ISBN: 978-81-308-0560-3 Editor: Ksenija Taški 5 ... 5.pdfBarrackpore, Kolkata-700120, India;...

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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


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


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


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


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


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.


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


(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


(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


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.


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


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


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


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.


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


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


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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