การวเิคราะห์ข้อมูลการถอดรหัสที่ส่งผลต่อการแสดงออกของยีน ที่แตกต่างกันในช่อดอกจากจีโนมข้าวลูกผสมและสายพันธ์ุพ่อแม่

กุลชนา เกศสุวรรณ์ ศูนย์วิจัยข้าวแพร่

ส านักวิจัยและพัฒนาข้าว กรมการข้าว

A genome-wide transcriptome analysis reveals the differentially expressed gene (DEGs) in panicle of hybrids and parents

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Rice genome

: a complete set of DNA of rice : consists of 12 chromosomes : ~ 383 Mb

: 30216 predicted number of genes

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Rice chromosome:

โครงสร้างภายในเซลล์ท่ีประกอบ ไปด้วยสารพนัธุกรรม ได้แก่ สายดีเอ็นเอท่ีจบักบัโปรตนี อยู่ในนิวเคลียสประกอบกนัเข้าเป็นยีน ท่ีท าหน้าท่ีต่างๆ ในสิ่งมีชีวิต

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Central Dogma

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DNA replication

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RNA synthesis or transcription

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Protein synthesis

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Gene expression

is .. the genotype give rise to the phenotype

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DNA Sequencing

double stranded DNA is dissociated

annealed with radioactive or fluorescent and synthesize new DNA

the reactions contains dideoxynucleotide bases

separated on a gel in side by side lanes

the sequence is read from the bottom up and it is the compliment of the bases

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RNA Seq workflow

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Materials:

• Two hybrids (PTT06001H & PTT06008H) • Two paternal lines (CK81 & CK120) • One maternal line (IR79156)

RNA Sequencing

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Methods:

Isolation of total RNA

Removing rRNA

Fragmentation of whole transcriptome RNA

Amplified library construction

Data analysis

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Results: isolation of total RNA

RNA isolation from different tissue samples; M = λ HindIII; lanes 1-3 RNA extracted from leaf; lanes 4-6 stem; lanes 7-9 root; and lanes 10-12 panicle

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Results: removing rRNA

Agarose gel electrophoresis analysis shows depletion of 18s and 28s rRNA bands as compared to a control sample. M = λHindIII; lane 1,3,5,7, and 9 are a total RNA from IR79156A, CK81, CK120, PTT06001H, and PTT06008H, respectively; lane 2,4, 6, 8, and 10 are RiboMinusTMRNA from IR79156A, CK81, CK120, PTT06001H, and PTT06008H, respectively

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Results: fragmentation of whole transcriptome RNA

The gel image from an Agilent 2100 with the RNA Pico Chip shows the fragmentation sizes from 35 nt up to several hundred nt. L= Ladder; lane 1-6 shows the fragmented RNA using RNaseIII from rice tissue samples; Lane 7-11 shows leaf tissue fragmented RNA from different rice varieties including, IR79156A, CK81, CK120, PTT06001H and PTT06008H, respectively

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Results: amplified library construction

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Results: cDNA size selection

Novex® Pre-cast gel stained by SYBR gold nucleic acid and illuminated under UV transilluminator. The gel containing 150-250 nt was vertically cut into four small pieces and stored at -80°C until use. L= 50 bp DNA Ladder, 1-6 = cDNA from rice tissues

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Results: cDNA amplification products

cDNA amplification products of rice tissues after library construction. M= Hyper Ladder I, 1-5 = amplified DNA from root, 6-10= amplified DNA from stem, 11-12= amplified DNA from leaf

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Data analysis: number of mapped reads

Sample

ID

Read

Length

Number of

Fragments

Passing Filters

Number of

Fragments Total

Number of

Mapped

Reads

Average

Percentage

Mapped

Reads

Unique

Start

Positions

Percentage

Number of

Base

Mismatches

Count Percent

Panicle1

(M)

75.00 24,047,578.00 24,047,578.00 2,244,306.00 9.32 26.73 0

1

2

1,642,871.00

308,724.00

105,864.00

73.20

13.76

4.72

Panicle2

(P1)

75.00 33,548,584.00 33,548,584.00 5,361,077.00 15.96 31.30 0

1

2

3,867,505.00

642,386.00

244,401.00

73.77

12.25

4.66

Panicle3

(P2)

75.00 51,508,873.00 51,508,873.00 9,747,526.00 18.91 26.94 0

1

2

7,309,012.00

1,203,449.00

411,555.00

74.98

12.35

4.22

Panicle4

(H1)

75.00 60,043,483.00 60,043,483.00 7,386,061.00 12.30 28.88 0

1

2

5,501,789.00

849,861.00

316,259.00

75.57

11.67

4.34

Panicle5

(H2)

75.00 43,037,114.00 43,037,114.00 5,196,982.00 12.06 38.30 0

1

2

3,936,617.00

616,649.00

217,491.00

75.75

11.87

4.18

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Data analysis: differentially expressed genes (DEGs)

P2 vs H2

P1 vs H1

M vs P2

M vs P1

M vs H2

M vs H1

3000

2500

2000

1500

1000

P2 vs H2

P1 vs H1

M vs P2

M vs P1

M vs H2

M vs H1

down

variety

no

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es

up

Individual Value Plot of no. of genesFold change = 2

Panel variable: regulation

Distribution of the number of DEGs showing a greater than 2 fold change in panicle. The two panels display pairwise data comparing two hybrids (H1, and H2) and their parents (M; maternal line, P1; paternal line1, P2; paternal line2). Left panel shows number of down-regulated genes, and right panel shows number of up-regulated genes B

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Data analysis: differentially expressed genes (DEGs)

Distribution of DEGs number greater than 10 fold changes in panicle. Two panels display pairwise data comparing two hybrids (H1, and H2) and their parents (M; maternal line, P1; paternal line1, P2; paternal line2). Left panel shows number of down-regulated genes, and right panel shows number of up-regulated genes

P2 vs H2

P1 vs H1

M vs P2

M vs P1

M vs H2

M vs H1

700

600

500

400

300

200

100

0

P2 vs H2

P1 vs H1

M vs P2

M vs P1

M vs H2

M vs H1

down

variety

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up

Individual Value Plot of no. of genesFold change = 10

Panel variable: regulation

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Data analysis: putative genes

No Gene ID M vs.P1

(Fold-changes)

M vs. H1

(Fold-changes)

Ratio Start End Description

1 Os02g0655700 2389.499 864.651 2.76:1 27343334 27347114 amino acid permease-like

2 Os02g0255700 434.598 434.598

1:1

8839102 8849958

tetratricopeptide repeat (TPR)-containing

protein-like

3 Os03g0454100 109.617 113.304 1:1 20150875 20157878 putative membrane protein

4 Os10g0495900 94.146 94.146 1:1 19367086 19370048 fiber protein Fb34, putative, expressed

5 Os02g0108100 87.806 136.139 1:1.5 458901 460810 hypothetical protein

6 Os03g0599600 85.768 85.768

1:1

23183921 23191096

putative chromosome condensation

regulator

7 Os11g0549300 69.232 18.258

3.8:1

22496224 22501632

Similar to Protein kinase domain containing

protein, expressed

8 Os07g0565200 67.546 67.546

1:1

23319655 23324107

Cell differentiation protein rcd1, putative,

expressed

9 Os08g0189100 67.195 28.926 2.3:1 5207392 5208392 germin-like protein

10 Os08g0564300 65.452 65.452 1:1 28352290 28357929 MDR-like ABC transporter

11 Os11g0297300 64.163 38.398 1.6:1 10979041 10982960 beta-D-xylosidase

12 Os09g0441400 63.322 47.368

1.3:1

17093839 17096387

putative elicitor-inducible cytochrome

P450

13 Os02g0658900 57.597 57.597 1:1 27519351 27523643 Sec20 family protein

14 Os08g0200100 54.812 54.812

1:1

5786600 5788138

Acyl-desaturase, chloroplast precursor,

putative, expressed

15 Os06g0486300 52.361 52.361 1:1 17473302 17476160 putative Mlo

Genes expressed in panicle and showing similar up-regulation between maternal line (M), paternal line1 (P1), and hybrid1 (H1)

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Data analysis: putative genes

Genes expressed in panicle and showing similar up-regulation between maternal line (M), paternal line1 (P2), and hybrid1 (H2)

No Gene ID

M vs.P2

(Fold-

changes)

M vs.H2

(Fold-

changes)

Ratio

Start End Description

1 Os02g0655700 2389.499 2389.499 1:1 27343334 27347114 amino acid permease-like

2 Os02g0255700 434.598 173.654

2.5:1

8839102 8849958

tetratricopeptide repeat (TPR)-containing

protein-like

3 Os02g0108100 136.139 44.878 3:1 458901 460810 Conserved hypothetical protein

4 Os03g0454100 113.304 113.304

1:1

20150875 20157878

TMS membrane protein/tumour differentially

expressed protein family protein

5 Os10g0495900 94.146 94.146

1:1

19367086 19370048

Protein of unknown function DUF1218 family

protein

6 Os07g0519600 86.551 344.030 1:4 20723574 20727202 Similar to taxane 10-beta-hydroxylase

7 Os03g0599600 85.768 85.768

1:1

23183921 23191096

Regulator of chromosome condensation, RCC1

domain containing protein

8 Os07g0565200 67.546 67.546 1:1 23319655 23324107 Similar to RCD1

9 Os08g0564300 65.452 65.452 1:1 28352290 28357929 ABC transporter-like domain containing protein

10 Os02g0658900 57.597 57.597 1:1 27519351 27523643 Sec20 family protein

11 Os08g0180000 56.681 22.207

2.5:1

4675295 4679429

mRNA capping enzyme, large subunit family

protein

12 Os06g0486300 52.361 52.361 1:1 17473302 17476160 MLO protein homolog 1

13 Os07g0110100 48.312 13.849

3.7:1

555107 557371

Esterase/lipase/thioesterase domain containing

protein (RAP-DB)

14 Os08g0371200 44.507 44.507

1:1

17387393 17411239

putative Lignostilbene-alpha,beta-dioxygenase

and related enzymes

15 Os12g0182366 43.250 43.250 1:1 4183260 4185772 Hypothetical conserved gene Bure

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Data analysis: Gene Ontology (biological process)

GO term distribution of DEGs in panicle common to the three two-way comparisons of hybrids and their parents. The GO Level 3 for the biological process category was constructed with the default settings

Up-regulated (H2 vs. M; H2 vs. P2; M vs. P2)

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Data analysis: Gene Ontology (molecular function)

GO term distribution of DEGs in panicle common to the three two- way comparisons of hybrids and their parents. The GO Level 3 for the molecular function category was constructed with the default settings

Up-regulated (H2 vs. M; H2 vs. P2; M vs. P2)

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Data analysis: KEGG map pathway

No

.

KEGG map pathway Enzyme EC number Gene ID

Up-regulated (M vs. P1 and H1)

1 Purine metabolism adenosine-triphosphate

guanylatel kinase

ec: 3.6.1.3

ec: 2.7.4.8

Os08g0564300

Os01g0237366

2 Fatty acid biosynthesis acyl-[acyl-carrier-

protein]

desaturase

ec: 1.14.19.2 Os08g0200100

3 Biosynthetic of

unsaturated

Fatty acid

acyl-[acyl-carrier-

protein]

desaturase

ec: 1.14.19.2 Os08g0200100

Up-regulated (M vs. P2 and H2)

1 Purine metabolism adenosinetriphosphate

guanylatel kinase

ec: 3.6.1.3

ec: 2.7.4.8

Os08g0564300

Os01g0237366

2 Pyrimidine metabolism aspartate

carbomoyltransferase

ec: 2.1.3.2 Os08g0248800

3 Alanine, aspartate and

glutamate metabolism

aspartate

carbomoyltransferase

ec: 2.1.3.2 Os08g0248800

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Data analysis: KEGG map pathway (purine metabolism)

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Summary and discussion

DEGs Up-regulated (> 2 fold) M vs. H1 = 2,902 P1 vs. H1 = 1,604 M vs. P1 = 3,168

M vs. H2 = 3,068 P2 vs. H2 = 1,171 M vs. P2 = 3,031

M vs. H1 = 155 P1 vs. H1 = 30 M vs. P1 = 254

DEGs Up-regulated (> 10 fold)

M vs. H2 = 173 P2 vs. H2 = 49 M vs. P2 = 207

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Summary and discussion

DEGs Down-regulated (> 2 fold) M vs. H1 = 3,145 P1 vs. H1 = 2,058 M vs. P1 = 2,945

M vs. H2 = 2,811 P2 vs. H2 = 1,140 M vs. P2 = 2,904

M vs. H1 = 603 P1 vs. H1 = 185 M vs. P1 = 644

DEGs Down-regulated (> 10 fold)

M vs. H2 = 529 P2 vs. H2 = 68 M vs. P2 = 565

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Summary and discussion

Putative genes Hybrid 1 36 common genes

- Os02g0655700 is highly expressed in M line

-7 genes of hybrid expressed more than paternal line Os11g0549300, Os08g0189100, Os11g0297300, Os09g0441400, Os08g0180000, Os08g0528100, Os02g0129800

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Summary and discussion

Putative genes Hybrid 2 41 common genes

- Os02g0655700 is highly expressed in M line

-4 genes of hybrid expressed more than paternal line Os07g0519600, Os11g0506300, Os03g0265200, Os09g0441400

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Summary and discussion

KEGG biochemical pathway

Up-regulated 5 pathway

Purine metabolism

Purimidine metabolism

Fatty acid biosynthesis

Lipid metabolism

Amino acid metabolism

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Summary and discussion

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Acknowledgements

Thank you

Acknowledgements

Prof. Jim M Dunwell

Director of Phrae Rice Research Center

Director of Phitsanulok Rice Research Center

The Royal Thai Government Scholarship

Director of Bureau of Rice Research and Development

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