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Improving Pearl Millet using Genomics and Molecular Breeding Tools

Rakesh K. Srivastava*, Pooja Katiyar, Mahesh D. Mahendrakar


1

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),


Patancheru, Hyderabad, Telangana, 502 324
(* Email id: r.k.srivastava@cgiar.org )

1. Introduction
Pearl millet [Pennisetum glaucum (L.) R. Br.] is a staple food for millions of poor people living in the
semi-arid tropical areas of Africa and Asia. The crop is predominantly grown in drier regions of West and
Central Africa (WCA), Eastern and Southern Africa (ESA) and the Indian subcontinent in South Asia (SA). It
is also emerging as an important fodder and feed crop in Brazil. Besides food for human consumption, the grain
is also used for poultry feed and some industrial purposes. Diseases that are considered economically important
include downy mildew, blast, rust, ergot and smut. Among these, downy mildew is the most destructive and
widespread in India and countries in Africa. Blast and rust are mainly foliar diseases and they affect the fodder
quality and production of both fodder and grain. In recent years, the blast has become more dangerous in many
regions of India. Abiotic stress such as drought, salinity and heat stress causes low yield or crop failure.

2. Importance of pearl millet as a grain and fodder crop


Pearl millet has high nutritive value, it contain carbohydrate ranges from 57 to 67.5g 100g-1 which is
higher than maize. Protein contain of pearl millet is 11.8 g 100g-1, it has higher level of iron (31-61ppm) and
zinc density (32-54ppm) in comparison to other cereals. Pearl millet is richer in fat content 5 mg 100g-1 as
compared to most grains. Pearl millet is a rich source of energy (361 KCal 100g-1). Pearl millet is popular
among livestock producers for grazing, silage, hay, and green chop. The grain can be used in poultry, cattle and
swine rations without adversely affecting feed ef ciency or weight gain. The crude protein content of green
pearl millet forage varies from 6 to 20%.

3. Development of genetic tools and molecular breeding in pearl millet


Molecular markers have been used to monitor DNA sequence variation by introducing favourable traits
from landraces and related species. Molecular markers have been utilized in pearl millet in various selections,
breeding methods and their modi cations for development of varieties, inbred lines and hybrids. As an outcome,
substantial advances in grain productivity and resistance to diseases have been accomplished in less time than
conventional breeding methods. Molecular markers such as amplied fragment length polymorphism (AFLP),
random ampli ed polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), simple
sequence repeat (SSRs/microsatellites), expressed sequence tag (EST), and single nucleotide polymorphism
(SNP) have been developed and used for diversity analysis, linkage mapping, and marker-assisted selection
(MAS) in pearl millet at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),
Patancheru, India. Marker techniques are further being used to assess genetic diversity at the molecular level
in germplasm collections and advanced breeding lines to assist in making appropriate choices of parents for
hybrid breeding, studying population structure, mapping and tagging genes/QTLs for agronomic traits and
disease resistance (Table 1).
3.1. Downy mildew
Downy mildew (DM) or green ear disease caused by Sclerospora graminicola (Sacc.) Schroet. is
considered as most destructive disease in most pearl millet growing areas of Asia and Africa (Andrews et
al., 1985) and causes 20-40% grain yield loss (Singh, 1995), and exhibits a high level of variation across the
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locations. Resistance to downy mildew has generally inherited as a complex trait. Quantitative Trait Loci
(QTL) for downy mildew resistance have been mapped (Jones et al., 1995) and results indicate both major
and minor genes for resistance, but a single major gene is hardly enough to provide high levels of resistance in
an inbred line of the hybrid (Hash et al., 2006). RFLP based map was developed by using cross LGD 1-B-10
ICMP 85410 and Tift 23DB WSIL F2 progeny of two single crosses involving four parent were screened
for resistance to pathogen populations collected from pearl millet in Patancheru in India, Maduguri in Nigeria,
Bengou in Niger and Doffane and Dimbetaba in Senegal (Jones et al., 1995) in this studies major QTLs
explaining 19.4 and 48.9% of the variation were detected on linkage group (LG1) for the Indian pathogen
population, on LG2 for the Senegal population, and on LG4 for the population from Niger and Nigeria. None
of the QTLs was effective against all populations tested. Other RFLP based QTLs was mapped by Jones et al.,
(2002) by crossing 7042 (S) with P7-3 (R). The F2 progeny was analyzed for downy mildew resistant QTL.
Two QTLs was detected on LG1 and LG2, QTLs on LG1 explained the highest phenotypic variances of 31%
to 60%, while another QTL on LG2 explained 10% and 16%. The estimated inheritance of resistance varied
between partially dominant to completely dominant for the QTL on LG1 and between additive and recessive
for the QTL on LG2. Linkage map was developed for the cross of ICMP 451, which is downy mildew resistant
with H77/833-2, which is susceptible to downy mildew. H77/833-2 is an elite male parent of grain hybrids
(including a popular hybrid HHB 67) in India with characteristics of seedling thermo tolerance, high tillering
capacity and earliness, but highly susceptible to downy mildew disease. Using genotypic and phenotype data
for this cross, downy mildew resistance (DMR) QTLs from the resistant parent was mapped to LG1 and LG4.
These QTLs were effective against pathogen isolates from India, Sudan, Mali, Nigeria and Niger. Interestingly,
the susceptible parent also carried resistance QTLs on LG3 and 7 that were effective against isolates from
Nigeria and India (Breese, 2000). Marker-assisted backcrossing (MABC) was successfully employed to
introgress LG1 and 4 DMR QTLs to the pollen parent of HHB 67 (Sharma, 2001). The commercial pearl
millet hybrid HHB 67-Improved incorporating resistance to downy mildew through marker-assisted breeding
was released collaboratively by ICRISAT and the Chaudhary Charan Singh Haryana Agricultural University
(CCSHAU), India in January 2005 (ICRISAT, 2005). HHB 67-Improved is currently cultivated in more than
875,000 ha in Northern and Western India.
Recently, four major QTLs were mapped for resistance to three (Sg 445 from Gujarat, Sg 519 from
Haryana, and Sg 526 from Rajasthan) new virulent isolates of downy mildew. Using new sources of DMR
QTLs, pollen parents of GHB 538, HHB 67- Improved (second cycle improvement), and seed parent of RHB
127 is being undertaken at ICRISAT. The DMR QTL introgression lines and their test cross hybrids are being
tested in multilocation trials at representative test locations in India. Some of the DMR QTL introgression lines
and the test cross hybrids have shown promise in terms of DMR, grain and stover yields, and other agronomic
traits.
3.2. Pyricularia leaf spot (Blast)
Blast (Pyricularia grisea) leaf spot is a major foliar disease of pearl millet in India, and it is negatively correlated
with green-plot yield, dry matter yield, and digestible dry matter. In the recent past, blast has assumed a disease
of major importance in India. Using a biparental RIL mapping population, two QTLS were mapped on LG 4
and 7. Using anking markers, the major LG4 QTL has been moved to the seed parent of a popular hybrid
HHB 146. The improved lines and test cross hybrids have been tested in the multi-location trials under the
All India Coordinated Pearl Millet Improvement Project (AICPMIP). The QTL introgression lines and test
cross hybrids appear promising in terms of foliar blast resistance, grain and stover yields and other traits of
agronomic importance.
3.3. Terminal drought
Drought can affect pearl millet in differential stages of growth, early during plant establishment, in vegetative
developmental stage (intermittent drought) or at the end of growing season in reproductive stage (terminal
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drought), leading to reduced grain production or crop failure. A major QTL for terminal drought tolerance has
been identi ed and validated on pearl millet LG 2 (Bidinger et al., 2005) by using segregating populations
derived from two independent crosses (H 77/833 2 PRLT 2/89-33 and ICMB 841 863B). A QTL associated
with drought tolerance (DT) of grain yield was obtained on LG 2, explaining up to 32% of the variation
in drought tolerance response of grain yield. In the same interval on LG 2, QTLs associated with drought
tolerance of seed mass, harvest index, grain numbers and panicle number also co-mapped. QTLs on other
linkage group was detected which were associated with grain yield determining component traits on LG1,
and LG6 was linked to increased grain lling. A Drought tolerance QTL for stover and biomass yield in the
most severe stress environment was also mapped to this interval on the LG 2. The PRLT 2/89-33 allele at
this QTL interval was associated with increased drought tolerance of grain yield and all the component traits
described above. Increased harvest index and biomass yield conferred by the PRLT 2/89-33 allele. Signi cant
differences in transpiration rate were observed between the drought-tolerant (PRLT 2/89-33 and 863B) and
drought-sensitive parents (H 77/833-2 and ICMB 841) of the mapping families used in mapping the DT-QTL
on LG 2, and among the NILs (ICMR 01029, ICMR 01031, and ICMR 02041) introgressed with this DT-QTL
(Kholova et al., 2010). Different marker type are been used in mapping drought tolerance QTL in pearl millet
such as conserved intron spanning primers (CISPs), EST-SNPs, Conserved orthologous sequences (COS),
EST-SSRs, and GBS/ddRAD-SNPs.
3.4. Other traits
Signi cant progress has been made in generation of genetic stocks to map traits of economic importance.
These include the conventional RIL mapping populations, chromosome segment substitution lines (CSSLs),
TILLING population, and association mapping panel (pearl millet inbred germplasm association panel,
PMiGAP). These mapping populations have been used to map traits like rust, grain iron and zinc density,
panicle length, panicle diameter, grain size, forage quality, etc.

4. Conclusions
With limited genetic and genomic resources, in the past one decade noteworthy progress has been made
in trait mapping, validation and QTL transfer to elite recurrent parent genetic backgrounds in pearl millet at
ICRISAT. Besides signi cant improvement for the target trait values, the advance generation QTL introgression
lines have been found to be comparable or better than the elite recurrent parents for the background agronomic
traits. With the advent of the whole genome sequencing of pearl millet lines including the mapping population
parents, and PMiGAP, it would be possible to precisely map genes/QTLs of small effects, and move into an era
of genomic selection and genomics-assisted breeding in pearl millet.

References
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Table 1. Details of mapping population parents, genetic maps, and trait mapping in pearl millet
S. Trait/
Mapping population parents
No linkage map

Marker

Linkage map LGD 1-B-10 ICMP 85410

RFLP,SSRs 287.7

Downy
81 B P6 ICMP 451 P8
mildew
Downy
W504-1-1 P310-17-B
mildew
Downy
IP18293 Tift 238 D1
mildew
Downy
PT 732B-P2 P1449-2-P1
mildew
Drought
ICMB 841-P3 ICMB 863-P2
tolerance
Downy
ICMB 89111-P6 ICMB 90111mildew
P6
Consensus
a) ICMB 841-P3 863B-P2
linkage map b) H77/833-2 PRLT 2/89-33
c) 81B-P6 ICMP 451-P8
d) PT 732B-P2 P1449-2-P
Linkage map H77/833-2 PRLT2/89-33

RFLP

695.7

LG4

RFLP

421.0

LG4

RFLP

513.5

RFLP

539.0

LG2,LG4 and
LG6
LG4,LG2,LG6
and LG7
LG2 and LG5

3
4
5
6
7

Map Length QTL location on Reference


(cM)
linkage group

RFLP,SSRs 617.4

Liu et al.
(1994)
Devos et al.
(2000)
KolesnikovaAllen (2001)
Azhaguzel
(2001)
Nepolean
(2002)
Yadav et al.
(2004)
Gulia(2004)

RFLP,
SSRs
Genomic
SSR, ESTSSRs

747.9

LG4

898

Rajaram et al.
(2013)

DArT,
SSRs
10 Panicle
(81B 4025-3-2-B)-11-5-2-2-B-2 SSCPlength,

SNPs
Panicle
HHVBC 11 D2 HS-302-3-1-6-8diameter and 2-6-2-B
grain size
11 Downy
LGD 1-B-10 (B70Tift 756)1- RFLP
mildew
4-5
12 Grain yield
ICMB841-P3 ICMB 863-P2
RFLP, SSR

1148

1019

LG2,LG6 and
LG3

Supriya et
al.(2011)
Vengadessan
(2013)

LG2, LG4 and


LG1
LG2, LG3 and
LG4

Jones et
al.(1995)
Bidinger et
al.(2007)

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