PCR Identification of Aroma, Blast Resistance, Kernel Length and Yield Genes in Pishori Rice grown in Kenya

1. PCR IDENTIFICATION OF AROMA, BLAST
RESISTANCE, KERNEL LENGTH AND YIELD GENES
ON PISHORI RICE GROWN IN KENYA
By
Aloo Denish and Tanui Laban
aloo.denish3@gmail.com
Supervisors: Dr. J. Njiru
Dr. S. Kiruki

2. 1.0. INTRODUCTION
• Rice (Oryza sativa) is one of the most consumed cereals
in world, it belongs to the grass species.
• In Kenya it is the third staple food.
• World wide about 40,000 varieties have been reported.
• Importance: ensures food security, creates
employment and opportunities for private investment,
thus improving livelihoods of people in both rural and
urban areas.
• In Kenya, about 80% of rice is cultivated by irrigation
and rain-fed(20%).

3. INTRODUCTION-Contd
• Varieties: through irrigation are basmati(pishori)
and sindano ; in rain-fed ecosystems include
NERICA and Dourado Precose.
• Pishori(basmati) is a special type of aromatic rice
with long slender grains, high cooked kernel
volume, less sticky soft fluffy texture after
cooking.
• Challenges: diseases such as blast,
counterfeit/adulterated products, quality and
quantity.

4. INTRODUCTION-Contd
• Pishori rice or basmati, is long slender-grained aromatic
rice with good yield, tolerance to diseases.
• Aroma: The Badh2 gene for expression of aroma is
located on chromosome 8, which encodes an enzyme
called betaine aldehyde dehydrogenase homologue
which regulates production 2-acetyl-1-pyrroline, a
principle compound responsible for aroma in rice.
• Blast disease: -fungal pathogen known as Magnaporthe
oryzae.
• An important way of controlling the disease is to identify
and map the blast resistance genes in blast resistance
varieties, isolate them then incorporate in blast
susceptible rice cultivars.
(kovach et al.,2009; Bourgis et al.,2008; Khush and Jena, 2009 ; Lieu et al., 2010)

5. 1.2. STATEMENT OF THE PROBLEM
• Rice is an important cereal consumed in the
Kenya -third from maize and wheat.
• Pishori rice is widely grown and preferred
• However, this species faces many challenges
ranging from diseases, counterfeit/adulterated
products.
• Pollination-high genetic modification of the
parent rice and it will cause loss of best
characteristics which market pishori rice.
• Need to preserve good genetic characteristics.

6. 1.3. OBJECTIVES
• To develop a molecular based protocol for
identification of pishori rice from counterfeit
and non-pishori rice.
• To identify aroma, blast resistance, kernel
length, kernel weight, and yield genes of
pishori rice.

7. 2.0 JUSTIFICATION
• Increasing human population and drought
poses food insecurity threat
• Need to preserve/promote good genetic
characteristics that will lead to high yield in
rice

8. 3.0. MATERIALS AND METHODS
3.1. Study site
Carried out at KEBS, Molecular and GMO laboratory
3.2. Source of Reagents and Rice
Mwea, Kenya. Non-pishori rice sample variety was
bought from the market. The reagents used were
availed by the KEBS.
3.3. Experimental Protocol
 CTAB Method
 Promega kit
 Agarose gel electrophoresis

9. 3.4. PCR analysis
• The multiplex PCR was performed.
• PCR mastermix was prepared with five
primers(aroma, yield, blast resistance, kernel
length, kernel weight).
• The PCR system was set up with rice DNA
templates for 35 cycles.
Visualization of the PCR products
• Agarose gel electrophoresis (2.0%)
• UV Transilluminator
(McChouch et al., 2002)

10. 3.5. Primers used
1. RMI6-Kernel length (R-5`AACACAGCAGGTACGCGC3`
;F-5`CGCTAGGGCAGCATCTAAA3`)
2. RM261-Yield (R-5`TGTACCATCGCCAAATCTCC3` F-
5`CTCTTCTCCCCTTGTGTGTCG3`)
3. RM459-Kernel weight (R-
5`CACTTTCTCTGCAGCACCAC3’ ` F-
5`CTGCAATGCTGCATGACC3)
4. RM22-Aroma, (R-5`CTGGGCTTCTTTCACTCGTC3’,
F-5`GGTTTGGGAGCCCATAATCT3`)
5. RM168-Blast Rest R-
5`GAAACGAATCAATCCACGGC3`; F-
5`TGCTGCTTGCCTGCTTCCTTT3’

11. 4.0. RESULTS AND DISCUSSION
4.1. Experimental Protocol
• CTAB buffer concentrations
• RICE 10% & 2% CTAB
Fig. 1: Agarose gel electrophoresis results for the extraction of genomic rice
DNA using 10% and 2% CTAB concentration
M, 10% 1O% 2% 2%

12. Pishori Rice Sample 1,2,3 &4-
CTAB method Promega kit
Fig. 2: Agarose gel electrophoresis results DNA extraction of
pishori rice sample using CTAB method and wizard magnetic
DNA purification system (promega kit)
M 1 2 3 4 M 1 2 3 4

13. A- Pishori Rice Sample 1,2,3 &4-
CTAB method Promega kit
Fig. 3: Agarose gel electrophoresis results DNA extraction of
pishori rice sample using CTAB method and wizard magnetic
DNA purification system (promega kit)
M 1 2 3 4 M 1 2 3 4

14. PCR Analysis
SSR MARKERS FOR PISHORI RICE
Fig. 4: Agarose gel electrophoresis results for PCR analysis for simple
sequence repeats markers in pishori rice

15. PCR PISHORI (Specific primers)
Fig. 5: ( M-ladder, 1-kernel length, 2-yield 3-aroma, 4-blast, 5-weight)
Agarose gel electrophoresis results for PCR analysis of the kernel length,
aroma, blast resistance, kernel weight and yield genes in pishori rice.
M 1 1 1 1 2 2 2 2 3 3 3 3 M
M 4 4 4 4 5 5 5 5 M

16. Non-Pishori rice sample 1,2,3&4
• CTAB method Promega kit
Fig. 6: Agarose gel electrophoresis results for extraction of non-
pishori rice DNA using CTAB and wizard magnetic DNA purification
system(promega kit)
M 1 2 3 4 M 1 2 3 4

17. PCR-NON-PISHORI RICE
Fig. 7: (L-ladder, 1-aroma, 2-blast, 3-kernel length, 4-yield)
Agarose gel electrophoresis results of PCR analysis for the kernel
length, aroma, blast resistance genes in non-pishori rice.

18. 5.0. CONCLUSION AND RECOMMENDATION
5.1. Conclusion
I. CTAB method was the best method for Pishori rice
DNA extraction
II. Pishori rice expressed genes for aroma, blast
resistance, kernel length and high yield.
III. Non- pishori rice did not express genes for aroma,
blast resistance and kernel length. However, the
gene associated with yield was expressed but the
intensity was low.

19. 5.2. RECOMMENDATIONS
• Aroma and blast resistance genes to be sequenced and
cloned to improve yield in non-pishori rice.
• PCR based technique should be used to identify genuine
Pishori rice.
• ACKNOWLEDGEMENT
• KEBS, CU, DPSC
• Supervisors
• God