1. Cassava Genome from Ancestor to Cultivar
GCP21-II S3
Cassava Genome from Ancestor to Cultivar
Wenquan Wang Ph. D
Chinese Cassava Genomics Consortium
Institute of Tropical Biosciences & Biotechnology, CATAS
Uganda, June 19, 2012

2. Biological characteristics of cassava
High photosynthesis
High starch accumulation
Extremely tolerance to drought and barren soil
Heterozygosity and somatic propagation.

3. Bottleneck in aspect of genetics for developing cassava industry
Less known genetic diversity in evolution
Lack knowledge for mechanisms of high photosynthesis and starch metabolism
Uncovering function of drought and barren soil tolerance
Less understanding adaptation to different kinds of diseases and pests of cassava plant
Lack tools for genotyping in cassava breeding

4. Genotypes used for whole genome sequencing
W14 (Manihot esculenta. ssp. flabellifolia) Semi-wild species
KU50 (Manihot esculanta Crantz) Cultivar (starchy)
S1.600 (Manihot esculanta Crantz) Cultivar (sugary )
W14
KU50

5. Characteristics of the three genotypes for sequencing
W14
KU50
S1.600
Regeneration
Seeds mainly
Stems
Tuber root
small
large
very large
Photosynthesis
middle
high
Fresh root yield
low
10 folds
5-10 folds
Starch content
4-5%
30%
5-6 folds
5% added 12-15% sugar, 2-3 folds

6. Net photosynthesis rate difference of W14 and KU50 in developing stages

7. contigs/scaffolds >10kb
Genome assembly of W14 and KU50
W14
KU50
all contigs/
scafolds
contigs/scaffolds >10kb
Fold genome coverage (Gb)
97.88
45.31
Number of contigs/scaffolds
54,426
15,234
62,763
7,441
Total span
475 Mb
302 Mb
416 Mb
167 Mb
N50
14 kb
21 kb
12 kb
23 kb
Largest contigs/scaffold
183 kb
123 kb
Average scaffold length
9 kb
20 kb
7 kb
22 kb
GC(%):
34.63%
34.47%
36.02%
36.07%

8. Repeats account and divergence rate in W14 and KU50
AM %
W %
KU %
12%
22%
17%

9. LTR in situ hybridization in all the chromosomes

10. Genome coverage in gene region
Transcript coverage 80.5%
KU50
W14
Transcripts coverage 97.1%
EST coverage 73.4%
KU50
W14
EST coverage 91.5%

11. Evaluation of assembly of W14
Miss match rate: 4.9/10000; mm and gap rate, 3.9/1000

12. Gene prediction in genomes of W14 and KU50
Gene Number:
43986
31480
Gene Length:
70 Mb
39 Mb
Coding Region Length:
46 Mb
28 Mb
Gene Density(%):
9.94%
9.95%
Mean Length of Intergenic:
4 kb
Maxmium Length of Intergenic:
52 kb
44 kb
Exon Number:
181,158
124,694
Exon Number/Gene:
4.13
3.97
Exon Length:
Mean Length of Exon:
252.73
225.44
Maxmium Length of Exon:
9 kb
6 kb
GC(%) of Exon:
44.09%
42.65%
Intron Number:
137266
93287
Intron number/Gene:
3.13
2.97
Intron Length:
31 Mb
Mean Length of Intron:
336.12
327.71
Maxmium Length of Intron:
11 kb
14 kb
GC(%) of Intron:
32.81%
33.40%

13. Annotation of genes predicted
Genome
W14
KU50
Predicated genes
Number
43,892
Percentage (%)
31,407
Swissprot
28,808
65.63%
19,240
61.26%
TrEMBL
38,784
88.36%
26,723
85.09%
InterPro/GO
39,918
90.95%
24,344
77.51%
KEGG
35,451
80.77%
24,247
77.20%
COG
18,205
41.48%
12,162
38.72%
NR/NT
38,802
88.40%
26,739
85.14%
Total annotated
41,934
95.54%
27,549
87.72%
Un-annotated
1,958
4.46%
3,858
12.28%

14. BAC library and physic map constructed in W14
Description
Index
BAC library coverage
BAC library insertion size
Number of BAC clones fingerprinted  
Number of high quality fingerprints used for assembly 
Number of contigs
Number of singletons 
Total length of the contigs 
 N50 contig length 
Longest contig
Average number of clones per contig   
93,000 clones, >10x
130kb
30,000 ?
2484  
984 Mb
kb  kb
2.16

15. Genome diversity decreasing in evolution
Heterozygosity of genome W14, KU50 and AM560
sample
# SNPs
SNPs density (1SNPs/n bp)
# gene SNPs
gene SNPs density
# SNPs in exon
SNPs In exon density (per SNPs/bp)
W14
1,377,370
1/257
295,358
1/270
220,600
1/272
KU50
806,271
1/286
109,701
1/336
43,610
1/422
AM560
(S3)
506,746
1/693
73,628
1/6170
46,524
1/5583

16. SNPs divergence in genome of wild ancestor W14 and cultivar KU50
Sample
# SNPs
SNPs density (SNPs/ bp)
# gene SNPs
gene SNPs density
# SNPs in exon
SNPs In exon density (SNPs/bp)
# intergenics SNPs
intergenics SNPs density ( SNPs/bp)
W14
4,812,287
6.94/
1000
1,574,460
1/294
563,588
1/676
3,237,827
1/160
KU50
3,620,860
4.57/
516,278
1/894
187,122
1/1947
3,104,582
1/229
S1.600
2,977,198
4.10/
517,321
1/893
186,413
1/1935
2,459,877
1/255

17. SNPs shared and distribution
Samples
# SNPs
# SNPs unique
# SNPs in gene
# SNPs in exon
# intergenics SNPs
# SNPs in repeat regions
W14
4,812,287
4,065,298
1,574,460
563,588
3,237,827
1,751,276
KU50
3,620,860
1,976,538
516,278
187,122
3,104,582
2,142,290
S1.600
2,977,198
1,375,917
517,321
186,413
2,459,877
1,737,544
W14-KU50
570,695
219,335
200,908
75,356
369,787
184,454
W14-S1600
527,654
176,294
205,509
76,873
322,145
162,873
KU50-S1600
1,424,987
1,073,627
281,464
101,783
1,143,523
770,687
W14-KU50-S1600
351,360
143,721
53,735
207,639
98,970

18. Indels divergence in genome of wild ancestor W14 and cultivar KU50
Sample
# indels
# indels density
# insertion
# deletion
average length
W14
390,652
0.80/1000
159,467
231,080
3.59
KU50
275,639
0.79/1000
132,396
143,200
3.65
S1.600
217,226
0.64/1000
103,964
113,207
4.07

19. SNP/Indels among four cassava genomes

20. Transcriptome for photosynthesis and starch metabolism in cultivar Arg7 and wild ancestor W14
Transcriptome sequenced samples:
C1 Arg7 Early root
C2 Arg7 Middle root
C3 Arg7 Later root
C4 W14 Middle root
C5 Arg7 Developing stem
C6 Arg7 Functional leaf
C7 W14 Functional leaf
C8 W14 Developing stem

21. Expression profiling of genes for starch and photosynthesis pathways

22. Comparative expression folds of genes for photosynthesis: Arg7/W14

23. Cell Wall metabolism, Arg7/W14, red-high expression in root of W14

24. Sucrose glycolysis in root of Arg7 is weak than in W14

25. Comparative expression folds of genes for starch metabolism in leaf and storage root: Arg7/W14

26. Expression folds of genes for starch accumulation in tuber root of KU50 than in W14

27. Phylogenetic tree of SuSy and INV

28. An efficient starch biosynthesis model in tuber root of cassava

29. miRNAs and drought tolerance in cassava
a set of 148 miRNAs in cassava have been predicted by sequencing of14 small RNA samples and referenced to genome which of 41 are novels and 107 are conserved.
miRNAs and targets related to drought and development of leaf and tuber root have been found.

30. Eleven drought and cold inducible miRNAs with interesting targets were revealed and confirmed by qPCR
miRNA
Targets
miR
Protein binding, Zinc ion binding; Transcription factor, DNA binding
miR
zinc ion binding, protein serine/threonine kinase, ATP binding
miR
Enzyme inhibitor activity, Pectinesterase activity; DNA-binding protein-related; FUNCTIONS IN: transcription factor activity
miR
Encodes a H3/H4 histone acetyltransferase; Encodes eukaryotic translation initiation factor.
miR
oxidative phosphorylation uncoupler activity, binding oxidoreductase activity, Iron ion binding
miR
Protein kinase family--kinase activity, small molecular g-protein
miR
Kinase activity; Nuclear protein required for early embryogenesis
miR
auxin induced gene (IAA1) encoding a short-lived nuclear-localized transcriptional regulator protein; acetylglucosaminyltransferase ; transferase activity

31. ABA biosynthesis pathway in tuber root of cassava

32. Expression of genes in carotene and ABA synthesis pathways

33. Comparative genomics among cassava, Jatropha and castor bean
Unique gene families:
Cassava
Jatropha
Castor bean 826
Shared gene families:
12041

34. Gene families in virion part and reproduction found only
Coherence in biological processes among cassava, Jatropha and castor bean of Eurphorbiceace
Gene families in virion part and reproduction found only
in cassava

35. Database: Cassava-genome.cn

36. Ongoing work
Genome fine mapping integrated assembly with physic map, BAC-end sequences and BAC-pooling sequences.
Chromosomes location with assembling BACs and scarfolds based on in situ hybridization
Functional verification of genes for important pathways
Development of SNP markers and molecular design breeding

37. Summary
Genome drafts of an ancestor and a cultivar in cassava were assembled and annotated.
Genome diversity decreased from wild ancestor to cultivar in domestication;Millions of SNPs were discovered and recommended for genotyping in cassava.
Advanced an efficient starch biosynthesis pathway in tuber root of cassava.

38. Acknowledgement CATAS
BX Feng, Z Xia, XC Zhou, KM Li, PH Li, M Peng, WQ Wang
BIG-CAS
JF Xiao, JX Liu, SN Hu
SCBG-CAS
Gong Xiao, Chi Song, Ying Wang
EMBRAPA, Brazil, Luiz C
UC Davis Mingcheng Luo
XJIEG-CAS
Bin Liu, Binxiao Feng
SIS-CAS
Jun Yang, Peng Zhang
Fudan U
Zhicheng Wu, Ruiqi Liao,
Shuigen Zhou
Copenhagen U, Demark
Rubini, Birger Muller
Nanjing Agricultural U
Qunfeng Lu