. olitorius)
Jute is the second most important plant fibre and most important bast
fibre crop of the world. It is known as the ‘golden fibre’ due to the shiny
golden appearance of the fibre and the economic importance of the crop
(Maiti et al., 2011). The jute fibre is coarser than cotton; therefore the
fibres are primarily used as sackings, bags, twines, yarn, ropes, burlaps and
carpets. Jute has many other versatile usage including geotextiles, paper
pulp, biofuel, fibre composites and diversified value added products.
C. olitorius or tossa jute (2n = 2x = 14) is the principal cultivated jute crop
occupying over 90% of the area. C. capsularis (2n = 2x = 14) produces
finer fibre, but lacks behind C. olitorius in production potential (Maiti
et al., 2011). A number of PCR based DNA marker systems have been
used in jute for genetic diversity analysis, evolutionary studies, linkage
map construction and QTL identification. Besides, a number of genetic
maps have been developed using SSR, SRAP, AFLP and SNP based
markers.
3.1.1. Genetic diversity and origin of jute
A few diversity studies have been documented in jute using RAPD,
ISSR, STMS, AFLP, RGA, POX and SSR markers. Marker based genetic
polymorphism study revealed low to moderate variability in both
C. capsularis and C. olitorius cultivars (Roy et al., 2006; Huq et al., 2009).
Genetic variability study of 81 jute genotypes with 45 SSRs established that
SSR markers can be used for genetic variability analysis in jute, although
polymorphism information and average number of alleles for individual
SSRs are low (Mir et al., 2008). Use of new marker systems like start codon
targeted (SCoT) markers generated higher variability in cultivars of
C. olitorius and C. Capsularis (Rana et al., 2012).
3.1.2. Marker development
A total of 2469 SSRs have been developed in C. olitorius from four
microsatellite-enriched genomic libraries (Mir et al., 2009). Validation of a
subset of these markers indicated that these SSR markers generate good
polymorphism for genetic mapping and QTL identification in jute. Further,
2229 polymorphic AFLP amplicons have been generated in C. olitorius and
DNA marker research in bast fibre crops 125
C. capsularis (Das et al., 2011). Das et al. (2012) reported additional 607
SSRs from 399 SSR containing ESTs.
3.1.3. Phylogeny and evolution
The two jute species, C. capsularis and C. olitorius have high botanical
similarity, but are proposed to originate from two different continents, Asia
and Africa, which is still a mystery to evolutionary biologists. Genetic
relationship of seven wild and two cultivated Corchorus species was examined
by Qi et al. (2003) using 27 RAPD primers. They observed closer relationship
between C. olitorius and C. capsularis in comparison to wild species.
C. olitorius, although originated in Africa is not used as a fibre crop in
African countries. Instead, it was domesticated as a fibre crop in the Indian
subcontinent. Kundu et al. (2013) used both nuclear and plastid SSR markers
to trace origin of cultivated jute and suggested C. aestuans as a progenitor of
cultivated jute. Among the wild jute species, C. aestuans is most widely
distributed in India. It is distributed throughout the India including Northern,
Southern, Eastern and Central India. Genetic diversity analysis using 82 SSR
markers in Indian Corchorus species show that average polymorphism
information content (PIC) is 0.31, indicating moderate genetic polymorphism
(Satya et al., 2011). However, the number of SSR alleles varied from 2 – 14
indicating high allele numbers. Cluster analysis revealed high genetic diversity
between the two cultivated species C. capsularis and C. olitorius. This genetic
difference may be the possible reason for very low crossability between two
cultivated jute species. Banerjee et al. (2012) investigated molecular diversity
of 292 genotypes of white and tossa jute by SSRs and found that Indian jute
genotypes form distinct clusters from the exotic germplasm collections. They
observed that genetic diversity of Indian accessions was higher in case of
C. capsularis, while the opposite was true for C. olitorius.
Satya et al. (2014) employed a new group of functional markers based
on plant phenylalanine ammonia lyase gene sequences (PALG) along with
peroxidase gene based (POG) markers and SSR markers to study genetic
diversity, population structure and geographical separation of C. olitorius
ecotypes. The markers exhibited very high amplicon number per marker,
high polymorphism (97%), and resolving power (13.8). The POG markers
exhibited high genetic polymorphism (89.2%) with high resolving power
(9.8). In comparison, the SSR markers exhibited less polymorphism (80.3%)
and much lower resolving power (2.3), suggesting gene based PALG and
POG markers have higher scope for genetic discrimination than SSR markers.
We then investigated the population structure of a set of world collection
of C. olitorius and other Corchorus species, and observed that SSR-based
Pratik Satya & Mridul Chakraborti 126
Figure 1. Genetic diversity in C. olitorius population and species relationship of
cultivated and wild Corchorus species, supported by high Bootstrap values indicated
at nodes.
population structure analysis was effective at species-level, but genic markers
based on POG and PALG were more efficient at sub-species level to
distinguish between geographically isolated populations of C. olitorius. The
POG marker based phylogenetic analysis clearly distinguished C. olitorius
from C. capsularis and wild Corchorus at species level. At the sub-species
level, three distinct groups of C. olitorious genotypes were identified, the
Indian C. olitorious germplasm, exotic African C. olitorius germplasm and
mutant C. olitorius accessions (Figure 1). C. capsularis exhibited closer
association with wild Corchorus species indicating the species has evolved
earlier than C. olitorius. Corchorus population of Indian origin, domesticated
for fibre production exhibited different allelic combinations of peroxidase
genes than the vegetable-type African accessions.
3.1.4. Genetic mapping and QTL identification
A preliminary genetic map based on 40 RAPD markers was developed
by Haque et al. (2008), which contained 18 linkage groups with total
distance of 463.7 cM. The second genetic map of C. olitorius placed 36 SSR
markers on six linkage groups spanning 784.3 cM (Das et al., 2012),
providing a better picture of marker distribution on linkage groups. The map
was constructed utilizing a RIL population of 120 genotypes developed from
JRO 524 x PPO4.
DNA marker research in bast fibre crops 127
The first complete genetic map representing all the seven linkage groups
was developed from a RIL population, which spanned a total length of 799.9
cM. The map contained 82 SSR markers with an average marker interval of
10.7 cM (Topdar et al., 2013). A total of 26 QTLs for bast fibre quality,
strength and fibre yield related traits were positioned on this map. Recently,
Kundu et al. (2015) developed a restriction site associated digestion (RAD)
linkage map of jute using next generation sequencing technology. The map
spanned a length of 358.5 cM, placing 503 RAD markers in seven linkage
groups. QTL mapping based on this genetic map identified nine QTLs,
identifying a few candidate genes within the QTL regions.
Only one genetic map is available for C. capsularis, containing SRAP,
ISSR and RAPD markers (Chen et al., 2014). The map was developed from
an F2 population derived by crossing ‘Xinxuan No. 1’ and ‘Qiongyueqing’
cultivars. The map covered a length of 2185.7 cM, with a mean marker
density of 18.7 cM. The large size of the map, despite small genome size of
the species indicates high gaps in the map.
3.2. Flax (Linum usitatissimum)
Flax is one of the oldest crops, being domesticated over 7000 years ago
possibly in Mediterranean or Indian region (Maiti et al., 2011). There are
two ecotypes, seed flax or the linseed, which is cultivated for seed and oil
and the fibre flax, which provides quality bast fibre. The linseed types are
shorter, branched with higher seed number and seed weight. The fibre type
or the flax type is taller, few branched with less seed. Flax fibre is highly
valued for making quality cloths, interior decoration and canvasses,
particularly in USA, Japan and Europe. European Union is the global leader
in fibre flax production followed by China. The flax fibre is much finer than
jute and kenaf fibre and is comparable to cotton fibre. Cultivation of the seed
crop or linseed is more compared to fibre flax. Canada is the leading
producer of linseed followed by USA, China and India.
3.2.1. Marker discovery
A set of 248 polymorphic EST-SSRs using a panel of 23 flax accessions
was identified by Cloutier et al. (2009) which initiated SSR identification in
flax. Bickel et al. (2011) developed a set of 92 SSR markers by amplified
fragment length polymorphism of sequences containing repeats (FIASCO)
method. Of these 46 were found polymorphic with 2–8 alleles and a PIC of
0.47. A set of 10 inter-retrotransposon amplified polymorphism (IRAP)
markers were developed by Smỳkal et al. (2011) from long terminal repeat
Pratik Satya & Mridul Chakraborti 128
(LTR) retrotransposon sequence of flax. Further, to facilitate genetic
mapping and gene discovery, a total of 55465 SNPs have been identified
from a reduced representation library of eight flax genotypes using next
generation Illumina platform (Kumar et al., 2012). Genotype Crepitam
Tabor contained the largest SNPs (21,704). The rate of SNP discovery was
much less in genic region than intergenic region indicating that the
intergenic region in flax has evolved much faster than the genic region. A
total of 4,706 SNPs were validated in a RIL population of 96 genotypes
using genotype by sequencing (GBS).
3.2.2. Genetic diversity
Wiesnerová and Wiesner (2004) used ISSR for fingerprinting of 53 flax
cultivars using 45 polymorphic loci. They obtained four groups and eight
sub-groups. In another study, 2,727 flax accessions were genotyped with 149
RAPD markers, revealing wide genetic difference among the geographically
isolated accessions (Fu, 2005). Uysal et al. (2010) used twenty four ISSR
primer pairs and studied fifty accessions including L. bienne (pale flax) and
L. usitatissimum accessions. It showed that pale flax was more closely
related to dehiscent type cultivated flax. Rajwade et al. (2010) have
classified 70 Indian flax genotypes using 12 ISSR primers. These 70 flax
genotypes were grouped in five clusters.
3.2.3. Evolution of fibre and seed flax
IRAP was used by Smỳkal et al. (2011) to study genetic diversity of 708
accessions of cultivated flax originating from 36 countries. They have
reported genetic overlapping between fibre and oil type flax and found high
diversity among the landraces. In another study, a core collection of 407 flax
accessions were developed from 3500 accessions maintained at Plant Gene
Resources of Canada (PGRC) and genotyped with 448 SSR markers (Soto-
Cerda et al., 2012). This core collection comprised of 92 fibre flax
accessions and 245 seed flax accessions. Population structure analysis
identified three major groups, the 2nd
group being admixture of group 1 and
group 3. Most of the fibre flax genotypes was in group 3, originating from
Eastern Europe.
3.2.4. Linkage mapping and QTL identification
The first linkage map of flax contained RFLP and RAPD markers
consisting of 15 linkage groups with 94 markers and covered about 1,000 cM
DNA marker research in bast fibre crops 129
(Spielmeyer et al., 1988). Later an AFLP map of 18 linkage groups,
developed with 213 AFLP markers spanning about 1,400 cM was
developed (Oh et al., 2000). A linkage map of flax containing 103 SSRs,
five SNPs, four genes (fad3A, fad2A, fad2B, dgat1) and one morphological
trait (seed coat colour) is also available (Cloutier et al., 2011). This study
also detected two major QTLs each for linoleic acid, linolenic acid and
iodine value and one major QTL for palmitic acid. A consensus genetic
map of 1551 cM comprising of 821 loci was later developed by Cloutier
et al. (2012) from three segregating populations originating from CDC
Bethune×Macbeth, E1747×Viking and SP2047×UGG5-5. They also
anchored 670 markers on 204 contigs of physical map, covering 74% of
the flax genome.
Three QTLs for resistance to powdery mildew disease were identified
from an F2:3 mapping population developed from susceptible cultivar
NorMan and the resistant cultivar Linda using 143 SSR markers (Asgarinia
et al., 2013). The QTLs were located on LG1, 7 and 9, explaining 97% of
the variation. The QTLs were further anchored to the physical map and 313
SNPs were identified in these QTLs.
3.3. Kenaf (Hibiscus cannabinus)
Kenaf is a crop species with diversified end use. It is also known as
kenaf, bimli jute, Guinea hemp, mesta and Deccan jute. The terms mesta and
Deccan jute also include another closely related fibre crop species
H. sabdariffa, which is commonly known as roselle. Kenaf and roselle are
cultivated as fibre, paper pulp or as biofuel crop in China, India, Indonesia,
Thailand, Russia, Vietnam, Malaysia, Brazil, Argentina, USA and European
countries (Webber and Liu, 2011).
3.3.1. Genetic diversity and population structure
Genetic diversity of kenaf germplasm has been studied by several
marker systems, such as RAPD (Cheng et al. 2002, 2004), AFLP
(Coetzee et al., 2008; Kim et al., 2010), ISSR (Huo et al., 2009; Satya
et al., 2013a), SRAP (Qi et al., 2011), SSR (Satya et al., 2013a; 2014)
and RGA (Satya et al., 2014). Qi et al. (2011) analysed SRAP variability
of 84 germplasm collected from 26 countries using 26 primer
combinations and found three groups: cultivated, wild and intermediate
types. A Comparative efficiency of SRAP and ISSR markers were tested
in the same population by Xu et al. (2013), revealing higher
Pratik Satya & Mridul Chakraborti 130
polymorphism for ISSR markers. The SRAP markers, however, produced
more number of amplicons.
Comparative evaluation of SSR and ISSR markers in generating
polymorphism and assessing genetic diversity in kenaf revealed higher
polymorphism for ISSR, but better marker resolvability for SSR markers
(Satya et al., 2013a). Phylogenetic association of kenaf, roselle and wild
Hibiscus species revealed closer association of kenaf with wild progenitors
than with roselle. The study established genetic relation of all the
cultivated genotypes/varieties in kenaf as well as in roselle and also
showed close resemblance of molecular and taxonomic classification in
Hibiscus section Furcaria.
Genic markers like RGA are expected to follow a different course of
evolution than the neutral SSR markers, because the RGAs are evolved
through host-pathogen coevolution process. This differential
evolutionary path of RGA from SSR can be used to study speciation and
geographical separation of species and ecotypes. In such a study, we
revealed the origin of the Indian and African ecotypes of kenaf showing
that Indian kenaf landraces have followed a distinct lineage being well
separated from the African kenaf ecotypes (Satya et al., 2014). At
species level also, the RGA markers were efficient to differentiate kenaf
(H. cannabinus) from roselle (H. sabdariffa) and wild Hibiscus species
(Figure 2). At sub species level, genetic admixture was observed within
both African and Indian ecotypes, indicating genetic exchange between
the two ecotypes at an early stage. The fibre type Indian cultivars shared
high genetic homogeneity with a group of exotic genotypes, mostly
comprising of cultivars developed in USA, Cuba and Guatemala,
indicating these cultivars have been developed utilizing exotic genetic
resources.
Figure 2. Genetic structure of cultivated kenaf (H. cannabinus) ecotypes from Africa
and India, roselle (H. sabdariffa) and wild kenaf (Hibiscus spp.) revealed using
resistance gene analog (RGA) markers.
DNA marker research in bast fibre crops 131
3.3.2. Wide hybridization and evolution of kenaf
Molecular marker analysis in kenaf and its wild relatives has helped to
understand their evolutionary relationship. Cross-species amplification using
jute specific SSR markers as well as exploitation of ISSR markers have
helped to predict H. surattensis as the progenitor of H. cannabinus (Satya
et al., 2012a). The analysis also confirmed that the evolution of tetraploid
species in the Genus Hibiscus section Furcaria has possibly taken place in
geographically isolated continents after land fragmentation, while the diploid
species might have arrived earlier (Figure 3). Genetic association of
H. cannabinus was found to be higher with H. radiatus than H. acetosella,
which support more recent origin of H. radiatus from H. cannabinus.
Moderate genetic similarity among these species suggests that transfer of
desirable genes from wild species to kenaf would be difficult due to low
genomic association.
The ISSR and SSR markers have also been utilized to confirm hybrid
identity of plants generated from interspecific hybridization and their
characterization (Figure 4). Both the marker systems were found to be
efficient to differentiate parents from the hybrids in crosses of H. cannabinus
x H. radiatus, H. cannabinus x H. acetosella and H. cannabinus x
H. surattensis (Satya et al., 2012a; 2013b). Moreover, marker polymorphism
has also confirmed genetic heterogeneity of interspecific hybrids developed
from diploid H. cannabinus and tetraploid H. acetosella and H. radiatus
(Satya et al., 2012b).
(Modified from Satya et al., 2012a)
Figure 3. Migration of diploid Hibiscus species in the period of Gondwanaland
followed by independent evolution of allotetraploid species
Pratik Satya & Mridul Chakraborti 132
Figure 4. Interspecific hybrids of different Hibiscus species. a, b, c, typical leaf
shape of interspecific hybrids and parental species; d, phylogenetic relationship of
Hibiscus species and allopolyploid interspecific hybrid of H. cannabinus and
H. radiatus.
3.3.3. Genetic map construction
A genetic map was constructed from F2 population derived from Alain
kenaf and Fuhong 992, which comprised of 307 SRAP, ISSR and RAPD loci
distributed in 26 linkage groups over a distance of 4924.8 cM (Chen et al.,
2011). Another genetic map was constructed from F2 population by crossing
Ga42 and Alain kenaf; the map spanned 2108.9 cM, comprising of 20
linkage groups, placing 65 SRAP, 56 ISSR and 13 RAPD markers (Zhang
et al., 2011). However, no QTL has been identified till date.
3.4. Ramie (Boehmeria nivea)
Ramie, rhea or China grass is a perennial fibre crop valued for high
quality textile grade bast fibre. The history of ramie cultivation is at least
five thousand years old in China and Indo-Malay peninsula (Maiti et al.,
2011). China is the major producer of ramie fibre with a production of 0.15
million metric tonnes. It alone occupies over 90% of area and textile
products of ramie. Ramie is also grown in Brazil, Japan, Laos PDR, India
and the Philippines. The fibre produced from ramie is strongest of all known
plant fibres bearing more than twofold strength of cotton fibre. The fibre has
resistance to bacterial degradation and higher tensile strength under
DNA marker research in bast fibre crops 133
hygroscopic condition. The root extract is also used as antioxidant, anti-
inflammatory and hepato-protective agent.
3.4.1. Marker development
A total of 1827 SSRs have been developed in ramie from 43990 EST
sequences (Liu et al., 2013). The SSRs were predominantly dinucleotide and
trinucleotide repeats, in which motif AG/CT was most abundant. Of the
1827 SSRs, 100 SSRs were validated in 24 ramie genotypes revealing 98%
amplification. These SSRs have further been used in phylogenetic analysis,
map construction, QTL identification and germplasm characterization. To
facilitate large scale genotyping, a direct PCR method was developed by
Satya et al. (2013c). Direct PCR methods save time and cost of experiments
with increased efficiency of PCR. Ramie contains high amount of gummy
complex polysaccharides, which reduces storability of DNA. NaOH was
used as extraction buffer and Tris/Tris–HCl/Tris–EDTA as dilution buffers.
The methods were successful to amplify DNA using ISSR and SSR markers.
The methods were also suitable for chloroplast DNA amplification using
primers for rbcL gene. Our results also showed that nature and quantity of
dilution buffer are important for increasing efficiency of direct PCR. The
NaOH based methods are simpler, cheaper and economic compared to other
direct PCR methods and work very well for tissues containing high amount
of complex polysaccharides and are suitable for batch processing for high
throughput genotyping within a short time period.
3.4.2. Phylogenetic and population genetic analysis
China and Indo-Malay region are considered to be the centre of origin
and diversity of ramie. SSR diversity for eight loci was examined in 50
ramie populations from China to understand evolution and domestication of
ramie in China (Liao et al., 2014). The results suggested that ramie
originated in Yunnan province and domesticated in the middle and lower
regions of the Yangtze valley, although this view needs further verification
as the data is based on a few SSR loci.
Start codon targeted (SCoT) markers were used by our group to trace
breeding history of Indian ramie cultivars and population structure of ramie
germplasm (Satya et al., 2015). The introduced ramie accessions exhibited
close genetic association with the domesticated genotypes, but not with the
Indian natural ramie populations. Initiation of ramie breeding in India relied
Pratik Satya & Mridul Chakraborti 134
primarily on the introduction of new cultivars from other countries. The
Indian ramie populations exhibited high SCoT polymorphism (>50%), high
genetic differentiation and gene flow. The study also indicated geographical
barrier for gene flow among Indian populations.
3.4.3. Genetic diversity and germplasm characterization
Quite a few marker systems have been used to characterize genetic
diversity in ramie, although in most cases the population size was low,
which is a bottleneck for providing definitive explanations on ramie
genetic diversity based on these studies. RAPD (Jie et al., 2002; Li et al.,
2006), ISSR (Hou et al., 2006), SSR (Zhou et al., 2005; Luan et al., 2014),
random amplified microsatellite polymorphism (RAMP) (Zhou et al.,
2004), and SRAP (Liu et al., 2008) marker systems have been used to
investigate genetic diversity in ramie cultivars and wild genetic resources.
Most of these studies indicate lower diversity within cultivars compared to
wild genetic resources. A core collection of ramie comprising of 22
accessions was constructed from 108 accessions using 21 SSR markers
(Luan et al., 2014). The SSR markers exhibited lower genetic diversity in
the core collection.
Recently, Satya et al. (2015) used SCoT markers to study the
population structure, breeding history and genetic diversity among
domesticated and undomesticated ramie in India. The SCoT primers are
based on conserved regions flanking the initiation codon sequences of
genes, with the principle of using a single primer like RAPD and ISSR.
These markers are expected to be linked to functional genes and
corresponding traits; so the PCR products can be converted to gene
targeted markers. SCoT marker also provides high accuracy and
reproducibility with high polymorphism. In this study, the SCoT markers
revealed high genetic polymorphism (87.5%) and moderate resolving
power (3.22). We observed that breeding of ramie cultivars in India relied
primarily on the introduction of new cultivars from other countries and the
Indian genepool has remained largely unexplored (Figure 5). The Indian
native ramie has higher adaptability; thus this gene pool is a valuable
reservoir for enriching genetic improvement programs. In natural
population, higher within-population diversity than diversity among
populations was observed, suggesting that these populations should be
conserved in situ for preserving genetic diversity.
DNA marker research in bast fibre crops 135
Figure 5. Genetic association studies show close association of Indian ramie
(Boehmeria nivea) populations, while improved clones are genetically similar to
exotic types
3.4.4. Genetic mapping and QTL identification
Only one genetic map is available in ramie which was developed using
SSR markers (Liu et al., 2014). The mapping population was developed by
crossing two heterozygous parental lines ZZ1 and QYZM, followed by
developing a F2 mapping population from selfed F1. Since the genotype
shows clonal propagation, a F2 agamous line (FAL) population was
constructed. The map spanned a length of 2,265.1 cM with 132 loci. A total
of 33 QTLs for the five fiber yield-related traits (fibre yield, stem length,
stem number, stem thickness and bark thickness) were identified on the map.
Majority of the QTLs exhibited overdominance, indicating heterozygous
advantage in ramie.
4. Future directions
Despite of the promise arising from several studies for advancement in
marker assisted breeding as well as map based cloning of genes, tangible
progress has not achieved for marker assisted selection. Even in crops like
Pratik Satya & Mridul Chakraborti 136
cotton where high genomic information is available, examples of MAS for
economic traits is scanty. Major impacts of molecular markers in fibre crops
have been the development of linkage maps and identification of QTLs.
Although QTL analysis is very important and a prerequisite for marker
assisted breeding, it is very difficult to identify and isolate the gene(s) for a
given QTL in a species without the availability of large scale of genomic
resources or to transfer QTLs in desirable background using linked markers.
The bast fibre crops lag much behind other major crops in terms of
availability of precise genomic resources for marker assisted selection
(MAS). However, new methods on large scale marker discovery based on
NGS technologies have enabled quick build-up of genomic resources within
a very short time. During the past one year, a good number of SNP based
maps have been constructed, and large scale EST database have been
developed form genomic and transcriptomic data, especially in jute, flax and
ramie. The pace of data discovery using NGS platform is astounding. One
can really feel surprised at the pace of development of NGS based genomic
resources. Five reports of transcriptome analysis in ramie have been
published within a span of 6 months during 2014-15; while before 2013, no
transcriptome database was available for this crop. The challenge in the next
generation plant breeding would be to effectively translate the huge genomic
and transcriptomic resources for developing genic or tightly linked markers
for accurate and rapid screening of breeding populations.
5. Conclusion
DNA marker research in the bast fibre crops are gaining attention in
present days, owing to discovery of large number of markers. Various
traditional and novel marker systems are being employed in these crops for
genetic diversity study, phylogeny, evolution, hybridity testing, paternity
identification, genetic mapping and QTL identification. However,
application of marker assisted selection in large scale has not progressed
much. The situation is similar for other major crops like wheat, cotton,
pulses and oilseeds, except in rice, where MAS has been effectively utilized
to transfer many traits of economic importance.
Genomic researches in bast fibre crops is highly challenging. Jute, as a
species is very suitable for genomic research, because the genome size is
quite small and the species is self-pollinated. But, extent of genetic
polymorphism is very low in jute, even for EST-SSR and SNP markers. This
poses a great challenge for suitable marker development for use in jute
breeding programmes. Flax, on the other hand is self-pollinated, but contains
DNA marker research in bast fibre crops 137
a large genome, which makes it difficult for high density genetic map
construction, marker identification and MAS. The kenaf is a diploid species
with big genome size, while another fibre crop roselle is allotetraploid with
bigger genome. Pollination control during plant breeding programmes in
ramie is extremely difficult. Ramie is a cross-pollinated species, where
clonal propagation increases chance of self-fertilization. There are several
limitations for phenotypic selection for perennial crops, such as the size of
breeding population, low heritability and complex inheritance pattern of
many economically important traits, complex G×E interaction and
requirement of multiple harvests per year which needs to be carried out for
several years. Hence, conventional MAS techniques like marker assisted
foreground and background selection may not be very useful in ramie. Thus
the bast fibre crops are genetically complex.
The population structure of the species, target traits, environment and
breeding methods are crucial to determine success of DNA marker
technology in fibre crops. Since genomic resources are scanty for these
crops, lessons learned from model crops like rice, maize and cotton should
be applied for development of genetic maps, identification of QTLs and
obtaining greater selection efficiency through various marker assisted
selection approaches in fibre crops.
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