Molecular & Physiological Evidence of Rht Genes Expression in Few

Indian Wheat Cultivars

WHEAT( Genus :Triticum )• Staple crop

• Wheat production must increase to meet the increasing demand

• Species of Triticum are grouped into three ploidy classes: 1. Diploid (14) 2. Tetraploid (28) 3.Hexaploid (42)• Common wheat (T. aestivum) is an allohexaploid : AABBDD

One strategy is to increase wheat productivity by optimizing plant architecture:• Determined significantly by stem elongation

•Decrease in stem stature increases yield:

Reduces risk of lodgingMore fertile florets per spikelet Increase in partitioning of assimilates to the grain Increase in harvest index

Increased grain numbers

•Decrease in stem stature is Achieved by:

Introduction of the "Dwarfing genes "

Dwarfing Genes Produce More Active DELLA Proteins

GA DELLA Signalling Mechanism‐Dwarfing gene present

Normal wheat

DELLA Protein

Rht genes cause changes in this domain

Dwarfing gene Mechanism

Rht-B1b ; Rht-D1b Single nucleotide substitutions that introduce premature stop codons in the N-terminal coding region

Rht-B1c

Cause an intragenic insertion, in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain

Rht-B1d ;Rht-B1e Introduce premature stop codons within the amino-terminal coding region

Rht-D1c Overexpression of the semidwarfing Rht-D1b allele

MATERIALS & METHODOLOGY

Plant young leaves samples were taken from wheat genotypes : 1. WH1105

2. HD3086 3. C323 4. C591 5. CB3136. CB3147. CB315

8.CB318 9. CB320

10.CB322 11.CB324 12.CB273 13. PBW321 14. DBW88 15. 5218

16. 5219.

DNA Extraction BY CTAB Method

Small pieces of young leave samples + 800µl CTAB buffer in pestle

and mortar ↓

Crush and then put the green soup so obtained in eppendorf ↓

Put on water bath at temp. 65 Cfor 1 hour ̊ ↓

Add chloroform: isoamyl alcohol (24:1) (800 µl) ↓

Place on revolver for 1 hour↓

Centrifuge at 10,000 rpm for 10 minutes ↓

Transfer the supernatant to clean sterile eppendorf & discard the pellet

↓

Add isopropanol (800 µl) & incubate in refrigerator for 1 hour

↓Centrifuge at 10,000 rpm for 10 minutes

↓Discard supernatant & add 300 µl of 70% ethyl alcohol to pellet

↓Centifuge at 10,000 rpm for 10 minutes

↓Discard supernatant & allow pellet to dry

↓Add 200µl of TE buffer & leave at room temperature for few

hours to allow DNA to dissolve. Tap with fingertips↓

Store at 4 C ̊

QUANTIFICATION OF DNA

1.BY GEL ELECTROPHORESIS

2.BY NANODROP

Agarose Gel ElectrophoresisAgarose was dissolved in TBE electrophoresis buffer

The mixture was heated till the agarose dissolved completely ( became transparent) It was cooled down to 55-60 C with constant stirring & 5 µl of EtBr ̊

The solution was then poured into already prepared gel mould and was left for 40-50 min for solidification; then combs are removed

In the meanwhile, DNA samples were prepared for loading by adding loading dye, Bromophenol blue to the DNA

After the gel solidified, the DNA samples were loaded into the wells

The gel was run & then visualized under UV transilluminator

2. BY NANODROP

The quantification of DNA was also conducted by using Nanodrop spectrophotometer

1.BY GEL ELECTROPHORESIS

•For quantification of DNA 0.8%gel was used

•For quantification 2 microlitre DNA sample + 8 microlitre bromophenol dye is loaded.

• Run at 80V/cm after loading

Sr.no Genotypes Quantity (ng)1 WH1105 1626.61

2 HD3086 50

3 C323 891.48

4 C591 75.71

5 CB324 501.82

6 CB313 378.66

7 DBW88 923.18

8 CB315 262.53

9 CB320 1448.59

10 PBW321 151.30

11 CB318 128.14

12 CB314 375.23

13 CB322 324.08

14 5218 2257.90

15 5219 1000.74

16 C273 905.68

IN VITRO Amplification Through Polymerase Chain Reaction (PCR)

Sr.no.

Primers used Sequence Tm

1. Rht-B1b F 5'-CAC TAC TAC TCC ACC ATG TTC GAT TCT CTG-3' 59.5°C

2. Rht-B1b R 5'- GCG GCA GGA GCA GCA GCC -3' 65.4°C

3. Rht-D1b F 5'- CCA CGA GAC GCT GGG-3' 59.5°C

4. Rht-D1b R 5'- CCT TCC TTC TCC TCC ACC TTG TAG-3'

58.4°C

Primers Used

Concentration of Various Components In PCR Reaction

Component Stock concentration

Quantity Final concentration

Sterile water - 7.6 µl -

PCR buffer 10X 2.0 µl 1X

MgCl2 25 mM 1.2 µl 1.5 mM

dNTP mix 1 mM 3.0 µl 0.15 mM

Forward Primer 5 µM 1.5 µl 0.375 µM

Reverse Primer 5 µM 1.5 µl 0.375 µM

Taq polymerase 5U/µl 0.2 µl 1U

Template DNA 20 ng/µl 3.0 µl 60 ng

Total 20 µl -

PCR

COCKTAIL

Steps Temperature (°C) Time (min)

Initial denaturation 95 5 min

Denaturation 94 20sec

Primer annealing 63 30 sec 42 times

Elongation 72 10 sec

Extension 72 2 min

Hold 4 ∞

Thermo-cycling Parameters for Rht B1-b

Steps Temperature (°C) Time (min)

Initial denaturation 95 5 min

Denaturation 94 20sec

Primer annealing 58 30 sec

Elongation 72 10 sec 42 times

Extension 72 2 min

Hold 4 ∞

Thermo-cycling Parameters for Rht- D1b

Visualization of PCR Products

•For visualization of PCR products 2.5% agarose gel was used.

•The PCR products were resolved by running gel at 120-150V/cm for 3-5 hours

• Visualization in UV light

Effect of Rht Genes on Coleoptile Length &

Root Length In Wheat Genotypes

Cigar method

The germination paper was taken and a line drawn in middle of itThe seeds were placed on the line ,after wetting the paper little bit

The paper was folded, rolled cigars were taped

The rolled cigars were wetted and laid in horizontal postion in a tray for 3 days

After 3 days ,the cigars were placed in vertical position in a jar

The coleoptile length was measured with ruler after germination on 10th and 15th day for all the genotypes

EFFECT OF EXOGENOUS APPLICATION OF GA3 ON COLEOPTILE LENGTH & ROOT LENGTH IN WHEAT GENOTYPES

•The cigar method in this case was performed as replicate for each variety

•But for a given variety one cigar was wetted with water while the other with the the gibberalic acid solution (GA3)(0.03 gm GA3 was dissolved in 1 litre of water)

• After germination in a growth chamber wetted with water or GA3 solution ;The coleoptile length was measured with ruler on the 10th and 15th day

•Data comparison

Effect of Rht Genes on Plant Height

The plant height of the genotypes were measured by a ruler in the field and the heights were noted down.

Results and Discussions

a) Rht -B1b

b) Rht -D1b

Results of Molecular Analysis

1. WH1105 2. HD3086 3. C323 4.C591 5.CB324 6.CB313 7.DBW88 8. CB315 9.CB320 10. PBW321 11.CB318 12.CB314 13.CB322 14.CB273 15.5218 16.5219

Results of CIGAR Method

Genotypes on the 10th day of Cigar Method

GENOTYPE COLEOPTILE LENGTH (cm)

ROOT LENGHT (cm)

HD 3086 1 7.5 13

2 10 13

3 8.5 15

4 7 12

5 8 12

MEAN 8.2 13

C323 1 12 12

2 12.5 12

3 12 10

4 14.5 9

5 10 9

MEAN 12.2 10.5

Measurements of the Coleoptile Length and Root Length on 10th Day

WH1105 1 10 8.5

2 6 8

3 6.5 8.5

4 11.5 10

5 10.5 10

MEAN 8.9 9

C591 1 12 9

2 17 9.5

3 17 9.5

4 15 8.5

5 18 8

MEAN 12.9 8.9

Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm) 1. HD3086 8.2 13

2. C323 12.2 10.5

3. WH1105 8.9 9

4. C591 64.5 8.9

Mean Coleoptile Length and Root Length on 10th Day

Genotypes on the 15th day of Cigar Method

GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm)

HD 3086 1 10.5 14

2 12.5 14

3 11.0 14

4 8 11

5 10 9

MEAN 10.4 12.4

C323 1 14 15

2 23 15.5

3 18 16

4 20 15.2

5 22 15

MEAN 19.4 15.34

Measurements of the Coleoptile Length and Root Length on 15th day

WH1105 1 15 15

2 9 11

3 14 13

4 15.5 14

5 15 12

MEAN 13.7 11

C591 1 28 15

2 21 14

3 28 14

4 30 15

5 27 15

MEAN 26.8 14.6

Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm) 1. HD3086 10.4 12.4

2. C323 19.4 15.34

3. WH1105 13.7 11

4. C591 26.8 14.6

Mean Coleoptile Length and Root Length on 15th Day

Effect of Exogenous Application of GA3 on Coleoptile Length & Root Length in Wheat Genotypes

Genotypes on the 10th day of Cigar Method (GA3 treated cigars)

GENOTYPE COLEOPTILE LENGTH (cm)

ROOT LENGHT (cm)

HD 3086 1 7.6 14

2 7.5 13

3 7 13

4 6.8 12

5 6 10

MEAN 6.98 12.4

C323 1 21 19

2 21.5 30

3 20.6 25

4 22 29

5 19.6 15

MEAN 20.94 23.5

Measurements of the Coleoptile Length and Root Length on 10th Day(GA3 Treated)

WH1105 1 9.1 19

2 11.2 15

3 10 14

4 10.5 13

5 9 15

MEAN 9.96 15.2

C591 1 24.6 30

2 25.2 29

3 26 32

4 24.5 17

5 25.6 21

MEAN 25.18 20

Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm) 1. HD3086 6.98 12.4

2. C323 20.94 23.5

3. WH1105 9.96 15.2

4. C591 25.18 20

Mean Coleoptile length and Root Length on 10th Day

(GA3treated)

Genotypes on the 15th day of Cigar Method

(GA3 treated )

GENOTYPE COLEOPTILE LENGTH (cm)

ROOT LENGHT (cm)

HD 3086 1 10.2 13.2

2 9.8 12.2

3 9 13.5

4 10 12.5

5 10.5 13

MEAN 9.9 12.8

C323 1 20 20

2 25 32

3 26.5 29

4 25.5 32

5 22.5 19

MEAN 23.9 26.4

Measurements of the Coleoptile Length and Root Length on 15th Day (GA3treated)

WH1105 1 12 23.2

2 9 19

3 12 18

4 14 15.5

5 16 20

MEAN 12.6 19.14

C591 1 29 32

2 29.5 31

3 28.5 32.5

4 27.5 19

5 26.5 25

MEAN 28.2 22.9

Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm) 1. HD3086 9.9 12.8

2. C323 23.9 26.4

3. WH1105 12.6 19.14

4. C591 28.2 22.9

Mean Coleoptile Length and Root Length on 15th day(GA3treated)

Sr.No GENOTYPE COLEOPTILE LENGTH (cm)

ROOT LENGHT (cm)

Water GA3 treated Water GA3 treated

1. HD3086 10.4 9.9 12.4 12.8

2. C323 19.4 23.9 15.34 26.4

3. WH1105 13.7 12.6 11 19.14

4. C591 26.8 28.2 14.6 22.9

Comparison of the Data of Water & Gibberalic Acid Treated Cigars

Effect of Rht Genes on Plant Height

Sr. no. Genotype Plant height(cm)

1. C323 125

2. C591 128

3. HD3086 103

4. WH1105 100

Plant Height Data For Genotypes C323,C591,HD3086,WH1105

Conclusion

1.Dwarfing genes PRESENT in all genotypes taken except C323, C591•Amplification of their DNA on PCR using dwarfing gene specific primers

• In Cigar method these genotypes showed shorter coleoptile length

• Thus, these varities are dwarf (high grain yields; less lodging)

2.Dwarfing genes ABSENT in C323, C591•No amplification of their DNA on PCR using dwarfing gene specific primers

•In Cigar method these genotypes showed longer coleoptile length

•Plant height data from field that C323, C591 have longer plant heights

• Thus, these are not dwarf (less grain yields; more prone to lodging)

3. Rht -B1b & Rht -D1b genes are the GIBBERLIN INSENSITIVE dwarfing genes

•As No increase in coleoptile length of dwarf varities in response to GA3application.

4. Rht -B1b & Rht -D1b genes have NO effect on the root length

• As by cigar method No Effect of Rht -B1b & Rht -D1b genes was found on the root length

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