Source: SEAMEO BIOTROP's Research Grant | 2021
Abstract:
Objectives
a.
Analysis of gene expression related to boktor
pest resistance and tumor rust
using RT-PCR
b.
Development of SNIP markers
for Genome-wide association selection (GWAS) on the
resistance properties of boktor pests and tumor
rust
c. Identification of Open Reading Frames (ORFs) from transcriptomic sequence analysis and Blastx.
Expected Output
a.
Optimization of the protocol
for the expression of multiple
gene sequences using
RT-PCR
b.
New SNIP markers for selection of
resistance properties related to boktor pests
and tumor rust
c.
The full length of the expressed
genes, especially those related to the resistance of sengon to tumor rust and boktor pestsIdentification of Open
Reading Frames (ORFs) from transcriptomic
analyses and BLASTX
Conclusion
The RT-PCR results for the TI4 gene
and the Actin 1 gene were good with the formation of a tight amplification curve
and the melting
curve only produced
one peak. The electrophoresis
results of TI4 and Actin 1 genes also produced a single band, which means the sample has good quality.
CT value in the TI4 gene ranged
from 24.33 to 26.99, while the CT value of
Actin 1 gene ranged from 22.36 to 24.69. Quantification using 2-ΔΔCT method
shows the level of TI4 gene expression in sengon
samples healthy (S) that is equal to 1.00 while
in the sample attacked by boktor (B) of 1.91. TI4 gene expression in sengon
attacked by boktor pests was higher than in healthy sengon.
Identification of gene encoding
α-Amylase enzyme in plants Falcataria
moluccana Miq, which was attacked
by boktor pests,
was successfully carried
out. RT-PC on Falcataria
moluccana amplified at temperature annealing 57 °C for Actin 1 and 61°C for the AAI gene.
The average CT value of the Actin 1 gene that was attacked by boktor
pests was 24.50 and those that were
not attacked by boktor pests was 23.51. While the average CT value in the AAI 2 gene that was attacked by boktor pests was 29.18 and those
that were not attacked by boktor
pests was 29.67. The value of 2^(-∆∆Ct) on trees that were attacked by boktor
pests had a higher value of 2.80 than
those that were not attacked by boktor
pests of 1.00.
The expression of tumor
rust-associated genes varies. In seedlings treated with infection, there were genes whose expression increased after infection was induced and there were genes that decreased expression
after infection. Meanwhile, in mature plants, the average expression of all genes was higher
in tumors than in healthy
wood and wood around the tumor. The higher expression of
certain genes in the wood around the tumor than in healthy wood indicated that there was an indication that the
gene was induced by infection with pathogens in the tumor tissue.
Sequence of the sengon reference
genome de novo using Ray assembler showed better genome
coverage than the two assembler other. Results assembly
from SOAPd enovo
and Ray showed low genome completeness due to the large number of
missing BUSCO genes (>70%). The annotation results
show that there are two sequences containing the AAI gene
and 19 sequences containing the TI gene assembly which
has been done.
Download full report