Plant Science 242 (2016) 278–287

Contents lists available at ScienceDirect

Plant Science
journal homepage: www.elsevier.com/locate/plantsci

From QTL to variety-harnessing the benefits of QTLs for drought, flood

and salt tolerance in mega rice varieties of India through a
multi-institutional network
Renu Singh a , Yashi Singh a , Suchit Xalaxo b , S. Verulkar b , Neera Yadav a , Shweta Singh a ,
Nisha Singh a , K.S.N. Prasad c , K. Kondayya c , P.V. Ramana Rao c , M. Girija Rani c ,
T. Anuradha c , Y. Suraynarayana c , P.C. Sharma d , S.L. Krishnamurthy d , S.K. Sharma d ,
J.L. Dwivedi e , A.K. Singh e , P.K. Singh f , Nilanjay g , N.K. Singh g , Rajesh Kumar g , S.K. Chetia h ,
T. Ahmad h , M. Rai i , P. Perraju j , Anita Pande k , D.N. Singh k , N.P. Mandal l , J.N. Reddy l ,
O.N. Singh l , J.L. Katara l , B. Marandi l , P. Swain l , R.K. Sarkar l , D.P. Singh l , T. Mohapatra l ,
G. Padmawathi m , T. Ram m , R.M. Kathiresan n , K. Paramsivam o , S. Nadarajan o ,
S. Thirumeni o , M. Nagarajan p , A.K. Singh q , Prashant Vikram r , Arvind Kumar r ,
E. Septiningshih r , U.S. Singh r , A.M. Ismail r , D. Mackill r , Nagendra K. Singh a,∗
a
National Research Centre on Plant Biotechnology, New Delhi, India
b
Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhatisgarh, India
c
Acharya N.G. Ranga Agricultural University, Maruteru, AP, India
d
ICAR-Central Soil Salinity Research Institute, Karnal, Haryana, India
e
Acharya Narendra Dev University of Agriculture and Technology, Faizabad, UP, India
f
Banaras Hindu University, Varanasi, UP, India
g
Rajendra Agricultural University, Samastipur, Bihar, India
h
Assam Agricultural University, Jorhat, Assam, India
i
Central Agricultural University, Umiam, Meghalaya, India
j
Jawahar Lal Nehru Krishi Vishwavidyalaya, Reewa, MP, India
k
Birsa Agricultural University, Ranchi, Jharkhand, India
l
ICAR-Central Rice Research Institute, Cuttack, Odisha, India
m
ICAR-Indian Institute of Rice Research, Hyderabad, India
n
Annamalai University, Annamalai Nagar, TN, India
o
Pandit Jawaharlal Nehru College of Agriculture & Research Institute, Karikal, Puducherry, India
p
ICAR-Indian Agricultural Research Institute, Aduthurai, TN, India
q
ICAR-Indian Agricultural Research Institute, New Delhi, India
r
International Rice Research Institute, Los Banos, Philippines

a r t i c l e i n f o a b s t r a c t

Article history: Rice is a staple cereal of India cultivated in about 43.5 Mha area but with relatively low average pro-
Received 20 May 2015 ductivity. Abiotic factors like drought, flood and salinity affect rice production adversely in more than
Received in revised form 10 August 2015 50% of this area. Breeding rice varieties with inbuilt tolerance to these stresses offers an economically
Accepted 13 August 2015
viable and sustainable option to improve rice productivity. Availability of high quality reference genome
Available online 20 August 2015
sequence of rice, knowledge of exact position of genes/QTLs governing tolerance to abiotic stresses and
availability of DNA markers linked to these traits has opened up opportunities for breeders to transfer the
Keywords:
favorable alleles into widely grown rice varieties through marker-assisted backcross breeding (MABB). A
Drought
Submergence
large multi-institutional project, “From QTL to variety: marker-assisted breeding of abiotic stress tolerant
Salinity rice varieties with major QTLs for drought, submergence and salt tolerance” was initiated in 2010 with
Marker-assisted backcross breeding funding support from Department of Biotechnology, Government of India, in collaboration with Interna-
(MABB) tional Rice Research Institute, Philippines. The main focus of this project is to improve rice productivity in
Quantitative trait loci (QTL) the fragile ecosystems of eastern, northeastern and southern part of the country, which bear the brunt of
Rice one or the other abiotic stresses frequently. Seven consistent QTLs for grain yield under drought, namely,
qDTY1.1 , qDTY2.1 , qDTY2.2 , qDTY3.1 , qDTY3.2 , qDTY9.1 and qDTY12.1 are being transferred into submergence

∗ Corresponding author.
E-mail address: nksingh4@gmail.com (N.K. Singh).

http://dx.doi.org/10.1016/j.plantsci.2015.08.008
0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.
 R. Singh et al. / Plant Science 242 (2016) 278–287 279

tolerant versions of three high yielding mega rice varieties, Swarna-Sub1, Samba Mahsuri-Sub1 and IR
64-Sub1. To address the problem of complete submergence due to flash floods in the major river basins,
the Sub1 gene is being transferred into ten highly popular locally adapted rice varieties namely, ADT 39,
ADT 46, Bahadur, HUR 105, MTU 1075, Pooja, Pratikshya, Rajendra Mahsuri, Ranjit, and Sarjoo 52. Further,
to address the problem of soil salinity, Saltol, a major QTL for salt tolerance is being transferred into seven
popular locally adapted rice varieties, namely, ADT 45, CR 1009, Gayatri, MTU 1010, PR 114, Pusa 44 and
Sarjoo 52. Genotypic background selection is being done after BC2 F2 stage using an in-house designed
50K SNP chip on a set of twenty lines for each combination, identified with phenotypic similarity in the
field to the recipient parent. Near-isogenic lines with more than 90% similarity to the recipient parent
are now in advanced generation field trials. These climate smart varieties are expected to improve rice
productivity in the adverse ecologies and contribute to the farmer’s livelihood.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction gence of rice plants for 10–15 days is another major constraint
prevailing mainly in rainfed lowlands. Most rice cultivars cannot
Genetic yield potential enhancement of rice under marginal survive complete submergence for more than a week [10,11]. In
environments is important for increasing production levels to meet India 30% of the rice growing area (12–14 Mha) is prone to flash
the current and future demands. Increasing population pressure, flooding resulting in severe losses with average productivity of only
climate change effects and depleting soil health pose major threats 0.5–0.8 t ha−1 [12]. The late duration varieties grown in rainfed low-
to rice production. Productivity of rice in India is nearly half of the lands usually face submergence stress at seedling stage followed
global figure. The yield gap is quite high in rainfed areas constituting by drought stress during flowering leading to huge yield losses.
more than half of the total area under cultivation, which are affected Similar to drought and submergence, soil salinity also has a devas-
frequently by drought, flood and salt stress. The modern high yield- tating effect on rice yields. It is estimated that 45 Mha of irrigated
ing varieties of rice developed during and after green revolution and 32 Mha of dry land area is affected by salt stress globally [13].
period had impact mainly in the areas with assured irrigation. Rain- In India, 6.3 Mha area is affected by salinity [14]. Rice plants are
fed agro-ecosystems could not reap the benefits of green revolution sensitive to salinity and can tolerate only up to 4 dS m−1 of salin-
varieties, therefore strategies focusing on these fragile ecologies ity [15]. Salt stressed plants face at least three major physiological
will help improve overall productivity. constraints, i.e., water deficit, ion toxicity and ion imbalance.
Breeders continue to develop resistant varieties for tolerance Significant efforts have been made in area of rice molecular
to abiotic stresses through conventional breeding approaches. breeding for abiotic stresses. QTL and genes have been identified
Landraces and traditional varieties have been used as donor of and introgressed into high yielding varieties imparting stress tol-
resistance genes but such introgression is often associated with erance [16]. Drought grain yield QTLs showing large effects, viz.
unwanted linkages. In contrast, precise improvement of popular qDTY1.1 , qDTY2.1 , qDTY2.2 , qDTY3.1 , qDTY3.2 , qDTY9.1 and qDTY12.1
varieties (mega varieties) of rice for one or more traits without have been identified by IRRI, explaining 31–77% of the phenotypic
alteration in quality and adaptive features, should deliver high variance [17–21]. These QTLs need to be pyramided in the locally
impact. The marker-assisted breeding offers fast track and pre- adapted high yielding varieties for enhancing their yield potential
cise introgression of genes/QTLs, allowing incorporation of genes under drought stress situations. Xu and Mackill [22] successfully
without unwanted linkages and minimum changes in the genetic identified a major QTL on rice chromosome 9 (Sub1) contributing
constitution of the recipient variety. Rice is a model crop for about 70% of the phenotypic variation for submergence tolerance,
marker-assisted breeding with the availability of high quality ref- besides several minor QTLs on other chromosomes [23–25]. The
erence genome sequence and an array of marker platforms and Sub1 region on chromosome 9 was narrowed to 0.16 cM using F2
information on QTLs/genes. QTLs and/or genes controlling abiotic population [26] and later Xu et al. cloned the Sub1A gene originating
stress tolerance have been identified and used to fast track rice from the flood tolerant genotype ‘FR13A’. The qSub1 QTL has three
genetic improvement across the world through marker-assisted ERF/APETALA2 domains containing genes. A survey for Sub1 genes
backcross breeding (MABB). It has been estimated that this method confirmed the presence Sub1B and Sub1C in all 21 rice accessions
results in significant incremental benefits in the range of $50–900 under study, whereas only a sub-set of Indica and Aus accessions
million per season depending on the crop and size of the country have the Sub1A gene [27]. Gene expression studies in Indica type
[1]. MABB can effectively compliment the conventional breeding varieties with Sub1A gene revealed that submergence tolerance is
efforts towards development of climate resilient improved rice correlated with the presence of Sub1A-1 allele and intolerance is
varieties for the Asian region. associated with the presence of Sub1A-2 allele or complete absence
Drought, flood and salinity are the three most devastating abi- of this gene. While the Sub1 gene has already been transferred to
otic stresses for rice cultivation in the rainfed environments. Nearly, several popular rice varieties, including Swarna, IR 64, Samba Mah-
34 Mha of rainfed lowland and 8 Mha of upland rice in Asia suffers suri, BR 11 and CR 1009, there is an urgent need to transfer this gene
from drought stress of varying intensities almost every year with into other varieties popular in the rainfed lowland areas.
13.6 Mha area affected in India alone [2,3]. Rice crop suffers max- Remarkable progress has been made in understanding the
imum losses due to shortage of water during reproductive stage molecular biology of salinity tolerance in plants. Many landraces
[4–8]. Even though the most popular Indian rice varieties such as of rice such as Pokkali, Nona Bokra, Dular, Azgo and Damodar are
Swarna, Samba Mahsuri, IR 64 and MTU 1010 are highly produc- known to possess remarkable tolerance to salt stress through com-
tive in the irrigated areas, these are also grown on large acreage in plex set of physiological mechanisms, including sodium exclusion,
rainfed environments due to their high yield potential and pre- tissue tolerance, compartmenting salts into the apoplasts, seques-
ferred grain quality. However, these varieties are susceptible to tration of salts into older tissues, stomatal responsiveness, and
drought and their improvement for drought tolerance would be upregulation of the antioxidant system during stress [28–30]. A
highly impactful [9]. Flash flooding leading to complete submer- number of QTLs have been identified in rice for the salt tolerance
280 R. Singh et al. / Plant Science 242 (2016) 278–287

Table 1
Recipient varieties, donor lines with parentage indicated in bracket, foreground and recombinant markers and research centres involved in the transfer of QTLs/gene for
abiotic stress tolerance into rice mega varieties.

Recipient QTL Donor Foreground marker Recombinant marker Institution

Swarna-Sub1 DTY 1.1 IR 86918-B-305 (N 22/IR 64) RM431 RM3825, RM12091 IGKV, Raipur
DTY 2.1 IR 81896-B-B-195 (IRRI RM521 RM5791,RM324 CRRI, Cuttack
DTY 3.1 132/2*Swarna) RM520 RM15791,RM16030
DTY 3.2 IR 86931-B-6 (N 22/Swarna) RM523 RM60,RM231 NRCPB, New Delhi
DTY12.1 IR 90019-17-159-B RM511 RM28099,RM28199 IGKV, Raipur
(IR 79971-B-102-B -B/3*Vandana)
Samba DTY 1.1 IR 86918-B-305 (N 22/IR 64) RM431 RM3825, RM12091 CAU, Umiam
Mahsuri-Sub1 DTY 2.1 IR 81896-B-B-195 (IRRI 132/2*Swarna) RM521 RM5791, RM324 IIRR, Hyderabad
DTY 2.2 IR 87728-367-B-B (IR77298-5-6-18/IR 64) RM555 RM279,RM492 CAU, Umiam
DTY 3.1 IR 81896-B-B-195 (IRRI 132/2*Swarna) RM520 RM15791, RM16030 IIRR, Hyderabad
DTY 3.2 IR 86931-B-6 (N 22/Swarna) RM523 RM60,RM231 NRCPB, New Delhi
IR64-Sub1 DTY 1.1 IR 86918-B-305 (N 22/IR 64) RM431 RM3825,RM12091 CRURRS, Hazaribagh
DTY 2.2 IR 87728-367-B-B (IR77298-5-6-18/IR 64) RM555 RM279,RM492
DTY 3.1 IR 81896-B-B-195 (IRRI 132/2*Swarna) RM520 RM15791, RM16030 BAU,
DTY 9.1 IR87728-59-B-B (Aday sel/IR64) RM24350,RM24390 RM566, RM24421 Ranchi
ADT 39 Sub1 CR1009-Sub1 Sub1BC2 RM23869, AU, Annamalai Nagar
ADT 46 Swarna-Sub1 RM8300 PAJANCOA, Karaikal
Bahadur AAU, Titabor
HUR 105 IR64-Sub1 BHU, Varanasi
MTU 1075 Swarna-Sub1 RM23869, RM464 ANGRAU, Maruteru
Pooja RM23869, RM8300 CRRI, Cuttack
Pratikshya CRRI, Cuttack
Rajendra Mahsuri RAU, Samastipur
Ranjit AAU, Titabor
Sarjoo 52 IR64-Sub1 BHU, Varanasi
ADT 45 Saltol FL478 (Pokkali/IR29) RM3412 RM8094, RM493 PAJANCOA, Karaikal
CR 1009 RM580, RM493 AU, Annamalai Nagar
Gayatri CRRI, Cuttack
MTU 1010 AP3206f, RM493 ANGRAU, Maruteru
PR 114 RM580, RM493 CSSRI, Karnal
Pusa 44 CSSRI, Karnal
Sarjoo 52 CSSRI, Karnal

NRCPB: National Research Centre on Plant Biotechnology, CRRI: Central Rice Research Institute, NDUAT: Narendra Dev University of Agriculture and Technology, RAU: Rajendra
Agricultural University, IIRR: Indian Institute of Rice Research, IGKV: Indira Gandhi KrishiVishwavidyalaya, CAU: Central Agricultural University, BAU: Birsa Agricultural
University, JNKVV: Jawaharlal Nehru Krishi Vishwavidyalaya, AU: Annamalai University, PAJANCOA: Pandit Jawaharlal Nehru College of Agriculture, ANGRAU: Acharaya N
G Ranga Agricultural University, BHU: Banaras Hindu University, AAU: Assam Agricultural University, CSSRI: Central Soil Salinity Research Institute

traits [31–38]. A major QTL, Saltol, responsible for selective ion (b) recombinant selection for breaking the linkage drag in the QTL
uptake and Na+ /K+ homeostasis at seedling stage has been mapped region using flanking SSR markers; and (c) background selection
on chromosome 1 using Pokkali/IR29 RIL population [31]. using a high density 50K SNP chip, to select for the highest recurrent
The ‘QTL to variety’ project aims to develop multiple stress toler- parent genome recovery in the derived lines. The DNA marker based
ant rice varieties that can contribute significantly to yield stability selection was supported by phenotypic selection for the introduced
and hence the livelihood security of Indian farmers in the rainfed trait in the greenhouse and for the background recovery in the field.
agro-ecosystems. Seven DTY QTLs for yield under drought are being
transferred in the background of flood tolerant versions of mega
2.2. SSR and SNP genotyping for foreground, recombinant and
rice varieties Swarna, Samba Mahsuri and IR 64. The Sub1 QTL for
background selection
submergence tolerance is being transferred into ten locally adapted
popular rice varieties possessing either intolerant allele or absence
The genomic DNA was extracted from leaf tissue by CTAB
of the Sub1A gene. FL478, a widely used donor line for salt toler-
method [39]. PCR reaction of 10 ␮l volume was set up using
ance is being used to introgress Saltol QTL into seven popular rice
20–30 ng template DNA, 5 pmol of each primer, 0.10 mM dNTPs, 1X
varieties sensitive to inland/coastal salinity.
ViBuffer S (16.0 mM (NH4 )2 SO4 , 50 mM Tris–HCl, pH 9.2, 17.5 mM
MgCl2 , 0.01% TritonTM X-100) and 0.5 U of Taq DNA polymerase
2. Materials and methods (Vivantis Technologies SDN. BHD., Malaysia). The PCR program
was as follows: Initial denaturation at 94 ◦ C for 4 min; denatur-
2.1. Plant materials and the MABB scheme ation in subsequent 35 cycles at 94 ◦ C for 30 sec, annealing at
55 ◦ C for 30 sec, extension at 72 ◦ C for 1 min; and final extension
The full list of recipient and donor parents used in the program is of 72 ◦ C for 10 min. The amplification products were resolved using
shown in Table 1. Fourteen crosses were made for the introgression MetaphorTM agarose gel electrophoresis.
of DTY QTLs into three recipient parents (RP), ten crosses were made High quality DNA (260/280 and 260/230 values in the range
for the introgression of Sub1 QTL into ten recipient varieties, and of 1.8–2.0) was used on Affymetrix chip. The steps involving DNA
seven crosses for the introgression of Saltol QTL into seven recipi- amplification, fragmentation, chip hybridization, and ligation was
ent varieties. The F1 s generated were checked for true hybrids using based on the Affymetrix SNP 6.0 protocol. Approximately 0.75–1 ␮g
DNA markers and then backcrossed with the respective RP to gener- genomic DNA was labeled overnight at 25 ◦ C using 3 volumes
ate the BC1 F1 seeds and the subsequent generations were handled of the BioPrime DNA labeling reactions. The labeled DNA was
as shown in the MABB scheme depicted in Fig. 1. The scheme com- ethanol precipitated, re-suspended in 40 ␮l H2 O and then added
prised of a 3-step selection strategy: (a) foreground selection for to the Affymetrix SNP 6.0 hybridization cocktail. Staining and
the target QTLs/gene using tightly linked or gene based markers; scanning was performed using GeneTitan integrated Affymetrix
 R. Singh et al. / Plant Science 242 (2016) 278–287 281

Fig. 1. Schematic representation of marker-assisted backcross breeding procedure for the transfer of major QTLs for abiotic stress tolerance into popular rice varieties.

platform (http://www.affymetrix.com). Foreground selection for thus keeping the fields free from standing water throughout the
the genes/QTLs was carried out using the gene based or tightly season. Sowing and transplanting for terminal stage drought (TSD)
linked markers for the QTLs/gene. The selection for the lines with experiments was delayed by approximately 20–25 days so as to
minimum linkage drag was done using both proximal and distal coincide with the reproductive-stage stress of the crop with the dry
markers (recombinant selection) as shown in Fig. 2. The sequence spell after the withdrawal of monsoon. The crop was irrigated till 4
of all the SSR primers used is given in Supplementary Table 1. weeks after transplanting and then drained. Precaution was taken
An in-house designed Affymetrix rice SNP genotyping chip (OsS- to select a field on higher topology with light soils, which loses its
NPnks) was used for background selection of twenty advanced soil moisture rapidly to allow the development of severe drought
backcross lines for each donor-recipient combinations. The assay stress in the field. The weekly rainfall pattern was recorded for the
includes 50,051 high quality non-redundant SNPs mostly repre- experiment for 2010–2014 (Fig. 3). The average rainfall received
senting single-copy (SC) genes from whole rice genome with an was 1121, 1320, 1589, 1365 and 1097 mm for the cropping season
average distance of 7.77 Kb between adjacent SNPs [40]. (June–November) of 2010–2014, respectively with 93–97 rainless
days per year. The consistent stress period was 28th September to
19th October. The temperature during the growth season ranged
2.3. Phenotyping for the target traits and background selection
from 25–28 ◦ C whereas it was in the range of 30–35 ◦ C at repro-
ductive stage. Also, parching water level, tensiometer reading and
2.3.1. Drought
soil moisture content (gravimetric method) was recorded at regular
The Swarna-Sub1-DTY1.1 population was phenotyped at IGKV,
intervals throughout the season [41].
Raipur during 2010–2014 in both wet monsoon and dry-summer
seasons. Raipur is located at 21◦ 16 N and 81◦ 36 E at an alti-
tude of 289.6 m above sea level. The BCn F1 and F2 were evaluated 2.3.2. Submergence
under irrigated condition while F3 and other advanced populations Thirty days old seedlings were transplanted at 20 × 15 cm spac-
were evaluated under three different conditions; irrigated, abso- ing in submergence ponds at all the seven centers involved in
lute rainfed and reproductive stage drought at crop establishment breeding for submergence tolerance (Table 1). Flash floods were
(transplanted and direct seeded) stage. Each trial was conducted artificially imposed at 15 days after transplanting for 15 days and
in RCBD with two replications, each genotype having 2–3 rows of percentage of plants survived was recorded at 10 days after de-
1.5 m length. The seed rate was maintained at 2.5 g/m2 for trans- submergence [42,43]. Advanced backcross families were screened
planted conditions and 6.0 g/m2 for direct seeding. The experiments by adopting this protocol from BC3 F2 to BC3 F5 generations. Fam-
were conducted in sandy or clay loam soils with soil pH ranging ilies exhibiting more than 75% plant survival were genotyped
from 6.8–7.4 and organic carbon 0.32–0.34%. Fields were selected with Sub1A gene specific marker. Sub1 positive plants possess-
for different conditions to manage in different ways. For irrigated ing maximum resemblance to the recipient parents were selected
condition continuous water was maintained in these experiments phenotypically in each generation. Phenotyping for seedling stage
from sowing/transplanting to 10 days before maturity. For rainfed submergence was carried out at all the centres involved in transfer
experiments fields were selected which are on higher elevation so of Sub1A. Thirty pre-germinated seeds were sown in plastic cups
that draining of water was easy, These fields were never irrigated which were submerged in submergence tank at 14 days after sow-
so as to allow complete drought like situation and rainwater was ing for 14 days and 10 days after de-submergence plant survival
drained just after rain so as to allow quick appearance of drought, was recorded. This method was adopted to assess submergence tol-
282 R. Singh et al. / Plant Science 242 (2016) 278–287

Fig. 2. Chromosome map showing phusical (Mb) position of QTLs/gene under introgression with the linked markers.

2.4. Evaluation of agro-morphological, grain and cooking quality

traits

The advanced backcross lines along with the recipient parents

were evaluated in a randomized complete block design (RCBD) with
two replications and the data for agro-morphological traits, viz.,
days to 50% flowering (DFF), days to maturity (DM), effective tiller
number (ETN), plant height (PH), panicle length (PL), grains per
panicle (GP), per cent spikelet fertility (SF), thousand grain weight
(TGW) and grain yield/plant (GY) were recorded on five plants for
each advance generation entry.
Fig. 3. Weekly rainfall in the wet seasons of 2010–2014 at IGKV, Raipur, India.

erance at seedling stage in advanced back cross families of BC3 F5 3. Discussion

generation.
3.1. Transfer of QTLs for yield under drought (DTY) into the
2.3.3. Salinity genetic background of Swarna-Sub1, Samba Mahsuri-Sub1 and
Salinity tolerance was evaluated at all the five centres involved IR64-Sub1
in breeding for salt tolerance (Table 1). The rice lines were grown in
hydroponics using nutrient solution and were screened for salinity 3.1.1. Swarna-Sub1-DTY lines
tolerance at seedling stage [44]. The experiment was carried out in It was aimed to transfer individually five different DTY QTLs viz;
saline (EC ∼10 dS/m) stress and control (EC ∼1.2 dS/m) by salinizing qDTY1.1 , qDTY2.1 , qDTY3.1 , qDTY3.2 and qDTY12.1 in the background
the nutrient solution by adding NaCl to obtain the desired salinity of Swarna-Sub1 which was released in India in 2009 and is 97.3%
level. Salinity (EC ∼10 dS/m) was included at the seedling stage (14 similar to Swarna as revealed by 50K SNP chip genotyping [40].
days after sowing) and the desired level of salinity was maintained Later these QTLs will be pyramided by intercrossing the individual
for the next 14 days. The modified standard evaluation system (SES) NILs.
of IRRI was used in rating the visual symptoms of salt toxicity on a
scale of 1–9 [45]. Each line was scored for symptoms after 14 days 3.1.1.1. qDTY1.1 . qDTY1.1 is reported to have consistent effect on
of salinization. grain yield (GY) under both reproductive-stage (RS) as well as non-
 R. Singh et al. / Plant Science 242 (2016) 278–287 283

stress (NS) conditions in Swarna and IR 64 backgrounds. Increase genome-wide 50K SNP chip after drought phenotype screening in
in the GY under both RS and NS in dry season (DS) and wet season 2015.
(WS) was observed mainly because of an overall increase in the
biomass (20). One of the recombinant inbred lines (RIL) IR86918- 3.1.1.3. qDTY3.1 . qDTY3.1 was identified along with qDTY2.1 from
B-305, derived from cross of a drought tolerant Indian Aus variety the Apo/2*Swarna cross [19]. This is the first QTL reported to
N22 with Swarna, was used as donor parent (DP) at IGKV, Raipur. have a large effect on yield in both lowland drought and aero-
The DP was crossed with Swarna-Sub1 to combine submergence bic environments, explaining about 31% of phenotypic variance for
and drought tolerance together. Foreground selection was car- the trait. qDTY3.1 significantly affects days to flowering, and plant
ried out in BC1 F1 generation using the peak marker for the QTL, height in the aerobic and lowland severe stress conditions. Line
RM431 that is located at 38.89 Mb position on the rice chromo- IR81896-B-B-195 was used as DP at CRRI, Cuttack. RM520 (posi-
some 1 [20], whereas recombinant selection was carried out with tion 30.7 Mb) was used as foreground marker, whereas RM15791
markers RM3825 and RM12091 flanking the peak marker at a dis- (position 28.56 Mb) and RM16030 (position 32.5 Mb) were used
tance of 2.4 Mb and 1.35 Mb, respectively (Fig. 2). At each stage the for recombinant selection. The advanced backcross lines are at the
lines were cross-checked for the presence of Sub1 QTL using inser- BC3 F4 generation.
tion/deletion (InDel) marker Sub1BC2 [46]. The advanced backcross
lines are in BC3 F2 generation with fixed recipient type alleles flank- 3.1.1.4. qDTY3.2 . qDTY3.2 was identified from a cross between Van-
ing the QTL, thus reducing the donor genome segment to less than dana/Way Rarem where the drought tolerance allele for this QTL
3.75 Mb. The qDTY1.1 locus is tightly linked to the green revolution is contributed by Vandana. This large-effect QTL shows high posi-
gene sd1. However, in a recent study Vikram et al. [47] have demon- tive effect on grain yield under severe upland and lowland drought
strated that DTY1.1 is about 200–400 kb distal to the sd1gene. The stress and explained 23.4% of the phenotypic variance for grain
DP used for introgression of qDTY1.1 in this project had the wild yield under severe lowland drought [48]. This QTL is reported to be
type Sd1 allele for tallness. Efforts were made to select recombi- in the close vicinity of HD9 locus controlling heading date in rice,
nant plants that have short stature along with drought tolerance. which is associated with earliness under non-stress conditions but
Total 712 plants were selected from a large population of 17,800 here the focus is entirely on the introgression of the QTL regardless
DTY1.1 positive BC1 F3 plants for similarity of their plant type with of the flowering duration. However, Vikram et al. [20] have reported
Swarna. These plants were tested with the foreground as well as that this QTL is responsible for grain yield under drought. Most
recombinant selection markers along with phenotypic evaluation importantly, this QTL also increases harvest index under drought
of drought tolerance in a rainout shelter. From this ten drought stress, which clearly reflects its drought responsiveness and wor-
tolerant lines were identified which had dwarf stature along with thiness for MABB. Line IR86931-B-6 was used as DP at NRCPB, New
phenotypic similarity with Swarna (Fig. 4). Another set of ten Delhi. RM523 (position 1.3 Mb) was used as peak marker for the
qDTY1.1 positive lines were selected from BC3 progeny but they presence of tolerance allele, whereas RM60 (position 1.0 Mb) and
were of tall stature due to linked Sd1 allele. The genotypic back- RM231 (position 2.4 Mb) were used for recombinant selection on
ground selection has been carried out on these 20 fixed lines using distal and proximal ends, respectively. The lines are still at BC1 F1
50K SNP chip to identify lines with highest similarity to Swarna stage due to late initiation of this cross.
(Section 3.4).
3.1.1.5. qDTY12.1 . qDTY12.1 is the largest effect QTL reported for
3.1.1.2. qDTY2.1 . qDTY2.1 is one of the major QTLs for drought tol- grain yield under drought stress for upland condition. This QTL was
erance under lowland and upland environments [19]. This QTL was mapped on chromosome 12 in Vandana/Way Rarem F3 derived
first reported in Apo/2*Swarna population with positive allele con- lines explaining 51% of the genetic variance [49]. The allele for
tributed by drought tolerant parent Apo, which is a well-known drought tolerance was contributed by the susceptible parent,
upland variety of Philippines. qDTY2.1 significantly affects plant Way Rarem. The QTL is reported to increase grain yield under
height in aerobic and lowland stress conditions. Line IR81896-B- drought stress by increasing the number of panicles, the biomass
B-195, a RIL derived from Apo/2*Swarna population was used as accumulation, and the harvest index while reducing days to flow-
DP for qDTY2.1 at CRRI, Cuttack. The foreground selection was done ering. Line IR90019-17-159-B was used as DP at IGKV, Raipur.
using peak marker RM 521 (position 10.8 Mb) and the recombi- Foreground selection was done with RM511 (position 17.4 Mb),
nant selection was carried out using RM5791 (position 10.74 Mb) whereas RM28099 and RM1261 at positions 15.8 and 17.5 Mb were
and RM324 (position 11.4 Mb). The advanced backcross lines are at used for recombinant selection [50,51]. The introgression lines are
BC3 F4 generation under field-testing. The lines with highest sim- at the BC3 F1 generation in 2015.
ilarity to the recipient genetic background will be selected using
3.1.2. Samba Mahsuri-Sub1-DTY lines
Samba Mahsuri-Sub1 is being introgressed with five different
DTY QTLs, namely qDTY1.1 , qDTY2.1 , qDTY2.2 , qDTY3.1 and qDTY3.2 .
Samba Mahsuri is a long duration drought susceptible variety
grown on large acreages of not only rainfed lowlands of eastern
India but also in adjoining areas of Nepal. Samba Mahuri-Sub1
developed at IRRI shows good submergence tolerance but has only
78.7% similarity to Samba Mahsuri [40][40]. All the QTLs mentioned
above have shown significant effects on grain yield under rainfed
lowland drought conditions.

3.1.2.1. qDTY1.1 . Line IR86918-B-305 was used as DP for the trans-

fer of the QTL qDTY1.1 into Sambha Mahsuri-Sub1 background at
CAU, Umiam. Foreground selection was carried out in BC1 F1 genera-
tion using the peak marker RM431, whereas recombinant selection
Fig. 4. Performance of backcross derived lines of Swarna-Sub1/qDTY1.1 in rainout was carried out with RM3825 and RM12091 markers flanking the
shelter under drought condition. QTL peak marker on the proximal and distal sides, respectively.
284 R. Singh et al. / Plant Science 242 (2016) 278–287

The presence of Sub1 QTL was assured using Sub1BC2 marker. The 3.1.3.3. qDTY3.1 . The recipient parent IR64-Sub1 was crossed with
advanced backcross lines are at BC3 F1 generation during 2015. But the donor line IR81896-B-B-195 at BAU, Ranchi for the introgres-
so far the undesirable linkage with the Sd1 gene for tall plant height sion of qDTY3.1 . The foreground selection was carried out using
has not been broken in this cross. RM520 and the recombinant selection was done with flanking
markers, RM15791 and RM16030. The advanced backcross inbred
3.1.2.2. qDTY2.1 . Line IR81896-B-B-195 was used as DP for QTL lines are at BC3 F4 generation undergoing field trial.
qDTY2.1 at IIRR, Hyderabad. The foreground selection was carried
out with peak marker RM521 (position 10.8 Mb), whereas RM5791
3.1.3.4. qDTY9.1 . qDTY9.1 was identified along with qDTY2.2 from a
(position10.74 Mb) and RM324 (position 11.4 Mb) were used as
cross between the tolerant parent, Aday sel and susceptible parent
recombinant selection markers. The advanced backcross lines are
IR 64 under both lowland drought stress and non-stress conditions
at BC3 F4 generation.
[51]. This region of chromosome 9 is reported to have QTLs for grain
yield under drought and shows associations with spikelet fertility,
3.1.2.3. qDTY2.2 . This QTL is suitable for lowland drought condi- plant height, plant water status and grain weight under non-stress
tions and was identified from a cross between tolerant parent Aday conditions along with the drought tolerance [50]. RM24350 and
sel and susceptible IR64 [52]. qDTY2.2 is reported to have effect RM24390 positioned at 15.42 Mb and 15.93 Mb, respectively were
on drought tolerance index, canopy temperature, osmotic adjust- used as foreground markers for the QTL region for introgression into
ment and leaf water content [48]. Line IR87728-367-B-B was used IR64-Sub1 background. The recombinant selection was done using
as DP for transferring this QTL into Samba Mahsuri-Sub1 at CAU, RM566 (position 14.70 Mb) and RM24421 (position 16.27 Mb) and
Umiam. The foreground selection was carried out with RM555 advance backcross inbred lines are at BC3 F3 generation for seed
(position 4.3 Mb) whereas recombinant selection was done with multiplication.
RM279 (position 2.8 Mb) and RM492 (position 7.3 Mb). Though the
distance between peak marker RM555 and the proximal recom-
binant marker RM492 is about 3 Mb but due to unavailability of 3.2. Transfer of Sub1A1 gene for flood tolerance in the genetic
polymorphic SSR markers it could not be reduced further, hence background of ten regional mega-varieties of rice
the background selection by field phenotyping and 50K SNP chip
will help identify line with minimum linkage drag. The advanced Ten mega-varieties of rice popular in the eastern, northeast-
lines for this cross are at BC3 F2 generation for seed multiplication. ern and southern regions of India for their excellent quality and
adaptability were targeted for the introgression of Sub1A gene for
3.1.2.4. qDTY3.1 . Line IR81896-B-B-195 used as DP for the transfer tolerance to complete submergence. Most of these varieties are
of qDTY2.1 into Samba Mahsuri-Sub1 background at IIRR, Hyder- released for rainfed lowland regions and usually face complete sub-
abad, also carried QTL qDTY3.1 . RM520 was used as foreground mergence at seedling and tillering stage. The Sub1A1 gene is known
marker and RM15791 and RM16030 were used for recombinant to provide tolerance to complete submergence at these early estab-
selection to minimize the linkage drag on both sides of the QTL lishment stages.
region. The advanced lines are at BC3 F4 generation undergoing field
evaluation. 3.2.1. ADT 39
ADT 39 was released in 1988 from Tamil Nadu for irrigated early
3.1.2.5. qDTY3.2 . Line IR86931-B-6 was used as DP for the trans- and medium ecosystems. This variety is resistant to blast, bacte-
fer of this QTL into Samba Mahsuri-Sub1 at NRCPB, New Delhi. rial leaf blight (BLB) and leaf spot diseases and the average yield is
RM523 was used as peak marker for foreground selection, whereas 5.8 t/ha. AU, Annamalainagar centre is introgressing Sub1 gene into
RM60 and RM231 were used as recombinant selection markers. The ADT 39 using CR1009-Sub1 as DP. Indel marker Sub1BC2 closely
backcross lines are still at BC1 F1 generation due to late start of this linked with the Sub1A gene, located between Sub1B and Sub1C
cross. genes and showing 38 bp size polymorphism between the DP and
RP [46], was used for foreground selection (Fig. 2). The recombi-
3.1.3. IR 64-Sub1-DTY lines nant selection was ensured with RM23869 (position 6.3 Mb) and
Four DTY QTLs, qDTY1.1 , qDTY2.2 , qDTY3.1 and qDTY9.1 which RM8300 (position 6.6 Mb). The advanced backcross lines are at
showed significant effects in IR64 background were selected for BC3 F1 generation. The program at AU has been discontinued and
transfer into IR 64-Sub1 background, which has 91.7% similarity to lines being transferred to PAJANCOA, Puducherry.
IR64 [40].

3.2.2. ADT 46
3.1.3.1. qDTY1.1 . At CRURRS, Hazaribagh, IRRI line IR86918-B-305
This variety was released in 2002 from Tamil Nadu and is suit-
was used as DP for the transfer of qDTY1.1 into IR64-Sub1 back-
able for irrigated medium ecosystem. The average yield is 6.5 t/ha. It
ground and SSR marker RM431 was used as peak marker for
is being improved at PAJANCOA, Puducherry using Swarna-Sub1 as
foreground selection. The recombinant selection for minimizing the
DP. Sub1BC2 was used for foreground selection and RM23869 and
linkage drag was done using RM3825 and RM12091 on the prox-
RM8300 are being used for recombinant selection. The advanced
imal and distal sides, respectively. The advanced backcross lines
backcross lines are at BC2 F2 generation.
from this cross are at BC3 F2 generation. However, these lines are
tall as the linkage between qDTY1.1 and sd1 gene is not yet broken
in this cross. 3.2.3. Bahadur
This semi-tall statured variety is recommended for rainfed shal-
3.1.3.2. qDTY2.2 . The QTL was transferred by crossing the recipient low lowland condition with an average yield of 5.0–5.5 t/ha. It was
parent IR64-Sub1 with the donor line IR87728-367-B-B at CRURRS, released from Assam state release committee in year 1991. Swarna-
Hazaribagh. RM535 was used as peak marker for foreground selec- Sub1 was used as DP at AAU, Assam. The foreground selection was
tion, whereas RM279 and RM492 were used for recombinant done with Sub1BC2 and recombinant selection was carried out
selection. The advanced backcross inbred lines are at BC3 F4 gen- using SSR markers RM23869 and RM8300. The advanced lines are
eration undergoing field trials. at BC2 F3 generation.
 R. Singh et al. / Plant Science 242 (2016) 278–287 285

3.2.4. HUR 105 3.3. Transfer of Saltol QTL for salt tolerance in the background of
HUR 105 was released in 2008 from Uttar Pradesh state release seven mega-varieties of rice
committee for scented rice growing areas. The average yield of this
variety is 6–7 t/ha. BHU, Varanasi is improving HUR 105 using IR 64- Seven mega-varieties from the saline prone (inland/coastal)
Sub1 as DP. The foreground selection was carried out using Sub1BC2 areas were targeted for the introgression of Saltol, a major effect
whereas RM23869 and RM8300 were used as recombinant mark- seedling stage salt tolerance QTL located on short arm of chromo-
ers. The lines are at BC3 F1 generation. some 1. This 1.5 Mb region spans from 11.2–12.7 Mb [53]. FL478,
a RIL derived from Pokkali/IR29 has been used as DP for all the
recipient parents described below.
3.2.5. MTU 1075
This highly popular semi-dwarf statured rice variety of southern
India was released from Central varietal release committee (CVRC) 3.3.1. ADT 45
in 2008 for irrigated medium rice ecosystem. The variety is resistant This variety was released in 2001 from Tamil Nadu state, suited
to major diseases/pests of rice, with an average yield of 5.6 t/ha. The for irrigated early ecosystems with an average yield of 6.1 t/ha.
variety is improved at ANGRAU, Maruteru using Swarna-Sub1 as ADT 45 is resistant to stem borer, BPH and gall midge disease.
DP. Sub1BC2 is being used as foreground marker whereas RM23869 This variety is being improved for salinity tolerance at PAJAN-
(position 6.3 Mb) and RM464 (position 6.5 Mb) are the recombinant COA, Karaikal. The foreground selection was carried out using peak
markers (Fig. 2). The advanced backcross lines are at BC3 F3 gener- marker RM3412 (position 11.56 MB). The recombinant selection is
ation. The background selection of 18 advanced lines was carried being done using RM8094 (position 11.23 Mb) and RM493 (posi-
out using 50K chip (Section 3.4). tion 12.26 Mb) (Fig. 2). The advanced backcross lines are at BC3 F2
generation.
3.2.6. Pooja
Pooja was released in 1999 from CVRC for rainfed shallow low- 3.3.2. CR 1009
land. This variety gives an average yield of 5 t/ha. The MABB for this This highly popular variety was released in 1983 by CVRC for
variety is being carried out at CRRI, Cuttack using Swarna-Sub1 as rainfed shallow lowlands with an average yield of 6 t/ha. The
DP. The foreground selection is being carried out using Sub1BC2 improvement of this variety for salinity tolerance is being car-
and the recombinant selection with RM23869 and RM8300. The ried out at AU, Annamalainagar (to be transferred to ANGRAU,
advanced lines are at BC2 F2 generation. Maruteru). The foreground selection is being done using RM3412
and the recombinant selection with RM580 (position 9.6 Mb) and
RM493 (position 12.26 Mb). The advanced backcross lines are at
3.2.7. Pratikshya
BC3 F1 generation.
Pratikshya was released from Odisha state in year 2005 for
irrigated medium ecosystem. The average yield of this variety is
4.8 t/ha. Swarna-Sub1 was used as DP for the transfer of Sub1 3.3.3. Gayatri
gene. Sub1BC2 was used as foreground selection marker whereas The variety was released in 1988 from Odisha state suited for
RM23869 and RM8300 were used as recombinant markers on rainfed shallow lowland. Gayatri gives an average yield of 5 t/ha.
either side. The advanced lines are at BC3 F1 generation. CRRI, Cuttack is using RM3412 as foreground marker and RM580
(position 9.6 Mb) and RM493 (position 12.26 Mb) as recombinant
markers. The advanced backcross lines are at BC3 F2 generation.
3.2.8. Rajendra Mahsuri
This high yielding variety was released in 2002 from Bihar state
for rainfed shallow lowland. It has resistance to brown plant hopper 3.3.4. MTU 1010
(BPH), leaf folder and moderately resistant to BLB. The average yield MTU 1010 was released in 2000 for irrigated early ecosystems
of this variety is 5–6 t/ha. RAU, Samastipur is involved in improving from Andhra Pradesh and has become one of the most popular rice
Rajendra Mahsuri for submergence tolerance using Swarna-Sub1 varieites in India with the highest breeder seed indent demand
as DP. The foreground selection is done using Sub1BC2 whereas of 46 tons during Kharif 2015. The average yield of the variety is
recombinant selection is carried out using RM23869 and RM8300. 4.0–4.5 t/ha. ANGRAU, Maruteru is using RM3412 for foreground
The advanced lines are at BC2 F1 generation. selection whereas AP3206f (position 11.2 Mb) and RM493 (position
12.26 Mb) are being used as recombinant marker. The advanced
lines are at BC3 F2 generation.
3.2.9. Ranjit
The variety was released for rainfed shallow lowland from
Assam state in year 1994 with an average yield of 5.0–5.5 t/ha. The 3.3.5. PR 114
introgression of Sub1A1 is being carried out at AAU, Assam using PR 114 was released from Punjab in year 2000 for irrigated
Swarna-Sub1 as DP. The foreground selection was carried out with medium ecosystems. The variety is resistant to BLB and gives an
Sub1BC2 whereas recombinant selection was done with RM23869 average yield of 6.7 t/ha. The variety is being improved for salinity
and RM8300. The lines are at BC2 F3 generation. Ranjit-Sub1 lines tolerance at CSSRI, Karnal. The foreground selection is being carried
have entered the AICRP field trials during 2015. out using RM3412 whereas RM580 and RM493 are the recombinant
markers. The advanced lines are at BC3 F3 generation.
3.2.10. Sarjoo 52
Sarjoo 52 was released in 1980 from Uttar Pradesh for irrigated 3.3.6. Pusa 44
medium ecosystem. The average yield of this variety is 5–6 t/ha. Pusa 44 was released from CVRC in 1993 for irrigated early
BHU, Varanasi has used IR 64-Sub1 as DP for the transfer of Sub1A ecosystem with an average yield of 5.5–6.0 t/ha. CSSRI, Karnal is
gene. The foreground selection was done with Sub1BC2 and the impoving this variety for salinity tolerance. RM3412 is being used
recombinant selection was carried out with RM23869 and RM8300 for confirmation of the QTL whereas RM580 and RM493 is being
on either side of the gene. The advanced backcross lines are at BC2 F2 used for confirming the lines with minimal linkage drag on both
generation. the sides of the QTL. The advanced lines are at BC3 F3 generation.
286 R. Singh et al. / Plant Science 242 (2016) 278–287

3.3.7. Sarjoo 52
This variety was released in 1980 from Uttar Pradesh for irri-
gated medium ecosystem. The average yield of this variety is
5–6 t/ha. The variety is being improved for salinity tolerance at
CSSRI, Karnal. The foreground selection is being done using RM3412
whereas the recombinant selection is done using RM580 and
RM493. The advanced backcross lines are at BC3 F3 generation.
In addition, crosses have been made to pyramid both seedling
and reproductive stage salinity tolerance to make variety tolerant
to the stress on all stages. A robust reproductive stage salin-
ity tolerance QTL qSSISFH8.1 was mapped on chromosome 8 in a
CSR27/MI48 RIL population, which increases the spikelet fertil-
ity percentage under high salt concentration [54]. Introgression of
both Saltol and qSSISFH8.1 will be useful for improving productivity
under inland salinity areas where only seedling stage tolerance is
not sufficient.

3.4. Background selection of advanced backcross lines using 50K

SNP chip

In our strategy for MABB, genotypic background selection was

Fig. 5. Per cent recurrent parent genome recovery on carrier chromosome in whole
postponed till BC3 F2 with the thinking that rapid gain of recipient genome (A) qDTY1.1 introgressed lines in Swarna-Sub 1 background (B) Sub 1A
background occurs during the first three backcrosses even without introgressed lines in MTU1075 background using 50K SNP chip.
any selection. Therefore, during early generations more attention
was paid on foreground selection for the QTL using peak marker
85–110 data points) [55] and therefore, use of SNP chip would be a
and recombinant selection using QTL flanking markers to minimize
better choice for the background selection in MABB.
the linkage drag. Background selection before BC3 was restricted
The project aims to develop abiotic stress tolerant improved
primarily to comparing the phenotypic appearance of backcross
versions of popular rice varieties adapted to eastern, northeast-
derived lines with the recipient parent (Fig. 1). In-house designed
ern and southern regions of India. So far most of the backcross
50K SNP chip is being used for genotypic background selection
lines are at BC2 –BC3 generations and background genotyping selec-
[40]. None of the MABB programs conducted so far has used such
tion is under progress. It is expected that these lines when released
high density SNP markers for background selection which will
as commercial varieties will provide yield stability in the rainfed,
help in selecting advanced backcross lines with maximum simi-
drought–flood prone and saline ecosystems of rice cultivation in
larity to recipient genome as well as detailed information on the
India. The selected lines will be tested along with the recurrent par-
transfer of non-target genomics regions from the donor parent.
ent for different parameters before entry into National coordinated
A high-density background selection will ensure minimum alter-
trials.
ations in the genome of highly popular recipient variety, which will
greatly help in their adoption by farmers. At present the background
selection has been completed for two crosses only, namely Swarna- Competing financial interests
Sub1/IR86918-B-305 for the introgression of drought tolerance QTL
qDTY1.1 (20 lines) and MTU 1075/Swarna-Sub1 for submergence The authors declare no competing financial interests.
tolerance (18 lines). These advanced backcross lines were identi-
fied based on agro-morphological, grain yield and foreground trait Acknowledgements
phenotyping. R1–R5 are in BC1 F5 whereas R6–R20 are in BC2 F2 gen-
eration (Fig. 5). A range of 82.33–94.01% overall recipient genome The research work was financially supported by Department
recovery and 71.23–90.84% similarity on carrier chromosome of of Biotechnology (DBT), Government of India and ICAR “National
the recurrent parent Swarna-Sub1 was observed. Among the lines Professor – B.P. Pal Chair” project.
having recombined the dwarf stature with the QTL DTY1.1 , line no.
R7 showed the highest recipient genome recovery (Fig. 5A).
Appendix A. Supplementary data
Similarly 18 advanced backcross lines (M1–M17 and M20)
from MTU 1075/Swarna-Sub1 cross at BC3 F4 generation, showed
Supplementary data associated with this article can be found, in
91.88–90.29% overall recipient genome recovery and 85.35–88.45%
the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.08.
similarity for chromosome 9 (Fig. 5B). Most of the donor lines
008.
for aforesaid QTLs used in this study were product of pre-
breeding/genetic enhancement activities from IRRI’s breeding
programme. These included FL478, a RIL derived from IR29/Pokkali References
cross harbouring Saltol for seedling stage salinity tolerance,
[1] Vida-Lina Esperanza B. Alpuerto, George W. Norton, Jeffrey Alwang, Abdelbagi
Swarna-Sub1, CR1009-Sub1 and IR64-Sub1, as donors for sub- M. Ismail, Economic impact analysis of marker-assisted breeding for tolerance
mergence tolerance, since these donors were available in highly to Salinity and Phosphorous deficiency in rice, Rev. Agric. Econ. 31 (2009)
adapted genetic background, we did not face serious problem of 779–792.
[2] M.C.S. Wopereis, M.J. Kropff, A.R. Maligayab, T.P. Tuong, Drought-stress
negative linkage drag. The background selection using 50K chip responses of two lowland rice cultivars to soil water status, Field Crops Res.
was found to be highly informative as compared to low-density 46 (1996) 21–39.
SSR markers as it provided better insight of the donor segments [3] R.E. Huke, E.H. Huke, Rice Area by Type of Culture, South, Southeast, and East
Asia, IRRI, Los Banos, Philippines, 1997.
still remaining in the advanced backcross lines. Also, it is much [4] T.C. Hsiao, The soil—plant atmosphere continuum in relation to drought and
more cost-effective (US$ 84/genotype with 50,051 SNP data points) crop production, in: Drought Resistance in Crops with Emphasis on Rice, IRRI,
as against the SSR markers (US$ 70/genotype with approximately Manila, Philippines, 1982, pp. 39–52.
 R. Singh et al. / Plant Science 242 (2016) 278–287 287

[5] J.C. O’Toole, Adaptation of rice to drought prone environments, in: Drought [30] F. Moradi, A.M. Ismail, Responses of photosynthesis, chlorophyll fluorescence
Resistance in Crops with Emphasis on Rice, IRRI, Los Banos, Philippines, 1982, and ROS scavenging system to salt stress during seedling and reproductive
pp. 195–213. stages in rice, Ann. Bot. 99 (2007) 1161–1173.
[6] G. Pantuwan, S. Fukai, M. Cooper, S. Rajatasereekul, J.C. O’Toole, Yield response [31] G.B. Gregorio, Tagging salinity tolerance genes in rice using amplified
of rice (Oryza sativa L.) genotypes to drought under rainfed lowlands: 2. fragment length polymorphism (AFLP), PhD. thesis, University of the
selection of drought resistant genotypes, Field Crops Res. 73 (2002) Philippines, Los Baños, 1997.
169–180. [32] N.T. Lang, S. Yanagihara, B.C. Buu, Quantitative trait loci for salt tolerance in
[7] J. Lanceras, G. Pantuwan, B. Jongdee, T. Toojinda, Quantitative trait loci rice via molecular markers, OMonRice 8 (2000) 37–48.
associated with drought tolerance at reproductive stage in rice, Plant Physiol. [33] V.D. Tuan, Y. Fukuta, M. Yano, T. Ban, Mapping quantitative trait loci for
135 (2004) 384–399. salinity tolerance in rice, OMonRice 8 (2000) 27–35.
[8] R. Venuprasad, M.E. Bool, C.O. Dalid, et al., Genetic loci responding to two [34] P. Bonilla, J. Dvorak, D. Mackill, K. Deal, G. Gregorio, RLFP and SSLP mapping of
cycles of divergent selection for grain yield under drought stress in a rice salinity tolerance genes in chromosome 1 of rice (Oryza sativa L.) using
breeding population, Euphytica 167 (2009) 261–269. recombinant inbred lines, Philippines Agric. Sci. 85 (2002) 68–76.
[9] A. Kumar, J. Bernier, S. Verulkar, H.R. Lafitte, G.N. Atlin, Breeding for drought [35] J.M. Niones, Fine Mapping of the Salinity Tolerance Gene on Chromosome 1 of
tolerance direct selection for yield. response to selection and use of drought Rice (Oryza sativa L.) Using Near Isogenic Lines, MS, Dissertation, University of
tolerant donors in upland and lowland-adapted populations, Field Crops Res. the Philippines Los Baños, Laguna, 2004.
107 (2008) 221–231. [36] H.X. Lin, M.Z. Zhu, M. Yano, J.P. Gao, Z.W. Liang, QTLs for Na+ and K+ uptake of
[10] M. Palada, B.S. Vergara, Environmental effect on the resistance of rice the shoots and roots controlling rice salt tolerance, Theor. Appl. Genet. 108
seedlings to complete submergence, Crop Sci. 12 (1972) 209–212. (2004) 253–260.
[11] S.W. Adkins, T. Shiraishi, J.A. McComb, Submergence tolerance of rice—a new [37] M.J. Thomson, M.D. Ocampo, J. Egdane, M.A. Rahman, A.G. Sajise, et al.,
glasshouse method for the experimental submergence of plants, Physiol. Characterizing the Saltol quantitative trait locus for salinity tolerance in rice,
Plant 80 (1990) 642–646. Rice 3 (2010) 148–160.
[12] M.K. Bhowmick, M.C. Dhara, Sudhanshu Singh, et al., Improved management [38] M.R. Islam, M.A. Salam, L. Hassan, et al., QTL mapping for salinity tolerance at
options for submergence-tolerant (sub1) rice genotype in flood-prone rainfed seedling stage in rice, Emir. J. Food Agric. 23 (2011) 137–146.
lowlands of West Bengal, Am. J. Plant Sci. 5 (2014) 14–23. [39] H.G. Murray, W.F. Thompson, Rapid isolation of high molecular weight DNA,
[13] FAO, 2008, FAO land and plant nutrition management service, http://www. Nucleic Acids Res. 8 (1980) 4321–4325.
fao.org/ag/agl/agll/spush [40] N. Singh, P.K. Jayaswal, K. Panda, P. Mandal, V. Kumar, et al., Single-copy genes
[14] G. Singh, D.S. Bundela, M. Sethi, K. Lal, S.K. Kamra, Remote sensing and GIS for based 50K SNP chip for genetic studies and molecular breeding in rice, Sci.
appraisal of salt-affected soils in India, J. Environ. Qual. 39 (2010) 5–15. Rep. 5 (2015), http://dx.doi.org/10.1038/srep11600, 11600.
[15] R. Munns, M. Tester, Mechanisms of salinity tolerance, Annual Rev. Plant Biol [41] S.B. Verulkar, S.K. Verma, Screening protocols in breeding for drought
59 (2008) 651–681. tolerance in rice, Agric. Res. 3 (1) (2014) 32–40.
[16] B.C.Y. Collard, D.J. Mackill, Marker-assisted selection: an approach for [42] T. Toojinda, M. Siangliw, S. Tragoonrung, A. Vanavichit, Molecular genetics of
precision plant breeding in the twenty-first century, Philos. Trans. R. Soc. submergence tolerance in rice: QTL analysis of key traits, Ann. Bot. 91 (2003)
Lond. B Biol. Sci. 363 (2008) 557–572. 243–253.
[17] J. Bernier, A. Kumar, R. Venuprasad, D. Spaner, G.N. Atlin, A large-effect QTL [43] A. Vishnuvardhan Reddy, M. Girija Rani, P.V. Satyanaryana, Y. Suryanaryana,
for grain yield under reproductive-stage drought stress in upland rice, Crop N. Chamundeswari, Physiological and molecular responses of rice genotypes
Sci. 4 (2007) 507–516. for different types of flooding, Curr. Biotica 8 (2015) 345–350.
[18] R. Kumar, R. Venuprasad, G.N. Atlin, Genetic analysis of rainfed lowland rice [44] S. Yoshida, D.A. Forno, J.H. Cock, K.A. Gomez, Laboratory Manual for
drought tolerance under naturally-occurring stress in eastern India, Physiological Studies of Rice (IRRI), International Rice Research Institute,
heritability and QTL effects, Field Crops Res. 103 (2007) 42–52. Manila, Philippines, 1976.
[19] R. Venuprasad, C.O. Dalid, M. Del Valle, D. Zhao, M. Espiritu, Identification and [45] G.B. Gregorio, S. Dharmawansa, R.D. Mendoza, Screening rice for salinity
characterization of large- effect quantitative trait loci for grain yield under tolerance, in: IRRI Discussion Paper, International Rice Research Institute,
lowland drought stress in rice using bulk-segregant analysis, Theor. Appl. Manila, Philippines, 1997, pp. 221–230.
Genet. 120 (2009) 177–190. [46] E.M. Septiningsih, A.M. Pamplona, D.L. Sanchez, C.N. Neeraja, G.V. Vergara,
[20] P. Vikram, B.P. Mallikarjuna Swamy, S. Dixit, H.U. Ahmed, M.T. Sta Cruz, et al., Development of submergence tolerant rice cultivars: the Sub1 locus and
qDTY1.1 , a major QTL for rice grain yield under reproductive-stage drought beyond, Ann. Bot. 103 (2009) 151–160.
stress with a consistent effect in multiple elite genetic backgrounds, BMC [47] P. Vikram, M. Swamy, S. Dixit, R. Singh, B.P. Singh et al. Did the green
Genet. 12 (2011) 89. revolution select against drought tolerance in rice? Personal communication.
[21] K.H. Ghimire, L.A. Quiatchon, P. Vikram, B.P.M. Swamy, S. Dixit, et al., [48] R.B. Yadaw, S. Dixit, A. Raman, K.K. Mishra, P. Vikram, et al., A QTL for high
Identification and mapping of a QTL (qDTY1.1 ) with a consistent effect on grain yield under lowland drought in the background of popular rice variety
grain yield under drought, Field Crops Res. 131 (2012) 88–96. Sabitri from Nepal, Field Crops Res. 144 (2013) 281–287.
[22] K. Xu, D.J. Mackill, A major locus for submergence tolerance mapped on rice [49] J. Bernier, A. Kumar, R. Venuprasad, D. Spaner, G.N. Atlin, A large-effect QTL
chromosome 9, Mol. Breed 2 (1996) 219–224. for grain yield under reproductive-stage drought stress in upland rice, Crop
[23] S. Nandi, P.K. Subudhi, D. Senadhira, et al., Mapping QTLs for submergence Sci. 47 (2007) 507–516.
tolerance in rice by AFLP analysis and selective genotyping, Mol. Gen. Genet [50] S. Dixit, B.P. Mallikarjuna Swamy, P. Vikram, H.U. Ahmed, M.T. Sta Cruz, et al.,
255 (1997) 1–8. Fine mapping of QTLs for rice grain yield under drought reveals sub-QTLs
[24] T. Toojinda, M. Siangliw, S. Tragoonrung, A. Vanavichit, Molecular genetics of conferring a response to variable drought severities, Theor. Appl. Genet. 125
submergence tolerance in rice: QTL analysis of key traits, Ann. Bot. 91 (2003) (2012) 155–169.
243–253. [51] K.K. Mishra, P. Vikram, R.B. Yadaw, B.P.M. Swamy, S. Dixit, et al., qDTY12.1 : a
[25] M. Siangliw, T. Toojinda, S. Tragoonrung, A. Vanavichit, Thai jasmine rice locus with a consistent effect on grain yield under drought in rice, BMC Genet
carrying QTLch9 (SubQTL) is submergence tolerant, Ann. Bot. 91 (2003) 14 (2013).
255–261. [52] B.P.M. Swamy, H.U. Ahmed, A. Henry, R. Mauleon, S. Dixit, et al., Genetic,
[26] K. Xu, X. Xu, P.C. Ronald, D.J. Mackill, A high-resolution linkage map in the Physiological, eand Gene expression analyses reveal that multiple QTL
vicinity of the rice submergence tolerance locus SUB1, Mol. Gen. Genet. 263 enhance yield of rice mega-variety IR64 under drought, Plos One 8 (2013)
(2000) 681–689. e62975.
[27] K. Xu, X. Xia, T. Fukao, P. Canlas, R. Maghirang-Rodriguez, et al., Sub1A is an [53] R. Alam, M.S. Rahman, Z.I. Seraj, et al., Investigation of seedling-stage salinity
ethylene response factor-like gene that confers submergence tolerance to tolerance QTLs using backcross lines derived from Oryza sativa L. Pokkali,
rice, Nature 442 (2006) 705–708. Plant Breed. 130 (2011) 430–437.
[28] A.R. Yeo, T.J. Flowers, Salinity resistance in rice (Oryza sativa L.) and a [54] A. Pandit, V. Rai, S. Bal, S. Sinha, V. Kumar, et al., Combining QTL mapping and
pyramiding approach to breeding varieties for saline soils, in: N.C. Turner, J.B. transcriptome profiling of bulked RILs for identification of functional
Passioura (Eds.), Effect of Drought on Plant Growth. Salts in Soils, CSIRO, polymorphism for salt tolerance genes in rice (Oryza sativa L.), Mol. Genet.
Melbourne, Australia, 1986, pp. 161–173. Genomics 284 (2010) 121–136.
[29] A.M. Ismail, S. Heuer, M.J. Thomson, M. Wissuwa, Genetic and genomic [55] A. Khanna, V. Sharma, R.K. Ellur, A.B. Shikari, S.G. Krishnan, et al.,
approaches to develop rice germplasm for problem soils, Plant. Mol. Biol. 65 Development and evaluation of near-isogenic lines for major blast resistance
(2007) 547–570. gene(s) in Basmati rice, Theor. Appl. Genet. 128 (2015) 1243–1259.