WORK DONE REPORT-2015 B. Srikanth Senior Research Fellow - - PowerPoint PPT Presentation

• B. Srikanth

Senior Research Fellow Biotechnology Division

National Initiative on Climate Resilient Agriculture

Co principal Investigator

• Dr. C N Neeraja

Principal Scientist, Biotechnology Division, Indian Institute of Rice Research, Hyderabad-500030.

WORK DONE REPORT-2015

Principal Investigator

• Dr. S. R. Voleti

Principal Scientist, Head- Plant physiology, Indian Institute of Rice Research, Hyderabad-500030.

“Identification of genes associated with nitrogen use efficiency in rice (Oryza sativa L.)” Introduction  Rice is the most important staple food and source of calorie intake for two thirds of Indians and play a key role in the national food and livelihood security. The present trend of rice production is in line with the country’s requirement, but India’s rice production needs to be enhanced to about 120 million tons from the current level of 105 million tons by the year 2025 A.D. Nitrogen fertilizer and its environmental impacts  The production of high-yielding crops is associated with the application of large quantity of nitrogen (N) fertilizers.  N is the most important macro nutrient for plant growth and developing high yields. Urea is the major N fertilizer and it contributes about 80% to the total fertilizer consumption in India. Incomplete capture or poor conversion of fertilizer N also causes global warming through nitrous oxide emissions. Thus, development of rice genotypes with Nitrogen Use Efficiency is need of the hour for cost effective, economically sustainable and environment friendly agriculture.

N Uptake Efficiency N Taken UP N Supplied N Lost N Utilization Efficiency N Structural N Functional N Lost Tissue N flowering Tissue N Maturity

Yield potential Efficient C:N metabolism Efficient root system Biological Nitrification Inhibition Nitrate Transporter Enhanced Efficiency of Enzymes Enhanced Translocation of Nonstructural C & N to grains Rane et al 2009

Nitrogen Use Efficiency  Nitrogen use efficiency (NUE) can be defined as genotypes which can uptake and accumulate higher N content and able to yield better under low N conditions.  NUE is a complex trait, it can be influenced by both internal and external factors.  In India, the current average NUE in the field is approximately 33%. Improving NUE will ensure lower level of N fertilizer usage thus reduce environmental contamination. Objectives

• 1. To screen rice genotypes for nitrogen use efficiency with zero and recommended

dose of nitrogen application. 2. To identify genes/genomic regions associated with nitrogen use efficiency in rice. 3. To study the expression

• f

identified candidate genes during zero and recommended dose of nitrogen application. There fore, It is important to screen large number of genotypes under differential N conditions and identify the genotypes with high NUE.  Identification of genes associated with NUE is important for the development of efficient breeding varieties with high NUE and reducing nitrogen inputs from farming to the environment.

N content in straw, grain and plant NUE Indicators

155 rice genotypes (11 categories)

Agro-physiological , biochemical and yield attributes were recorded Leaf length, width, area, Chlorophyll content Days to 50% flowering , days to maturity, plant height, number of tillers, number of productive tillers, number of filled grains/ panicle, spikelet sterility, and grain yield/plant Under low N conditions 25 best performers were identified ( Utilized for breeding)

Screening of rice genotypes for NUE in low and recommended N conditions (Kharif 2011 and Rabi 2012)

N-0

N-100

Candidate gene based association mapping (CGAM) for identification of genes and genomic regions associated with NUE

SC29 20 SC29 12 SC25 82 SC25 86

Polymorphism survey with 12 genotypes Diversity analysis (Neibour joining method) QQ-Plot MLM

Two way analysis of variance (ANOVA) revealed significant variability among genotypes. Wide genetic variability was observed for 22 yield, yield related parameters and 13 NUE indicators in 155 genotypes under low and recommended N conditions. Significant interactions for the treatments (low and recommended N) have been

• bserved for leaf length, filled grain weight and grain yield suggesting the importance of

N in filled grain weight and thereby yield. Significant interactions for seasons and treatments were observed for majority of the traits suggesting the complexity of yield and NUE. Correlation coefficient of the different yield and yield related parameters along with NUE indicators confirmed the significance of panicle weight, straw yield, total dry matter, total grain weight and N content in plant under low N conditions. Several significant, common correlations across the seasons (kh11 and rb12) were

• bserved

28 5,743 11,457 31 48,179 96,326 Grain_yield__kg_ha vs. Filled_gr.wt_g_hill… 28 5,743 11,457 0.6 41.7 82.7 Grain_yield__kg_ha vs. HI… 28 5,743 11,457 11.9 34.5 57.0 Grain_yield__kg_ha vs. Leaf_Len_cm___Rep… 28 5,743 11,457 0.070 0.200 0.330 Grain_yield__kg_ha vs. Leaf_Thickness__Rep… 28 5,743 11,457 0.71 1.26 1.80 Grain_yield__kg_ha vs. N.W_cm___Rep… 28 5,743 11,457 0.73 1.26 1.79 Grain_yield__kg_ha vs. N__in_Grain… 28 5,743 11,457 0.280 0.500 0.720 Grain_yield__kg_ha vs. N__in_Straw… 28 5,743 11,457 2.4 10.1 17.9 Grain_yield__kg_ha vs. No._of_PB_Tillers… 28 5,743 11,457 2.4 13.5 24.7 Grain_yield__kg_ha v No._of_Tillers… 28 5,743 11,457 3 76 149 Grain_yield__kg_ha vs. P_Height… 28 5,743 11,457 1.6 9.7 17.8 Grain_yield__kg_ha vs. Pa.No_hill… 28 5,743 11,457 0.4 16.9 33.4 Grain_yield__kg_ha vs. Pan.Wt__G__hill… 28 5,743 11,457 11.5 34.5 57.5 Grain_yield__kg_ha vs SPAD__Rep… 28 5,743 11,457 0.0 7.3 14.5 Grain_yield__kg_ha vs. Unfilled_gr.wt_g__hill… 28 5,743 11,457 0.2 18.1 35.9 Grain_yield__kg_ha vs. To_Gr_Wt__g__hill… 28 5,743 11,457 3.4 25.9 48.4 Grain_yield__kg_ha vs. TDM… 28 5,743 11,457 1.14 1.70 2.26 Grain_yield__kg_ha vs. Total_N__in_plant… 28 5,743 11,457 169 4,148 Grain_yield__kg_ha vs. STRAW.WT_kg_ha… 28 5,743 11,457 0.0 5.7 11.5 Grain_yield__kg_ha vs. Grain_yield__t_ha… 28 5,743 11,457 4.4 51.9 99.5 Grain_yield__kg_ha vs. GF…

Correlation graphs (Kharif 2011)

66 31,604 63,142 7.3 26.4 45.5 Filled_gr.wt_kg_ha vs. Leaf_Len_cm___Rep… 66 31,604 63,142 0.50 1.15 1.79 Filled_gr.wt_kg_ha vs. N.W_cm___Rep… 66 31,604 63,142 0.080 0.155 0.230 Filled_gr.wt_kg_ha vs. Leaf_Thickness__Rep… 66 31,604 63,142 14.0 35.7 57.5 Filled_gr.wt_kg_ha vs. SPAD__Rep… 66 31,604 63,142 45.7 82.9 Filled_gr.wt_kg_ha vs. P_Height… 66 31,604 63,142 2.1 8.7 15.4 Filled_gr.wt_kg_ha vs. No._of_Tillers… 66 31,604 63,142 2.1 8.4 14.7 Filled_gr.wt_kg_ha vs. No._of_PB_Tillers… 66 31,604 63,142 0.7 7.4 14.2 Filled_gr.wt_kg_ha vs. Pa.No_hill… 66 31,604 63,142 0.7 14.2 27.7 Filled_gr.wt_kg_ha vs. Pan.Wt__G__hill… 66 31,604 63,142 0.2 11.2 22.3 Filled_gr.wt_kg_ha vs. Filled_gr.wt_g__hill… 66 31,604 63,142 23.8 61.7 Filled_gr.wt_kg_ha vs. GF… 66 31,604 63,142 0.6 15.3 30.0 Filled_gr.wt_kg_ha vs. To_Gr_Wt__g__hill… 66 31,604 63,142 6 2,667 5,328 Filled_gr.wt_kg_ha vs. Unfilled_gr.wt__kg_ha… 66 31,604 63,142 0.06 3.71 7.36 Filled_gr.wt_kg_ha vs. Grain_yield__t_ha… 66 31,604 63,142 3.1 42.1 81.1 Filled_gr.wt_kg_ha vs. HI… 66 31,604 63,142 965 4,916 Filled_gr.wt_kg_ha vs. STRAW.WT_kg_ha… 66 31,604 63,142 5.8 25.5 45.2 Filled_gr.wt_kg_ha vs. TDM… 66 31,604 63,142 0.10 0.88 1.66 Filled_gr.wt_kg_ha vs. N__in_Grain… 66 31,604 63,142 0.200 0.400 0.600 Filled_gr.wt_kg_ha vs. N__in_Straw… 66 31,604 63,142 0.42 1.24 2.06 Filled_gr.wt_kg_ha vs. Total_N__in_plant…

Correlation graphs (Rabi 2012)

AROMATIC GERMPLASM UPLAND AEROBIC HEAT TOLERANT IRHTN

N-100 -TOP N-100 -TOP N-100 -TOP N-100 -TOP N-100 -TOP N-100 -TOP

Basmati 242 IC-462350 IR72876-62-2-2-2 IR 82639-B-B-3-3 NCR 606 IR 1561-228-3-3 Basmati Kamon IC-462316 IR 82635-B-B-47-1 IR 82639-B-B-140-1 ARC 14088 GIZA 178

N-100 -LEAST N-100 -LEAST N-100 -LEAST N-100 -LEAST N-100 -LEAST

Kola Joha 3 IC-463176 IR 82590-B-B-102-4 IR88633:20-125-B-1 KHASRAN (ACC 76382) Ratnasundari IC-466475 IR 83754-B-B-46-4 IR88633:5-54-B-1 PARO DUMBJA(WHITE) (ACC 75200)

DRR RELEASED RESTORER LINES A LINES B LINES HYBRIDS

N-100 -TOP N-100 -TOP N-100 -TOP N-100 -TOP N-100 -TOP

Pooja BCW-56 DRR10A DRR12B DRRH-3 Sasyasree KMR-3 DRR13A IR80555B PA6444

N-100 -LEAST N-100 -LEAST N-0 -LEAST N-0 -LEAST N-0 -LEAST

Swarna PSBRC80 DRR13A IR80561B PA6201 Rpbio 226 IR82039-B-B-140-1 DRR12A DRR10B DRRH-3

Several best and poor performers were identified in each category based on yield

A total of 325 marker trait associations were observed in the present study. In kh11 season, a total of 161 marker trait associations were observed in which 94 under recommended N and 67 under low N conditions. In rb12 season, a total of 164 marker trait associations were observed in which 113 under recommended N and 51 under low N conditions.

16 genes /genomic regions associated with NUE were identified on different chromosomes (CHR.1,2,3,4,8 and 10).

Expression profiles of twelve genes in panicle samples of Basmati 370 and Thuroorbhog under low N

• 10
• 5

5 10 15 20 25 30 35 40 Basmati370 Thuroorbhog

• 10
• 5

5 10 15 20 25 30 35 40

LOC_Os02g52730 Os03g0712800 Os04g0659100 Os01t0704100-01 Os03t0235900-01 Os02t0770800-01 Os04t0304400-01 LOC_Os01g57690 LOC_Os10g42960 LOC_Os08g20190 LOC_Os01g48960 LOC_Os02g41680

• 10
• 5

5 10 15 20 25 30

LOC_Os02g52730 Os03g0712800 Os04g0659100 Os01t0704100-01 Os03t0235900-01 Os02t0770800-01 Os04t0304400-01 LOC_Os01g57690 LOC_Os10g42960 LOC_Os08g20190 LOC_Os01g48960 LOC_Os02g41680

Basmati370 Thuroorbhog

Differential expression of OsNIA2, PAL and OsAMT1;1 genes in shoot and root samples of Somaly 2- 023-3-5-1-2-1, GQ-25 and IR55178 under low N condition.

• 8
• 6
• 4
• 2

2 4 6 Shoot Root

OsNIA2

2 4 6 8 Shoot Root

PAL OsAMT1;1

• 10
• 5

5 10 Shoot Root

*The recommended N condition used as control, and low N condition as treated.

Differential expression of OsAMT1;1, OsNIA2, OsGS1;3, and OsSPL14 genes in ten rice genotypes under low N conditions.

• 30.00
• 20.00
• 10.00

0.00 10.00 20.00 30.00 PS1 PS2 PS3

OsAMT1;1

• 20.00
• 15.00
• 10.00
• 5.00

0.00 5.00 10.00 15.00 PS1 PS2 PS3

OsGS1;3

• 10.00
• 5.00

0.00 5.00 10.00 15.00 20.00 PS1 PS2 PS3

OsNIA2 *The recommended N condition used as control, and low N condition as treated. OsSPL14

• 20.00
• 15.00
• 10.00
• 5.00

0.00 5.00 10.00 15.00 PS1 PS2 PS3

• 150
• 100
• 50

50 100 150 200 250 300 350 SC3725 SC3726 SC3728 SC3729 SC3730 SC3731 SC3732 SC3733 SC3734 SC3735 SC3737 SC3738 SC3739 SC3740 SC3741 SC3742 SC3743 SC3745 SC3746 SC3748 SC3749 SC3751 SC3752 SC3753 SC3754 SC3755 SC3756 SC3757 SC3758 SC3759 SC3760 SC3761 SC3762 SC3763 SC3764 SC3765 SC3766 SC3767 SC3768 SC3769 SC3770 SC3771 SC3772 SC3773 Basmati370-0 IC463254-0

Expression analysis of short listed genes (Transcriptome analysis) Fifty key genes related to NUE were shortlisted; primers designed for shortlisted genes and validated by Real-time PCR analysis in four panicle samples collected from both low and recommended N conditions. Expression patterns of transcriptome identified genes under low N conditions

• 4
• 2

2 4 6 SC3141 SC2508 SC3163 SC3177 SC2498 SC3178 Basmati370 Thuroorbhog

Diffrential expression of genes under low N conditions

Ammonium transporter protein Low affinity nitrate transporter NRT1.2 valyl-tRNA synthetase Glutamate synthase [NADH] Nitrate induced proteins Ferredoxin- dependent glutamate synthase

SC3141 SC2508 SC3163 SC3177 SC2498 SC3178 Basmati370

• 2.87908

1.082519

• 1.18559
• 3.19454
• 1.23594
• 1.0538

Thuroorbhog

• 2.64466
• 1.33153
• 1.68539
• 3.01693

3.37975 4.616875

Expression profiling of Meta analysis genes AAT gene sequencing

Sequence was retrieved and candidate gene based primers were designed and given for synthesis. CG based primers were made stocks and workings. Kept PCR using BPT5204 and Varadhan and amplification of the primers were confirmed. Involved in AAT gene sequencing works.

Rabi2015 Harvesting works

• RIL Collection in D4 plot (18 RIL populations)
• Line and single plant harvested.
• Yield parameters were recorded for GFGP samples in low N, N-50 and N-100 conditions.
• GFGP samples 6 plants were harvested
• Recommended N RIL population single plant harvest was done.
• 50% flowering dates are noted down for all mapping populations.
• Seed for NICRA BIOTECH promising lines was given to Dr Voleti sir.
• Field observations and management.

Kharif 15 Sowing , planting and harvesting works D4 Plot Best plants selections, RIL collection, line harvesting completed in D4 Plot. NICRA (Zero, 50 and 100 N plots)

• Selection of best plants in GFGP populations
• Yield parameters such as Plant height, number of tillers and number of productive tillers were recorded
• IA-RIL population selections
• Field and Net house plants observation.

Kharif 16 Sowing , planting and harvesting works D4 Plot Best plants selections, RIL collection, line harvesting completed in D4 Plot. NICRA (Zero, 50 and 100 N plots)

• Selection of best plants in GFGP populations
• Yield parameters such as Plant height, number of tillers and number of productive tillers were recorded
• IA-RIL population selections
• Field and Net house plants observation.

Several Crosses were attempted during this season using Good and poor performers on popular varieties

Regular Field Observations

1.

• B. Srikanth, I. Subhakara Rao, K. Surekha, D. Subrahmanyam, S.R. Voleti, C.N. Neeraja. Enhanced expression
• f OsSPL14 gene and its association with yield components in rice (Oryza sativa) under low nitrogen
• conditions. Gene (2016), volume 576, 441-450. http://dx.doi.org/10.1016/j.gene.2015.10.062.

2.

• B. Srikanth, I. Subhakara Rao, K.N. Swamy, P. Vijayalakshmi, K. Surekha, S. Zehra Moiz, M. Lakshmi Narasu,

M.S. Archana Giri, S.R. Voleti, C.N. Neeraja. Physiological and molecular analysis of GQ-25, a promising genotype for Nitrogen use efficiency. Progressive Research- An International Journal (2015), volume 10 (Spl- II), 651-656. 3.

• B. Srikanth, I. Subhakara Rao, S. Zehra Moiz, K.N. Swamy, K. Surekha, D. Subrahmanyam, L.V. Subba Rao, P.

Vijayalakshmi, S.R. Voleti, C.N. Neeraja. Physiological and Molecular characterization of three rice genotypes with differential nitrogen use efficiency. Asian Journal of Bioscience (2016), Volume 11(1), 162- 171.DOI:10.15740/HAS/AJBS/11.1/162-171. 4. I Subhakara Rao, B. Srikanth, D. Subrahmanyam, S.R. Voleti1 V. Ravindra Babu, K.R.S. Sambasiva Rao, P. Sudhakar, A. Krishna Satya, C.N. Neeraja. Identification of Promising Lines for Yield from IR64/ Akhikari Recombinant Inbred Lines under Low Nitrogen. Progressive Research – An International Journal (2016) Print ISSN: 0973-6417, Online ISSN: 2454-6003 Volume 11 (Special-II): 1481-1483. 5.

• P. Vijayalakshmi, T. Vishnukiran, B. Ramana Kumari, B. Srikanth, I. Subhakar Rao,K.N. Swamy, K. Surekha, N.

Sailaja, L.V. Subbarao, P. Raghuveer Rao, D. Subrahmanyam, C.N. Neeraja, S.R. Voleti. Biochemical and physiological characterization for nitrogen use efficiency in aromatic rice genotypes. Field Crops Research (2015), volume 179:132-143. Available at: http://dx.doi.org/10.1016/j.fcr.2015.04.012. 6. Swamy KN, Kondamudi R, Vijayalakshmi P, Jaldhani V, Suchandranath Babu Munnam, Kiran TV, Srikanth B, Subhakar Rao I, Sailaja N, Surekha K, Neeraja CN, Subba Rao LV, Raghuveer Rao P, Subrahmanyam D and Voleti SR. A comparative study on nitrogen response among Upland, IRHTN, DRR and other released rice groups. African Journal

• f

Agricultural Research (2015), volume 10(48), 4364-4369. DOI: 10.5897/AJAR2015.10323. 7.

• T. V. Kiran, P. Vijayalakshmi, Y. V. Rao, K. N. Swamy, R. Kondamudi, B. Srikanth, I. Subhakar Rao, M.

Suchandranath Babu, V. Jaldhani, D. Prema Latha, C. N. Neeraja, K. Surekha, P. R. Rao, D. Subrahmanyam, L. V. Subbarao and S. R. Voleti*. (2016). Effects of Nitrogen Limitation on Antioxidant Enzymes, Chlorophyll Content and Grain Yield of Rice Genotypes. Asian Research Journal of Agriculture, 1(2): 1-10, 2016, Article no.ARJA.28503.

Publications/ papers

“RICE IS LIFE”