(Oryza sativa L.) UNDER AEROBIC CONDITIONS

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... to discriminate between both the parents are RM281, RM582, RM275, RM224, RM8, RM10, RM19, RM175,. RM226, RM258, RM269, RM304 and RM431.

Agric Res J 55 (1) : 20-26, March 2018 DOI No. 10.5958/2395-146X.2018.00003.0

AGRONOMIC AND MOLECULAR MARKER EVALUATION OF SELECTED F4 PLANTS OF RICE (Oryza sativa L.) UNDER AEROBIC CONDITIONS Kuldeep Kumar*, Kanika Rani, Rahul Kumar Meena, Mahavir, Rajinder Kumar Jain and Sunita Jain Department of Molecular Biology, Biotechnology and Bioinformatics, CCS Haryana Agricultural University, Hisar- 125 004, Haryana ABSTRACT A study was carried out to evaluate segregating F4 aerobic x lowland progenies PAU201/MAS25, PAU201/MAS26 and PAU201/MASARB25 of rice for various physio-morphological and root traits and SSR markers linked to the traits promoting aerobic adaptation. In all three populations, enormous variation was observed for the physiomorphological traits. A significant positive correlation was observed between yield per plant and plant height, effective number of tillers per plant, length/breadth ratio, 100 grain weight, and root length and/or root biomass. Promising plants were selected which had higher grain yield and/or root length and biomass. These were analyzed for the presence of desirable alleles using SSR markers which were previously found to be linked to aerobic traits. Most of these selected plants had the desirable alleles for the markers analyzed. Key words: Aerobic rice, Polymorphism, QTL, Root biomass, SSR markers

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ice (Oryza sativa L.) requires 3000-5000 liters of water to produce one kg of grain which is almost 2-3 times higher than any other cereal crop such as wheat and maize (IRRI, 2009). The declining availability of water and its increasing cost threaten the traditional way of producing irrigated rice in India. To reduce water consumption, water saving technologies for rice cultivation such as alternate wetting and drying (AWD) and aerobic rice need to be introduced that aim to reduce non beneficial water flows from rice fields during crop growth, such as seepage, percolation and evaporation (Bouman et al., 2005). However, water requirement of these production systems is also very high as land preparation consists of soaking, followed by wet ploughing or puddling of saturated soil.

thickness (Qu et al., 2008) and root dry weight (Kanbar and Shashidhar, 2004). Sandhu et al. (2013) reported that QTLqDRW8.1 RM (152-310) flanks to dry root weight; qRL8.2 RM (310-547) and qRL9.1 (524-257) flank to root length; and qRT1.1 RM (488-237) flanks to root thickness. Dixit et al. (2014) identified three QTLs viz. qDTY3.1 (RM168-RM468), qDTY6.1 (RM586-RM217), and qDTY6.2 (RM121-RM541) for grain yield under drought. QTL qDTY3.1 and qDTY6.1 showed consistent effect across seasons under lowland drought-stress conditions. Research efforts have been initiated to transfer the QTLs responsible for aerobic adaptation in the aerobic rice varieties MAS25, MAS26 and MASARB25 to a high yielding rice variety PAU201 so that the water requirement of the high yielding variety can be reduced. The present study was conducted to dissect the presence of these QTLs using SSR marker in F4 progenies of rice under aerobic conditions.

Aerobic rice requires no standing water and the farmers can skip irrigation if soil moisture status is sufficient for the crop. It is a water saving system, as rice can be cultivated with 600 to 700 mm of total water in summer and entirely on rainfall in wet season (Hittalmani, 2007a, 2007b). Varieties suitable for this type of cultivation also possess ability to withstand intermittent drought spells with minimum yield loss (Anonymous, 2008). The adapted rice varieties are grown in fertile aerobic soils that are non-puddled and have no standing water. Supplementary irrigation, however, can be supplied in the same way as to any other upland cereal crops (Wang et al., 2002; Bouman et al., 2005).

MATERIALS AND METHODS Plant material The experimental plant material comprised of seeds harvested from the F3 population (i.e. F4 generation) of PAU201/MAS25, PAU201/MAS26 and PAU201/ MASARB25 crosses. Selected F3 seeds were sown both in the net house as well as in the field conditions to raise the F4 plant. PAU201 is a lowland high-yielding rice cultivar unadapted to cultivation in aerobic conditions (developed at PAU, Ludhiana), with upland and aerobic adapted genotypes MAS25, MAS26 (both developed at the University of Agricultural Sciences, Bangalore) and MASARB25 (developed at IRRI, Philippines), respectively, were used in our study. The populations were developed from crosses involving MAS25, MAS26 and MASARB25 as male parents and PAU201 as female parent.

Genetic variations for adaptation of rice under aerobic conditions in upland have already been deciphered. Many SSR markers have been reported to be linked to QTL promoting aerobic adaptation in rice such as yield under drought (Venuprasad et al., 2009; Vikram et al., 2011), maximum root length (Steele et al., 2007), basal root *Corresponding author : [email protected] Date of receipt : 24.11.2017, Date of acceptance : 18.01.2018

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Field experiment

manually. For fresh root weight, the roots were blotted gently with a soft paper towel to remove any free surface moisture and the roots were weighed immediately. The thickness of the root crown was measured using a vernier caliper. Root length was measured using a centimeter scale. For dry root weight, the roots were dried in an oven set to low heat (50°C) for 2 days, and then cooled in a dry environment. Once cooled, they were weighed on a weighing scale.

F4 generation was sown in the replicates of three lines at the Rice Research Station, Karnal, India, during kharif season, 2015 by direct-seeded aerobic cultivation practices that involved dry seeding at approximately 2 cm depth in dryploughed and harrowed aerobic plots with row spacing of 25 cm. Aerobic fields were irrigated for about 1 week with a 2-3 cm water layer to facilitate crop establishment; thereafter, the fields were re-irrigated once at a 10-day interval. Five best plants among the three replicate of each line were harvested at maturity and the data were recorded on agronomic traits, plant height, number of effective tillers per plant, panicle length, number of panicles per plant, number of grains per panicle, 100 grain weight, grain length/breadth ratio and grain yield per plant. Grain yield per plant was recorded after harvesting, threshing and drying to moisture content adjusted to 14 per ecnt.

Statistical analysis Mean and standard deviations were used as the parameter of variability and phenotypic correlation coefficient was calculated using OPSTAT statistical tool.

Genotyping of mapping population For genotyping, promising plants from respective crosses under both field and net house conditions were selected. Performances of plant for yield and root traits were the main criteria for selection in field and net house, respectively. The DNA marker work was conducted in the Department of Molecular Biology and Biotechnology, CCSHAU, Hisar, India. Genomic DNA was isolated from young leaves using the CTAB method (Saghai-Maroof, 1984). DNA quantity was estimated by ethidium bromide staining on 0.8% agarose gels using a standard containing 100 ng/μl λ genomic DNA. PCR amplification was essentially carried out as described earlier by Jain et al. (2006).

Pot experiment F4 generation was sown in the replicates of five plants, with one plant per pot. Manual weeding was done whenever required. The pots were irrigated with one liter of water for the first 15 days, and then with one liter after every third day up to panicle emergence. After 20 and 40 days, the pots were supplemented with Yoshida solution (Yoshida, 1976). The data on root morphological traits i.e. root length, root thickness, fresh and dry root weight from five plants from each line at maturity were recorded and analyzed. For the measurement of root traits, plants were removed from the soil

A total of 30 SSR most of which were reported to be linked to trait promoting aerobic adaptation (Table 1) were

Table 1. Details of the markers found to linked with different traits promoting yield attributes and aerobic adaptation of rice Markers used

Linked with QTL

Reported by

Trait

RM17

gw12.1

Thomson et al. (2013)

Grain weight

RM259

qDTY1.2

Sandhu et al. (2014)

Grain yield

RM282

gpp3.1

Septiningsih et al. (2003)

Grains per panicles

RM285

grm1.1

Thomson et al. (2013)

Percentage germination

RM306

rfw1c

Li et al. (2005)

Root fresh weight

RM310

qRL8.2

Sandhu et al. (2013)

Root length

RM526

qGY2.2

Sandhu et al. (2013)

Grain yield

RM410

qPH9.1

Sandhu et al. (2013)

Plant height

RM231

qgy3.1

Lanceras et al. (2004)

Grain yield

RM6

bm2.1

Swamy et al (2014)

Vegetative biomass

RM211

qDTY2.2

Sandhu et al. (2014)

Grain yield

RM217

qDTY6.1

Dixit et al. (2014)

Grain yield

RM287

rn11 and brt11c

Li et al. (2005)

Total root number and basal root thickness

RM162

ph6.1

Thomson et al. (2013)

Plant height

RM336

dth7.1

Thomson et al. (2013)

Day to heading

RM13

QRbm5

Yue et al. (2005)

Relative biomass

RM175

rn3

Li et al. (2005)

Total root number

Besides these markers, other markers which were used to discriminate between both the parents are RM281, RM582, RM275, RM224, RM8, RM10, RM19, RM175, RM226, RM258, RM269, RM304 and RM431.

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screened for polymorphism between the parents of all the three populations. The markers were obtained based on published rice genome maps and their physical position (Mb) on the indica genome (www.gramene.org) was used as a reference. A total of 5, 6 and 3 markers showed polymorphism in PAU201/MAS25, PAU201/MAS26 and PAU201/MASARB25 F4 populations, respectively, and were run on 10, 16 and 14 selected plants, respectively. Genetic similarities between the cultivars were measured by the similarity coefficient based on the proportion of shared electromorphs using the ‘Simqual’ subprogram of the NTSYS-PC (Version 2.02 Exeter Software, Setauket, NY, USA) software package (Rohlf, 1993). The resultant distance matrix data were used for two-dimensional scaling of rice genotypes by principal component analysis (PCA).

yield and yield components when compared for both aerobic rice varieties and lowland indica rice varieties under aerobic conditions. In all the three crosses, effective numbers of tillers, panicle length and number of grains per panicle were found to be positively correlated with yield, panicle length and grain weight (Table 2). In net house conditions, a total of 21, 41 and 23 plants (grown in replicate of five but only given numbers survived) were grown from seed of 6, 12 and 12 F3 plants selected on the basis of performance for root trait and yield attributes of PAU201/MAS25, PAU201/MASARB25 and PAU201/ MAS26 to develop F4 progenies, out of which 3, 6 and 4 plants were found to be promising in terms of root length, root thickness and root biomass when compared with performance of aerobic rice varieties and low-land indica rice varieties. Under water-limited conditions in net house, most of the plants of both crosses showed transgressive segregation for root traits and grain yield. Several F4 plants had greater root length, dry root weight, grain yield per plant and grain length-breadth ratio than the respective aerobic rice parents (Fig. 1). Almost all of the promising plants selected after evaluation of root traits showed significantly better performance than parents for all the root traits analyzed. In all the three crosses root length and plant height were found negatively correlated, root length and root thickness were negatively correlated with effective number of tillers per plant, but surprisingly panicle length was found to be positively correlated with the fresh and dry root weight in the

RESULTS AND DISCUSSION Morphological traits Populations derived from the above crosses showed large variation for physio-morphological and root traits indicating a greater diversity among these populations. In field conditions, a total of 30, 60 and 60 plants (replicate of five) were grown from seed of 6, 12 and 12 F3 plants selected on the basis of performance for yield attributes of PAU201/ MAS25, PAU201/MASARB25 and PAU201/MAS26 to develop F4 progenies, out of which 7, 10 and 10 plants, respectively, were found to be promising in terms of grain

Table 2. Phenotypic correlation coefficient between different physio-morphological traits in F4 population of respective crosses of rice grown in the field under aerobic conditions Traits

Cross variety

PH (cm)

ET

PL (cm)

GW (g)

Y (g)

NG

PH (cm)

MAS25 MASARB25 MAS26

1.000 1.000 1.000

ET

MAS25 MASARB25 MAS26

0.247 0.245 0.236

1.000 1.000 1.000

PL (cm)

MAS25 MASARB25 MAS26

0.037 0.255 0.164

-0.078 0.087 0.366

1.000 1.000 1.000

GW (g)

MAS25 MASARB25 MAS26

0.254 0.452 0.046

-0.140 0.085 0.310

0.401 0.328 0.269

1.000 1.000 1.000

Y (g)

MAS25 MASARB25 MAS26

0.481 0.376 0.248

0.620 0.794 0.816

0.194 0.287 0.534

0.411 0.339 0.178

1.000 1.000 1.000

NG

MAS25 MASARB25 MAS26

0.326 0.096 0.011

-0.063 0.115 0.006

0.272 0.090 0.395

0.415 0.065 -0.206

0.628 0.551 0.510

1.000 1.000 1.000

LB

MAS25 MASARB25 MAS26

-0.276 0.063 0.094

-0.037 -0.039 0.365

0.058 0.429 0.269

0.102 0.325 0.120

-0.198 0.067 0.326

-0.102 -0.189 0.212

LB

1.000 1.000 1.000

PH, RL, FRW, DRW, ET, RT, PL, GW, Y and LB stand for plant height, root length, fresh root weight, dry root weight, effective number of tillers , root thickness, panicle length, 100-grain weight, yield and grain length: breadth ratio, respectively.

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Fig. 1. Variation for root trait in PAU201/MAS26(a), PAU201/MASARB25(b) and PAU201/MAS26F4(c) populations grown in net house. A stands for MAS25, MASARB25 and MAS26, B stands for PAU201 while C, D and E stand for the progenies derived from respective crosses.

net house under water limited aerobic conditions (Table 3).

six SSR loci (RM582, RM17, RM336, RM275, RM13 and RM175). Notably four out of six net house plants analyzed for RM175 (rn3, Li et al. 2005) as shown in Fig. 2b and root morphological traits, shows allele specific to MASARB25, also there performance for root traits was comparable to it. In PAU201/MAS26 cross all the F4 plants had MAS26 specific allele or PAU201 specific allele or alleles specific for both the parents at three SSR locus (RM281, RM287 and RM306). A total of 4 plants were used for both molecular profiling with RM287 (rtn11 and brt11c, Li et al., 2005) as shown in Fig. 2c and root morphological studies, 2 plants shows allele specific to MAS26. These plants performed well for the all the root traits analyzed.

Variation and genetic relationship Most of the markers used were found to be linked to aerobic traits previously (Table 1). In PAU201/MAS25 all the F4 plants had either MAS25 specific allele or PAU201 specific allele or alleles specific for both the parents at five SSR locus (RM17, RM281, RM282, RM285 and RM306) indicating heterozygous state or absence of both the alleles. Agarose gel displaying allelic polymorphism in PAU201/MAS25 F4 plants for SSR marker is shown in Fig. 2a. In PAU201/MAS25 population notably out of four net house plants analyzed for molecular profiling and root morphological data one exhibited RM306 (rfw1c, Thomson et al., 2013) allele same as MAS25 allele ( confirmed only by using single marker). Also the fresh root weight of this plant was the highest among the all F4 plants analyzed for this particular cross. In PAU201/MASARB25 cross all the F4 plants had MASARB25 specific or PAU201 specific allele or had both the allele at

Besides the molecular marker studies, NTSYS-PC and PCA analysis also showed similar results clearly depicting the closeness of some progenies towards the aerobic parents in all the three crosses. The results of NT SYS-PC and PCA are shown in Figs. 3(a)-3(f). Tight linkage of a marker with a QTL is the key for dissecting the inheritance of the particular

Fig. 2. SSR markers showing polymorphism between PAU201/MAS25 F4 population at RM306 locus (Fig. 2a), between PAU201/ MAS26 F4 population at RM287 locus (Fig. 2b) and between PAU201/MASARB25 F4 population at RM175 locus (Fig. 2c). In Fig. 2(a) P1 stands for MAS25, P2 stands for PAU201, A1-A7 are progenies grown in field while A8-A10 are progenies grown in net house, in Fig. 2(b) P1 stands for MAS26, P2 stands for PAU201, A1-A10 are progenies grown in field while A11-A14 are progenies grown in net house while in Fig. 2(c) P1 stands for MASARB25, P2 stands for PAU201, A1-A10 are progenies grown in field while A11-A16 are progenies grown in net house.

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Fig. 3a. Dendrogram (NTSYS-pc) showing genetic relationships between ten PAU201/MAS25 F4 plants and parental genotypes using allelic data at five SSR loci (RM17, RM281, RM282, RM285 and RM306) (E1-E7 are plants grown in field while E8-E10 are plants grown in net house)

Fig. 3b. Two-dimensional PCA scaling of ten PAU201/MAS25 F4 plants and parental genotypes using allelic diversity data at five SSR loci (RM17, RM281, RM282, RM285 and RM306). (E1-E7 are plants grown in field while E8E10 are plants grown in net house)

Fig. 3 c. Dendrogram (NTSYS-pc) showing genetic relationships between sixteen PAU201/MASARB25 F4 plants and parental genotypes using allelic data six SSR loci (RM582, RM17, RM336, RM275, RM13 and RM175) (D1-D10 show plants grown in field while D11-D16 are plants grown in net house.)

Fig. 3 d. Two-dimensional PCA scaling of sixteen PAU201/ MASARB25 F4 plants and parental genotypes using allelic diversity data at six SSR loci (RM582, RM17, RM336, RM275, RM13 and RM175) (D1-D10 show plants grown in field while D11-D16 are plants grown in net house.)

Fig. 3e. Dendrogram (NTSYS-pc) showing genetic relationship between fourteen PAU201/MAS26 F4 plants and parental genotypes using allelic data at three SSR loci (RM281, RM287 and RM306) (F1-F10 are field plants while F11-F14 are net house plants)

Fig. 3f. Two-dimensional PCA scaling of fourteen PAU201/ MAS26 F4 plants and parental genotypes using genetic diversity data at three SSR loci (RM281, RM287 and RM306) (F1-F10 are field plants while F11-F14 are net house plants)

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Table 3. Phenotypic correlation coefficient between different physio-morphological and root traits in F4 population of respective crosses grown in the net house under water limited aerobic conditions Traits

PH (cm)

RL (cm)

FRW (g)

RT (mm)

DRW (g)

ET

PL (cm)

GW (g)

Y (g)

PH (cm)

1.000 1.000 1.000

RL (cm)

-0.341 0.074 -0.478

1.000 1.000 1.000

FRW (g)

-0.191 0.252 0.270

0.369 0.283 0.121

1.000 1.000 1.000

RT (mm)

-0.026 -0.227 -0.535

0.485 0.375 0.234

-0.022 0.227 -0.320

1.000 1.000 1.000

DRW (g)

0.189 0.012 0.322

-0.050 0.095 -0.171

0.691 0.450 0.714

-0.035 0.601 -0.085

1.000 1.000 1.000

ET

0.140 0.291 0.567

-0.167 -0.051 -0.163

0.339 0.182 0.273

-0.103 -0.003 -0.468

0.523 -0.026 0.087

1.000 1.000 1.000

PL (cm)

-0.222 0.034 -0.391

0.794 0.758 0.840

0.761 0.594 0.420

0.535 0.778 0.456

0.469 0.592 0.232

0.182 0.121 -0.119

1.000 1.000 1.000

GW (g)

0.354 0.095 0.164

-0.168 -0.130 0.173

0.020 -0.078 0.151

-0.055 0.187 0.002

0.203 0.393 -0.089

0.235 0.063 0.383

-0.042 0.074 0.214

1.000 1.000 1.000

Y (g)

0.369 0.132 0.065

-0.136 0.188 0.108

-0.066 0.073 0.344

0.030 0.214 -0.305

0.134 0.029 -0.026

0.565 0.583 0.613

-0.034 0.281 0.127

0.669 0.246 0.685

1.000 1.000 1.000

LB

0.305 0.150 0.193

-0.005 0.071 -0.018

0.276 0.413 -0.129

0.224 0.269 0.139

0.471 0.303 0.232

0.333 0.386 0.008

0.309 0.339 0.055

0.186 0.111 -0.316

0.181 0.218 -0.411

LB

1.000 1.000 1.000

PH, RL, FRW, DRW, ET, RT, PL, GW, Y and LB stand for plant height, root length, fresh root weight, dry root weight, effective number of tillers , root thickness, panicle length, 100-grain weight, yield and grain length: breadth ratio, respectively.

Authors’ contribution

QTL. The molecular results signified that some of the plants in all the progenies studied showed positive results for the presence of the markers wich dictates the presence of the QTL to which the marker is linked. Progenies showing the positive results are good material for further generation studies.

Conceptualization of research work and designing of experiments (RKJ, SJ); Execution of field/lab experiments and data collection (KK, KR); Analysis of data and interpretation (RKM, M); Preparation of manuscript (KK).

Significant and positive association of these traits indicates that selection based on these traits would ultimately improve grain yield under drought stress situations. A high positive correlation of root traits with yield components is a clear indication that thicker and deeper roots facilitate easy uptake of water from deeper layers of soil and help the plants improve their water relationship and thereby yield. Similar results were also reported by Sheeba (2005) for root length, Anbumalarmathi (2005) for dry root weight and Sinha et al. (2000) for root thickness. The interrelationships between root morphological characters and yield-related traits clearly identified the importance of root length, fresh root weight, root thickness and root dry weight in breeding rice genotypes for water limited aerobic soils.

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