Molecular breeding in cotton - Icrisat

2 nd National Workshop on Marker Assisted
Selection for Crop Improvement
Molecular Breeding in Cotton:
Opportunities and Challenges
VIJAY N. WAGHMARE
Division of Crop Improvement
Central Institute for Cotton Research, Nagpur

Cotton Growing Regions of the World
80 countries
320 Lakh ha

Economic Position
• Cotton, popularly known as ‘White Gold’, is
important commercial crop in India.
• It contributes about 30% of the countries export
earnings.
• It provide employment to large section of societyon
farm, textile and allied industries.
• India stands first in global cotton acreage (10.17
m ha) and second in production (29.2 m bales),
while the productivity is at lowest (488 kg lint/ha)
among the major cotton growing countries.

Genetic Resources
• The genus Gossypium comprises of 50 species, among them 5
are tetraploids and 45 are diploids.
• India is the only country in the world growing all the four
cultivated species of cotton (two diploids- G. arboreum and G.
herbaceum; and two tetraploid- G. hirsutum and G.
barbadense) and successfully exploiting heterosis through
cultivation of intra and interspecific hybrids.
• India maintains large germplasm collection, at present
germplasm accessions holding stand at more than 10,000,
second largest, just next to the germplasm collection of USA
• The available land races, wild species are under utilized and are
good source for fibre quality such as strength; resistance to
insect pests and diseases.

Phylogenetic relationship of Gossypium species
Source: Wendel and Cronn (2003). Adv. Agron., 78:139-186.

Genetic variability in the germplasm of
G. hirsutum and G. arboreum.
A field view of G. barbadense

Wild resources of Gossypium

IMPROVEMENT IN YIELD
•In 2009-10, the average lint yield was 488 kg/ha as against 88
kg/ha in 1947-48.
•This increase in cotton yield could be achieved through
development of high yielding varieties and hybrids and their
production and protection technologies.
IMPROVEMENT IN FIBRE QUALITY
•Fibre quality parameters include fibre length, strength, fineness,
maturity and uniformity.
•Development and release of extra-long staple cotton varieties viz.
MCU5, MCU 5VT, Surabhi and Sharda in G. hirsutum; Sujata and
Suvin in G. barbadense and Varalaxmi and DCH 32 in hybrids are
notable landmarks in cotton quality.
•Long staple varieties have also been released in G. arboreum. The
long staple varieties of G. arboreum include PA 255, PA 402, DLSA 17
and AKA 8401.

Varieties from CICR

Field view of G. arboreum selections

Changes in area under the four Gossypium
species
1947 2009

Bt Cotton was approved in 2002 in India
1340 Bt Cotton Hybrids

Kg lint per hectare
Bt-cotton area & productivity in India
Bt cotton
1340 Bt hybrids
Bt Cotton area lakh ha

India ranks 24 th in Cotton productivity
World’s largest cotton area Second in production
Even with 85-90% Bt hybrid area 24th-30 th rank
in productivity. Only India relies on hybrids

Productivity in India and China

Challenges
• Competition from synthetic fibres.
• High incidence of insect pests -resulting into huge
economic loss.
• Stagnation and decline in seed cotton yield.
• Continually narrowing of genetic base of the
cultivated varieties.
• Lack of drought tolerant varieties/ genotypes to
mitigate water stress situation.
• Increasing demand of textile industries for quality
fibres particularly high fibre strength, length,
fineness and elongation of fibres.

Narrow Genetic Base
• Multani and Lyon (1995)- in number of Australian cotton
cultivars found 92.1 to 98.9% genetic relatedness.
• Iqbal et al (1997)- indicated narrow genetic base in
cotton cultivars.
• Brubaker and Wendel (1994)- reported RFLP diversity, it
was lowest in G. hirsutum cultivars than any other
reported taxon.
• Rahman et al. (2002)- reported 81.58 to 94.9 % genetic
relatedness in elite cotton cultivars.
• Dongre et al (2005); Rana and Bhat (2004, 2005) also
reported low level of genetic diversity among the cotton
varieties

• In India, there is no report of efforts made towards
molecular mapping in cotton
• Few reports on use of molecular markers are available
that are mainly on assessing genetic variability / diversity.
• Mostly SSR and RAPD markers were employed.
• At present, CICR is in the process of developing molecular
linkage map in diploid and tetraploid cotton. Mapping
populations such as F2, Backcross and RILs are being
developed for mapping fibre quality traits.
• A genome map is expected to be ready in next 6 months.
• Meanwhile, core set of germplasm has been established
in G.hirsutum that shall be used for association mapping.

Strategies
• Diversification of gene pool.
• Use of wild tetraploid species to generate more variability
in cultivated tetraploid cotton.
• Identification of diverse genotypes for development of
varieties and hybrids.
• Molecular characterization of representative set of
germplasm and wild sources.
• Development of saturated genetic linkage map of
cultivated diploid and tetraploid species.
• Mapping agonomically important traits that includesfibre
quality traits, genes/ QTLs responsible for WUE,
photosysnthesis and biotic and abiotic stresses and
application in plant breeding-
– Biparental mating
– LD based association mapping

Use of Molecular Markers in Cotton
• The first RFLPs based genetic linkage map of tetraploid
cotton was published in 1994 (Reinisch et al 1994).
• RFLPs have widely been used in cotton for genetic
diversity analysis and genome mapping.
• Reinisch et al. (1994) map was further saturated using
the same F2 (G. hirsutum x G. barbadense) population
and a detailed map was published (Rong et al. 2004).
The map consists of 2584 loci spaced at 1.75 cM in 26
linkage groups.
• Comparative map of tetraploid and its diploid
progenitors (Brubaker et al. 1999, Rong et al. 2004);
cultivated and wild tetraploid (Waghmare et al. 2005)
are now available.

• The HT map comprises 589 loci
• Total map length: 4259.4 cM

Genetic linkage maps
• At present, more than a dozen genetic linkage
maps are available.
• Collectively, these maps consists of > 6000
DNA markers that includes-
– 3,300 RFLP
– 700 AFLP
– >2000 SSR and
– 100 SNP

Mapping Genes in Cotton
• About 200 qualitative traits have been identified in diploid and
tetraploid cotton and several of them have been mapped to
specific chromosomes.
• Shappley et al (1998) identified more than 100 QTLs associated
with agronomic and fibre traits.
• Mei et al (2004) detected seven QTLs for fibre related traits, five of
them were mapped on A genome.
• Zhang et al.(2003) also identified QTLs for fibre quality traits.
• Lacape et. Al. (2003) studied 6 quality traits i.e. length, uniformity,
strengh, elongation, fineness and colour.
– Identified 50 QTLs i.e. LOD scores 3.2 to 4.00.
– Additional 30 QTLs –LOD above 2.5
Observations – QTLs detected for various traits often colocalized
within QTL rich regions.

Mapping Genes in Cotton (Related to adaptation)
• Genetic mapping has been used to identify QTLs responsible for
improved productivity under arid conditions (Agrama and Moussa
1996; Tuinstra et al 1996; Ribant et al 1997).
• QTLs that confer physiological variations associated with stress
tolerance-
– Osmotic adjustment - Morgan 1992; Morgan and Tan 1996
– WUE (measured as carbon isotope ration 13C/12C - Martin et al
1989; Mansur et al 1993.
– Ash content- Mian et al 1996; 1998.
– Abscisic acid levels- Quarrie et al 1994; Tuberosa et al 1998.
– Stomatal conductance - Ulloa et al 2000.
• Productivity and physiological differences were genetically mapped in
the same population and found productivity to be unrelated to δ13C
(Mansur et al 1993) or to relative water content (Teulat et al 1998).

a Wild type,
b Fuzzfibered lintless Li1;
c Fuzz-fibered lintless Li2;
d Sparsely linted in n2;
e Naked with lint in n2;
f Naked with lint in N1;
g Naked without lint in N1;
h Naked with lint in Fbl;
i Naked without lint in Fbl;
j Wild type,
k Naked without lint or
tufted in SMA-4

Genetic mapping of cotton fiber mutants.

Saranga et al (2001)
• Detected 161 QTLs for 16 measured traits
Traits QTLs LOD >3
Productivity DM 4
SC 14
HI 11
BW 15
BN 4
Physiological traits OP 12
d13 11
CT 4
Chl.a 3
Chl.b 4
Fibre Traits FL, UR, FS, FE, FF,
FC
79

QTLs Affecting Physiological Traits of Cotton
Traits
Osmotic
Potential (3)
Carbon
Isotope ratio
Chr./Linkage Marker LOD Score Gene
Group
(Dry) Action
Chr.06 PAR 3-32a - RA
Chr.25 PXP1-47 3.74 D
Chr.15 A 1109 - -
Chr.22 PAR 243 3.72 D
LG D04 A 1163b 3.50 D
LG D05 A 1220 5.60 A
• Reduced OP and SC found to share common basis. OP has
clearly been implicated for improved cotton productivity
under arid conditions.

Interval mapping of
root-knot nematode QTL
localized linkage map on
chromosome 11
Genotypes of recombinant lines and their
resistance . Solid bars and open bars represent
the M-120 RNR and Pima S-6 type, respectively.
Gray bars represent the interval in which
recombination has occurred

Summary of QTLs mapped for fibre quality traits in cotton
Fibre Quality Trait No of QTLs No. of Maps
Fibre length 107 20
Fibre uniformity 9 1
Short fibre content 13 2
Fibre strength 102 17
Micronaire 112 16
Fibre finness 33 2

• The available DNA markers linked to the fibre quality
QTLs promise to be used in markers assisted selection
(MAS).
• However, the genetic distances between DNA markers
and most of the QTLs are too far to be used in
molecular breeding.
• There is no published report of successful application
of MAS in cotton
• Thus, fine mapping of the QTLs is must.
• IT is difficult to discriminate co-localization and
coincidence when comparing one QTL with the others.
• Consistency and homology among the QTLs for the
same trait detected in different populations can be
deduced only from their map locations.

Consensus map assembly
Source: Genome Research,15:1198-1210 (2005)

Consensus map of cotton homoeologous group 3 (Chr.3, Chr14/17, and D3).

Conserved synteny between a segment of C06 and Arabidopsis duplicates 11 and 14.

Duplication in hypothetical ancestral cotton chromosomes.

Source: Genetics 176:2577-88 (2007)

C4
Chr. 16
C Map: Comparison between
individual map and a
Consensus map

Genetic map and Arabidopsis syntenic regions of
segment of cotton C12 chromosome

Genomic Resources available in public domain
SSR : 11938 in CMD data base
Summary of cotton ESTs
Gossypium species Available ESTs (As on
28.10.2010)
G. hirsutum 268797
G. arboretum 41768
G. raimondii 63577
G. herbaceum (africanum) 247
G. barbadense 1356
Total ESTs of Gossypium 378184

Whole Genome Sequencing
• As a long term goal of characterizing the spectrum of
diversity among 8 genomes types, D genome
‘Gossypium raimondii’ has been prioritized for
complete sequencing by International Cotton
Community under International Cotton Genome
Initiatives (ICGI).
• Advantages:
– It has the smallest genome (60% of the ‘A’ genome)
– Genome size 880 Mb
– Detailed genetic linkage map is available
– Recently, a physical map of D genome has also been
assembled.

•A whole genome physical map of G. raimondii was assembled.
•A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in
genetically anchoring 1585 contigs to a cotton consensus genetic map.

SRP003645: Whole genome sequencing of Gossypium
raimondii genome
Study Type: Whole Genome Sequencing
Submission: SRA024364 by Monsanto Company on 2010-09-29 15:19:51
Abstract: These data represent whole-genome shotgun sequences of the wild diploid
Peruvian cotton species Gossypium raimondii Ulbr., the closest extant
representative of the Dt subgenome of the domesticated allotetraploids G. hirsutum
L. (Upland Cotton) and G. barbadense L. (Pima Cotton). The data contained in these
accessions comprises roughly 135x genomic coverage of Illumina paired-end 2x100
sequence generated by Monsanto and Illumina as part of a multi-data type approach
to shotgun sequencing of a diploid cotton genome.
Description: n/a
Project: Gossypium raimondii [Monsanto]
Center Project: n/a
Download fastq for entire study(use Aspera plugin for fast download)

International Cotton Genome Initiatives (ICGI)
• Joint Genome Institute (JGI), USA has
completed sequencing of G. raimondii (D
genome) a putative progenitor of tetraploid
cotton.
• Sequence information of Gossypium genome
from two different sources will complement /
synergize the efforts and provide insight into
gene function and allelic variation between
Gossypium genomes.

Mirid Bugs
Creontiades biseratense Distant
Miridae; Hemiptera
Campylomma livida Reuter
(Ragmus morosus Ballard)
Miridae; Hemiptera
Hyalopeplus lineifer walker
Miridae; Hemiptera

Gall midge: New Report
Dasineura gossypii Fletcher,
1914 (Cecidomyiidae : Diptera)

Tea mosquito Helopeltis bradyi
(Water house) on Bt cotton
It is common pest of Guava/Cashew/tea etc
and called as Kajji bug or tea mosquito This
mirid is most dangerous pest causing > 90%
damage or yield loss whenever appeared on
cotton During current season Helopeltis bradyi
out break has been noticed in Hosalli (Uttar
Kannada District) on RCH -708 and MRC -6918
interspecific Bt cotton. The yield loss in both
the hybrids is > 85%.

SUMMARY
• In past two decades extensive genomic resources have been
developed in cotton.
• A large collection of robust, portable markers (SSR) are available in
public domain.
• In cotton, molecular markers have successfully been used to access
genetic diversity of germplasm resources, genetic linkage maps and
mapped important agronomic QTLs using bi-parental mapping
populations.
• However, considering the large genome of tetraploid cotton (2450
Mb), marker density appears to be very less to tag the economically
important QTLs with tightly linked markers.
• There is no published report of successful application of MAS in
cotton
• Thus, fine mapping of the QTLs is must.
• Alternately, LD based association mapping promises effective
utilization of natural genetic diversity of world wide cotton
germplasm resources.