Seminar PowerPoint Presentation on Transcriptomics in Ornamentals

2. SEMINAR –FLA 691
DATE: 20-03-2021
Vidyashree S
Roll No.: 11769
Discipline of Floriculture and Landscape Architecture
ICAR- Indian Agricultural Research Institute, New Delhi

3. Introduction

4. • Anatomical changes
• Succulent leaves, salt
glands
Avoidance
Plant Adaptive Mechanisms
• Molecular responses
• Induction of stress
responsive cellular
reactions
Tolerance

5. Stress
Perception of stress signal by receptors of plant
Signal transduction
Activation of transcription factors (CBF/DREB)
Kinases ROS
• CBF: Calcium Binding Factor
• DREBS: Dehydration Response
• Temperature 0 to 15 cause
chilling stress
• Less than 0 will cause freezing
stress
• Temperature 0 to 15 cause
chilling stress
• Less than 0 will cause freezing
stress
Activation of transcription factors (CBF/DREB)
Expression of genes related to cold stress
LEA Proteins, HSPs, synthesis of osmoprotectants
Tolerance/resistance to stress
Mechanism of temperature tolerance during stress condition
• DREBS: Dehydration Response
Element Binding Factor
• LEA: Late Embryogenesis
Abundant Protein
• HSP: Heat Shock Protein

6. Central Dogma of Molecular Biology

7. What is Genome?
Genome defined as the complete
set of chromosomal and extra
chromosomal genes present in an
organism, including virus.
Term coined by Hans Winkler
(1920)

8. What is the percentage of genome is
expressed in an individual or plant?
1 to 2 percent (x)
100 percent (√ )
Junk DNAs are considered to be gold
mine now a days, why?
mine now a days, why?
• Contains many regulatory sequences
i.e. non coding genes
• Contains repeat region

9. What is Transcriptome?
Transcriptome is the set of all RNA
molecules including mRNA, rRNA, tRNA
molecules including mRNA, rRNA, tRNA
and other non coding RNA produced in one
cell type or an organism at a given time
point.

11. Genome verses Transcriptome

12. Transcriptomic technologies
Transcriptomic technologies are the techniques
used to study an organism's transcriptome.
1970s
1970s 1980s
1980s 1990s
1990s Present
Present
1970s
1970s 1980s
1980s 1990s
1990s Present
Present
Northern blot qPCR Microarrays RNA-Seq

13. RNA-Seq based on NGS technology
RNA-Seq is a sequencing technique which uses next
generation sequencing (NGS) to reveal the presence and quantity
of RNA in a biological sample at a given moment, analyzing the
continuously changing cellular transcriptome.

14. Work flow for NGS

16. NGS Platforms

17. Illumina sequencing

18. Cluster generation by bridge amplification
Cluster generation
Adopter ligated DNA
Flow cell
DNA degenerated
and bind
Bridge amplification
Bridge formation
DNA amplication by
PCR
Degeneration and
washing away of
original strand

19. Sequencing
A
C
T
G
C
A T
C
T
G
G
A
T
G
G
C
T
T
G
G
G
C
A
C
C
A
A
C
T
C

20. Quality analysis of Transcriptomic data
• Q scores (Phred quality score): indicate probability of error in
base call (Q10, Q20, Q30).
• N50 value: It is the minimum contig length needed to cover 50 %
of the genome.
• Differentially expressed analysis: to find out the changes in
level of expression between experimental results.
Under normal condition Under Cold stress
Gene A
Gene B
For cold tolerance
Gene A= 4/2 = +2
fold up regulated
Gene B = 2/4 = -2
fold down regulated
Both gene A and B are
differentially expresses.

21. Utilization of
genes responsible
for temperature
for temperature
stress tolerance in
ornamentals

22. Chrysanthemum
Over expression of chrysanthemum CgHSP70 gene produced plants
with increased peroxidase activity and proline content allowing
transgenic lines to recover after heat stress.
Song et al. (2014)
Song et al. (2014)
Enhanced cold tolerance in chrysanthemum with successful
introduction of ICE1 gene isolated from C. dichrum without any
abnormalities in existing plant characters.
Chen et al. (2012)

23. Petunia
Warner (2011)
Enhanced frost tolerance in Petunia with successful introduction of
CBF3 gene isolated from Arabidopsis thaliana without any
abnormalities in existing plant characters.
Orchid
Orchid
Cold tolerance in Phalanenopsis orchid is increased with successful
introduction of LTP gene isolated from rice without changing existing
plant characters.
Qin et al. (2011)

24. China Rose
Enhanced freezing tolerance in china rose with successful introduction
of DREB1C gene isolated from Medicago tranculata without any
abnormalities in existing plant characters.
Chen et al. (2010)
Bahia grass
Drought tolerance in bahia grass (Paspalum notatum) is increased with
successful introduction of HsDREB1A gene isolated fromwild barley
(Hordeum spontaneum).
James et al. (2008)

26. CASE STUDY - 1
Objective:
 To provide insights into the molecular mechanisms of D. grandiflorum in
response to low temperature
 Exploring new candidate genes for chilling-tolerance and freezing-tolerance
chrysanthemum molecular breeding

27. Materials and method
Plant material Dendranthema grandiflorum var. jinba
Treatments
T1: normal condition as control,
T2: 4 °C for 24 h,
T3: 4 °C for 24 h, followed by -4 °C for 4 h,
T4: -4 °C for 4 h
1. RNA preparation
Method
1. RNA preparation
2. Library preparation for transcriptome
sequencing
3. Annotation of transcriptome assembly and gene
4. Carry out differential expression analysis
5. Validation of RNA-Seq data by qRT-PCR

28. Analysis of proline content
Phenotypic comparison of chrysanthemum Analysis of soluble sugar content

29. Length range Transcript Unigene
Total clean reads 86,444,237
Q30 96.33 %
Overview of RNA sequencing and length distribution of the
transcripts and unigenes
Length range Transcript Unigene
Total number 1,99,754 93,837
N50 length
(bp)
1,321 1,155
Mean length
(bp)
878.8 719.19

30. Similarity of D. grandiflorum var.
Jinba sequences with those of other
species.
In total, 20,400 unigenes were grouped
into 25 functional classifications based
on KOG database.
KOG functional classification of consensus sequence

31. A total of
19,534 unigenes were
successfully annotated
and classified into 52
functional groups of
three major GO
GO classification of the annotated unigenes
categories.

32. CP1: T1 vs T2
CP2: T1 vs T3
CP3: T1 vs T4
Venn diagram and histogram of DEGs during low temperature stresses.
a. Venn diagram showing DEGs expressed at each of the three low temperature
treatments.
b. The numbers of DEGs identified in comparisons between pairs of libraries

33. • Low temperature signal transduction: They identified DEGs
encoding 3 key proteins (ABA receptor, Protein phosphatase 2C
and Serine/threonine protein kinase), 1 ABF (ABA-responsive
element binding factor) and 11 DRE-binding factors (DREB) of
ABA signaling pathway. And also they found 6 PLD (Phospholipase
D) DEGs to produce lipid which are involved in signal transduction.
D) DEGs to produce lipid which are involved in signal transduction.
• TFs responding to low temperature: Among TFs, DREBs/CBFs
played pivotal roles in improving low temperature tolerance of
plants. A total of 10 DREBs (Dehydration-responsive element-
binding factors) were found in these study.

34. • Cold resistant genes responding to low temperature: They found
a total of 12 genes encoding LTPs (Lipid-transfer proteins) and 7
genes encoding FADs (Fatty acid desaturase) to tolerant low
temperatures by enhancing the stabilization of cell membrane and
16 LEA (Late-embryogenesis-abundant) protein genes for coding
antifreezing proteins and also found 9, 21 and 14 genes encoding
4
antifreezing proteins and also found 9, 21 and 14 genes encoding
HSPs in DEGs of CP1, CP2 and CP3.
• They found P5CS (Pyrroline-5-carboxylate synthetase), which
participates in the synthesis of proline, was only found to be up-
regulated in T2 and T3. ProDH (Proline dehydrogenase), which
participates in the degradation of proline was only found to be up-
regulated in T4.

35. CASE STUDY - 2
2020
Objective:
 Understanding the cold response of this species at the molecular level should
lead to the characterization of candidate genes important for genetic
improvement.

36. Materials and method
Plant material Manila Grass (Zoysia matrella)
Treatments
CK: Control (25/20 °C)
CT-2h: (4 °C for 2 h)
CT-72h: (4 °C 72 h)
Method
1. RNA preparation
2. Library preparation for transcriptome
sequencing
3. Annotation of transcriptome assembly and gene
4. Carry out differential expression analysis
5. Validation of RNA-Seq data by qRT-PCR

37. Physiological change in
leaves under cold stress
Phenotypic comparison of Manila grass
under cold stress

38. Overview of RNA sequencing and length distribution of the
transcripts and unigenes
Length range Transcript Unigene
Total clean reads 27,402,090
Q30 93.76 %
Length range Transcript Unigene
Total number 2,86,574 82,605
N50 length (nt) 2,236 1,471
Mean length
(nt)
1,480 787

39. Species distribution
of the BLAST hits
In total, 17,710 unigenes were
grouped into 25 functional
classifications based on KOG
database.
KOG function classification of unigenes

40. A total of
25,532 unigenes were
successfully annotated
and classified into 52
functional groups of
three major GO
Gene Ontology (GO) classification of unigenes
categories.

41. • There were 324 DEGs
between CK and
CT_2h, including 205
up-regulated unigenes
and 119 down-
regulated ones.
• There were 5,851
Number of DEGs between normal condition and cold treatments
DEGs between CK
and CT_72h, including
2,944 up-regulated
unigenes and 2,907
down-regulated ones.

42. • They identified the expression of 16 unigenes annotated as CIPK (CBL-
interacting protein kinase) were different significantly after a 72 h-cold
exposure, among which CIPK2, CIPK6, CIPK7, CIPK16, CIPK19,
CIPK21, CIPK22, CIPK23, CIPK24, and CIPK31 were included and
were up-regulated.
• 156 unigenes belonging to TFs. Among them, MYB-related TFs were
identified as cold regulated genes having important roles in the cold
response in Manila grass.
• They also identified 5 DREB1/CBFs genes in Manila grass. They were
significantly up-regulated at the 2 h-cold time point and recovered at the
72 h-cold time which regulates a large spectrum of COR genes,
collectively called the CBF regulon.

43. • They found that 2 genes annotated as ‘cold acclimation protein
COR413-PM1’ and 1 gene annotated as ‘dehydrin COR410’
respectively were up-regulated at the 72h-cold point, but 1 chloroplastic
COR413 inner membrane protein (COR413IM1) was down-regulated.
• They also found that well-established ICE-CBF-COR cold response
pathway might functionally act in Manila grass.

44. CASE STUDY - 3
2015
Objectives:
• To characterize the leaf transcriptome of tree peony leaf using high-throughput
sequencing.
• To identify heat shock protein genes which can be use to improve heat stress-
tolerance in tree peony.

45. Materials and method
Plant material Tree peony (Paeonia suffruticosa)
Treatment
Leaves were collected from healthy plants in july
when the plants were experiencing temperatures
of approximately 35 to 37 .
Two replicated samples were frozen immediately
in liquid nitrogen and stored at -80 .
in liquid nitrogen and stored at -80 .
Method
1. RNA preparation
2. Library preparation for transcriptome
sequencing
3. Annotation of transcriptome assembly and
gene
4. Carry out differential expression analysis
5. Validation of RNA-Seq data by qRT-PCR

46. Overview of RNA sequencing and length distribution of the
transcripts and unigenes
Length
range
Transcript Unigene
Total
number
1,34,151 93,714
N50
N50
length
(nt)
1217 1130
Mean
length
(nt)
692.52 639.7

47. Gene Ontology (GO) enrichment classification of differentially expressed genes
• Total 17,340 unigenes were assigned to 3 main GO categories. including molecular
functions, biological processes, and cellular components
• 14,292 unigenes involved in biological processes- 87.2 % in metabolic process.
• 8524 unigenes involved in cellular components- 76.8 % in cell
• 15,810 unigenes involved in molecular function- 74.5% in binding function.

48. KOG function classification of unigenes
• In total, 7,618 unigenes were grouped into 25 functional classifications based on KOG
database.
• The largest groups are Signal transduction mechanisms (14.8 %) and general function
prediction (14.1 %) only followed by posttranslational modification, protein turnover,
chaperones (12.8 %) and so on.

49. Tree peony Hsp genes with complete open reading frames (ORFs)

50. • In this study, they identified 24 putative Hsps.
• Among them, 5 are members of Hsp90, 5 are members of Hsp70, and
14 are members of sHsp.
• The newly identified Hsps will increase the understanding of how the
tree peony responds to the environment and can also be exploited as
novel genes for molecular breeding of tree peony-related species with
novel genes for molecular breeding of tree peony-related species with
improved thermal tolerance.

51. Common mistakes during
transcriptome study
1. Sampling: Several issues e.g. Improper cold
stress studies.
2. Replication: How many?
How many?
3. Software: How do you select them.
4. Validation: Choosing of reference gene.

52. Conclusion
• Transcriptome has paved the way for a comprehensive
understanding of how genes are expressed and
interconnected.
• Over the last three decades, methodological breakthroughs
have repeatedly revolutionized Transcriptome profiling and
redefined what is possible to investigate.
• Integration of Transcriptomic data with other omics is
giving an increasingly integrated view of cellular
complexities facilitating holistic approaches to biomedical
research.

54. Future prospects
• We are still a step away from understanding the plant
response to multiple abiotic stresses in fields as the
information generated has to be collected and understood
in detail.
• Most of the research work has been done only in few
• Most of the research work has been done only in few
model plants. There is urgent need to generate data from
non-model plant species.
• There is need to identify new types of long ncRNAs