Diapositiva 1 - Academia Nacional de Ciencias

SESION DE INCORPORACION DE LA
DRA. GISELLA ORJEDA FERNANDEZ
COMO ACADEMICA TITULAR
Miraflores, 19 de junio de 2014
Sede de la Universidad de Piura – Campus Lima
Avances de la genética moderna
y contribuciones peruanas
Gisella Orjeda
Universidad Peruana
Cayetano Heredia
Secuenciando el genoma de la papa y
descifrando su organizacion y funcion
www.potatogenome.net
Participants
BGI-Shenzhen, China (Sanwen Huang, Ruiqiang Li, Xun Xu, Wei Fan, Peixiang Ni, Hongmei Zhu, Desheng Mu,
Bicheng Yang, Jian Wang and Jun Wang); Center Bioengineering RAS, Russia (Boris Kuznetsov); Central
Potato Research Institute, India (Swarup Chakrabarti, V.U. Patil, Shashi Rawat and S.K. Pandey); Chinese
Academy of Agricultural Sciences, China (Sanwen Huang, Zhonghua Zhang and Dongyu Qu); University of
Dundee, United Kingdom (Dan Bolser and David Martin); ENEA, Italian National Agency for New
Technologies, Energy and the Environment, Italy (Giovanni Giuliano and Gaetano Perrotta); Imperial College
London, United Kingdom (Gerard Bishop); International Potato Center (CIP), Peru (Merideth Bonierbale, Marc
Ghislain and Reinhard Simon); Institute of Biochemistry and Biophysics (PAS), Poland (Wlodzimierz Zagorski,
Jacek Hennig, Pawel Szczesny, Piotr Zielenkiewicz and Robert Gromadka); Instituto Nacional de TecnologÌa
Agropecuaria (INTA), Argentina (Gabriela Massa, Leandro Barreiro and Sergio Feingold); Instituto de
Investigaciones Agropecuarias (INIA), Chile (Boris Sagredo, Alex Di Genova and Nilo MejÌa); Michigan State
University, USA (Robin Buell, David Douches, Steven Lundback, Alicia Massa, and Brett Whitty); New Zealand
Institute for Plant & Food Research, New Zealand (Jeanne Jacobs, Mark Fiers and Susan Thomson); Scottish
Crop Research Institute, United Kingdom (Glenn Bryan, David Marshall, Robbie Waugh and Sanjeev Kumar
Sharma); Teagasc Agriculture and Food Development Authority, Ireland (Dan Milbourne, Istvan Nagy and
Marialaura Destefanis); Universidad Peruana Cayetano Heredia, Peru (Gisella Orjeda, Frank Guzman, Michael
Torres, Tomas Miranda, German de la Cruz, Roberto Lozano and Olga Ponce); University of Wisconsin, USA
(Jiming Jiang and Marina Iovene); Virginia Polytechnic Institute & State University, USA (Richard E. Veilleux);
Wageningen University, The Netherlands (Bas te Lintel Hekkert, Christian Bachem, Erwin Datema, Jan de Boer,
Richard Visser, Roeland van Ham, Theo Borm and Xiaomin Tang)
19 Institutos
En Peru: UPCH y UNSCH
• Bac by Bac
• Sanger
• 454
• Heterocigote
tuberosum di-haploid
DM1-3 51644
RH89-039-16
Secuenciamiento del genoma
• WGS
• Solexa
• 454
• Sanger
• Homocigota
phureja double monoploid
The Potato Genome Sequence Initiative
Poster presented @ SOL meeting- Scotland. 2009
DM1-3 51644 vs RH89-39-16
Unified view of assembly
Assembly (assemblies, contigs)
Scaffolding
•178 Mb
(21% nr)
•1,644BACs
DM1-3 51644
RH89-039-16
Results
•681 super
Scaffolds
•727 Mb
Haplotype diversity and inbreeding depression.
X Xu et al. Nature 000, 1-7 (2011) doi:10.1038/nature10158
Guzman, F.; Sharma, S.K.; Sagredo, B.; Mejia, N. ;Di
Genova, A.; Bolser, D.; Jacobs, J.; Thompson, S.;
Feingold, S.; D'Ambrossio, J.; Herrera, M.;
Bonierbale, M.; Ghislain, M.; Martínez, D.; Eguiluz,
M.; Lozano, R.; Ponce, O.; De la Cruz, G.; Buell , R.;
de Boer, J.; Bryan, G.; Orjeda, G.
WGS DM1-3 51644
Marker
cM
667007
2.8
Solcap_stsnp_C1_86
3
4.5
Genetic map with STS markers
PM022
5.6
10
Markers
RH89-039-16
DM1-3 51644
AFLP markers
SSR´s de Scaffolds y de
previos mapas
Whole Genome
profiling from AFLP
sequences
DArT’s
SNP’s
Población
de mapeo
DM genetic
and physical
mapping
S.phureja x S. goniocalyx
DM
(DM 1-3 516 R44)
X
F1
DI
(CIP No. 703825)
X
186 individuals
DI
Markers for DM Genetic mapping
• 4836 STS markers for the map
– 2174 DArTs
– 1920 SNPs
– 358 SSRs from DM scaffolds and previous maps
Diversity Array Technology (DArT)
Created by Dr Andrzej Kilian
To overcome limitations of
other markers.
• Based on microarray DNA
hybridization
• No need of nucleotidic
sequence info
• High throughput
• Generates reproducible
data at low cost
Microsatellite selection
From 500 bigger
scaffolds
Series PM
Construcción del mapa genético
Microsatelites from DM sequence
DM Scaffold
Microsatellite
identification
(Sputnik)
dinucleotidic Microsatellite (AT)5
Position 42 846
Primer design
(Primer 3)
Microsatellite PM series
Microsatelites from previous maps
Potato-TXB 1992 MAP
MPI MAP
BCT and BCP MAP
BCT MAP
(Tanskley et al, 1992)
(Milbourne et al,
1998)
(Feingold et al, 2005)
(Ghislain et al, 2009)
Series
STM
STI
TG
POMAMO
Meyer, Gebhardt
2005
SNPs in DM (SolCap)
Query
SNPs
Filtered SNPs
Atlantic
224748
150669
Premier
265673
181800
Snowden
258872
166253
DM Scaffold
A/C SNP
Construcción del mapa genético
Genotyping population with SSr
DM
DI
DM/DI
Population Format: CP
(hk x hk, lm x ll, nn x np)
Map Construction
(JoinMap 4.0)
Construcción del mapa genético
population name:
population type:
nr. of loci:
nr. of individuals:
(DMxDI) x DI
CP
2619
169
(Individuals excluded: 6, 14, 18, 46, 74, 86, 88, 92, 93, 101, 102, 103, 104, 105, 154, 185 & 186)
Marker type: DArTs, SSRs y SNPs
Construcción del mapa genético
blue = DArTs
yellow = SNPs
green = SSRs
Blue and magenta
=AFLP
intensity of green
=AFLP marker
density per bin
Pseudomolecula del
cromosoma 1
integrada con los
mapas genticos de
DM y RH.
G3 November 1, 2013
vol. 3 no. 11 2031-2047
Genome wide identification and mapping of NBSencoding resistance genes in Solanum phureja
Lozano, G.R.; Guzman, E.F.; Torres, C.M.; Orjeda, G.
Unidad de Genómica
Universidad Peruana Cayetano Heredia
Facultad de Ciencias y Filosofía Alberto Cazorla Talleri
Departamento de Bioquímica, Biología Molecular
y Farmacología
Lima, Perú
E-mail: roberto.lozano@upch.pe
Protein families: Pfam
Whole Genome Shotgun Assembly V3
BGI pipeline
Whole Genome Annotation
CDS
GFF
PEP
RAW-HMM of PFAM NBS
HMMERsearch
Initial set of potato proteins
encoding NBS domains
Selection of high quality NBS encoding
proteins (e value < 1E-60)
HMMERbuild
Potato specific HMM for NBS domain
HMM-papa
Aligned using
HMMERalign
Table 1. Number of Solanum tuberosum group phureja genes that encode NBS-domains with homology to plant
resistance proteins.
Lozano R, Ponce O, Ramirez M, Mostajo N, et al. (2012) Genome-Wide Identification and Mapping of NBS-Encoding Resistance
Genes in Solanum tuberosum Group Phureja. PLoS ONE 7(4): e34775. doi:10.1371/journal.pone.0034775
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034775
Figure 3. Distribution of Solanum tuberosum group phureja sequences that are predicted to encode NBS
resistance proteins.
Lozano R, Ponce O, Ramirez M, Mostajo N, et al. (2012) Genome-Wide Identification and Mapping of NBS-Encoding Resistance
Genes in Solanum tuberosum Group Phureja. PLoS ONE 7(4): e34775. doi:10.1371/journal.pone.0034775
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034775
Figure 4. Rpi-vnt1 cluster structure.
Lozano R, Ponce O, Ramirez M, Mostajo N, et al. (2012) Genome-Wide Identification and Mapping of NBS-Encoding Resistance
Genes in Solanum tuberosum Group Phureja. PLoS ONE 7(4): e34775. doi:10.1371/journal.pone.0034775
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034775
Figure 5. Alternative splicing of a TIR-NBS-LRR resistance gene.
Extraccion de mRNA de DM 14 semanas
Amplifico clono y secuencio cDNA
Blast con Scaffold de DM
Lozano R, Ponce O, Ramirez M, Mostajo N, et al. (2012) Genome-Wide Identification and Mapping of NBS-Encoding Resistance
Genes in Solanum tuberosum Group Phureja. PLoS ONE 7(4): e34775. doi:10.1371/journal.pone.0034775
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034775
Identification and description of miRNAs
from the complete potato genome
sequence
Mostajo, B.N.; Lozano, R; Orjeda, G.
Universidad Peruana Cayetano Heredia – Facultad de Ciencias - Unidad de Genómica – Lima, Perú
E-mail: nelly.mostajo@upch.pe
miRNA
MicroRNAs (miRNAs)
son RNA de -22 nt
que pueden jugar
importantes roles
regulatorios en
plantas y animales
usando como blanco
de su acción los mRNA
y silenciandolos por
cleavage o represion
de la traduccion
Figure 1 Examples of Metazoan miRNAs Shown are predicted stem loops involving the mature miRNAs (red) and flanking
sequence. The miRNAs* (blue) are also shown in cases where they have been experimentally identified (Lim et al. 2003a)
David P Bartel MicroRNAs : Genomics, Biogenesis, Mechanism, and Function. Cell, Volume 116, Issue 2, 2004, 281 - 297
Relevancia
Using 36.9% of the genome
miRNA
Family
Total found
Reported previously
New
Candidates after filtering
433
56
61
25
372
31
69
14
Filtering criteria:
1) not reported previously
2) Found in >3 species
3) Identity
Example of a miRNA
MIR162a
8 nuevas familias de miRNA con
estructuras predichas,posicionados y
validados experimentalmente
Distribution of the new miRNAs
in potato
Distribución de los miRNAs en el genoma - 1
I
II
-
0.0
19.7
MIR166J
25.3
MIR408
III
0.0
3.1
MIR156G MIR156H
0.0
28.9
MIR164C MIR164D
MIR164E
29.9
MIR319B MIR319E
MIR159B MIR159C
41.2
MIR162 MIR162A
MIR162B
49.5
MIR169E MIR169G
MIR169J MIR169O
56.4
-
-
0.0
Distribución de los miRNAs en el genoma - 3
MIR394B
IX
0.0
MIR166B MIR166D
MIR166E
39.8
IV
-
0.0
25.4
MIR166E
MIR171F MIR171H
58.3
59.8
79.8
MIR166B MIR166F
MIR166J MIR169H
MIR169I
105.3
MIR169K MIR169L
MIR169M MIR169N
MIR169D MIR169E
MIR169F
MIR399G MIR399I
-
106.8
XII
-
0.0
6.7
42.3
91.8
XI
67.8
72.7
110.1
66.8
16.4
MIR171C MIR171M
45.0
MIR394A
51.1
MIR398A
69.6
-
MIR398B MIR398C
MIR167C MIR167D
MIR167E MIR167F
MIR167G
MIR171I
-
MIR171D MIR171D
MIR396A MIR396B
MIR396C
-
-
Distribución de los miRNAs en el genoma - 2
V
0.0
3.9
VI
MIR160A MIR160B
MIR160C MIR160D
MIR160E MIR160F
0.0
34.8
63.6
-
VII
-
MIR166D MIR166F
MIR166G MIR166H
MIR166I
65.0
67.9
MIR162A
MIR171E MIR171G
78.9
MIR167D
85.8
-
0.0
29.5
65.3
VIII
-
0.0
-
8.1
MIR319B MIR319C
MIR159A
51.1
MIR166B MIR166C
65.7
MIR156F MIR156I
MIR171B MIR171D
MIR171D MIR171M
-
Co-localisation of
miRNA and
Resistance
QTL’s in potato
Articulo enviado a “Silence” pero….
Continuará
Transcriptome analysis of
native potato accessions under
drougth stress using RNAseq
Roberto Lozano, Yerisf Torres, Carlos Merino, Robin
Buell, Noemi Zuñiga, Gisella Orjeda
Universidad Peruana Cayetano Heredia (UPCH)
Michigan State University (MSU)
Instituto Nacional de Innovacion Agraria (INIA)
Native potatoes
.4000 varieties
.Underutilised
Not very commercial
.Rich Source of genetic
diversity
.Grown at very high
altitudes
4000 masl
“PapaSalud” Project –
NEIKER- Spain in collaboration with
several LA institutes
• http://www.neiker.net/neiker/papasalud/
• Objective:
– Select and develop “Native potatoes” with higher
quality, performance and that are adapted to
different environmental stresses.
* T3: Agronomic Evaluations and post-harvest
behaviour.
CODE
Variety
Species
Country
NKD-130
MURO SHOCCO
S.andigena
NKD-131
PUCA HUAYRO
NKD-132
CHAUCHA
Drougth
Presion [bar]
PH 1 *
PH 2 *
dif
Perú
2
5
3
S. chaucha
Perú
2
5
3
S. phureja
Perú
3
5
2
NKD-134
PULU
S.andigena
Bolivia
3
11
8
NKD-135
SOCCO HUACCOTO
S. andigena
Perú
2
4
2
NKD-137
SIPANCACHI
S.andigena
Bolivia
3
6
3
NKD-138
LARAM AJAWIRI
S. ajanwiri
Bolivia
5
5
0
NKD-139
JANCKO AJAWIRI
S. ajanwiri
Bolivia
3
5
2
NKD-140
MORAR NAYRA MARI
S. stenotonum
Perú
4
7
3
NKD-141
UNKNOWN
S.andigena
Perú
3
7
4
NKD-143
WILA HUAKA LAJRA
S.andigena
Bolivia
3.5
10
6.5
NKD-145
PUCA QUITISH
S.andigena
Perú
3
5
2
NKD-149
YANA PPOCCOYA
S. stenotonum
Perú
5
6.5
1.5
NKD-152
MORADA TURUNA
S. stenotonum
Perú
4
6
2
NKD-155
KASHPADANA AMARILLA
S. goniocalix
Perú
3
3
0
NKD-156
HOLANDESA
S.andigena
Colombia
4
7
3
NKD-157
UNKNOWN
S. andigena
Colombia
3.5
8
4.5
NKD-158
POLUYA
S. stenotonum
Perú
3
5
2
NKD-159
CAMUSA
S. andigena
Venezuela
3
12
9
NKD-160
CHIMBINA
S.andigena
Perú
3.5
5
1.5
NKD-161
NEGRITA
S.andigena
Perú
4
4
0
NKD-162
YEMA DE HUEVO
S. phureja
Colombia
2.5
4.5
2
NKD-163
COLOR UNCKUNA
S. chaucha
Perú
4.5
12
7.5
Testigo
MONALISA
S.tuberosum
España
4.5
10
5.5
PH1: Hydric potential of the “just watered” plant at 20’C and 16
light hours.
PH2: Hydric potential of the plant after 15 days without being
watered at 20’C and 16 light hours.
General Objective.• Identify the responsible genes of the drougth tolerance phenomena
in Solanum tuberosum ssp. andigena
Specific objectives.• Characterize the physiological response of tolerant and susceptible
plants against drougth stress.
• Compare the expression profiles of these two potatoes in different
moments after drought
• Identify candidate genes that are related with the drougth response.
Plant Material
Accesion Number
CIP 703671
CIP 703248
Condition under drougth
Tolerant
Susceptible
Common Name
Negrita
Wila Huaka Lajra
Genus
Solanum
Solanum
Species
tuberosum subsp. andigena
tuberosum subsp. andigena
Country of origin
Perú
Bolivia
Latitud
-7.08
-17.63
Longitud
-78.33
-65.73
MASL (feet)
3100
3800
In-vitro growing
plants
Methods
Grown in
aeropony
Negrita
S. t. andígena
CIP.703671
Wila Huaka La
S. t.andígena
CIP.703248
Physiology
evaluations and
sampling
RNA extraction
Phenol protocol
RNASeq
Tolerant
Susceptible
Michigan State University
Transcriptome
Analysis
12 plants of each
variety
T0 : control
T1 : 30 min after last water
T2 : 2 h and 30 min after last water
T3: Recovery
?????
?
Aeropony System:
Otazú, 2010 (Manual de producción de
semilla de papa usando aeroponía)
Fertilizante
Fórmula
g/l
Nitrato de Potasio
KNO3
Nitrato de amonio
NH4NO3
Sulfato de Potasio
K2SO4
0.087
Sulfato de Magnesio
MgSO4
0.247
Fosfato de Potasio
KH2PO4
0.136
Superfosfato triple de Calcio
Ca(H2PO4)2
0.117
Fetrilon Combi
Micronutrientes
0.101
0.08
0.0125
Diseño del Experimento
T0
Toma de
muestra
T1
Hoja
Toma de
muestra
Raíz
3 Réplicas
Las plantas
muestreadas se
descartan para el resto
del experimento
T2
Hoja
Toma de
muestra
Raíz
Las plantas
muestreadas se
descartan para el resto
del experimento
Hoja
Raíz
Las plantas
muestreadas se
descartan para el resto
del experimento
12 plantas
inciales por
cada
variedad
T3
Toma de
muestra
Hoja
Raíz
3 meses: Irrigación normal
Sequía: minutos
Sequía: horas
Recuperación
Sampling times
Photosynthetic rate
(PR)
T0 (Control)
T1(early
response)
T3(recovery
response)
100% PR
~80% PR
T2 (late
response)
~25%: tolerant
~50-60%:susceptible
~40%: tolerant
~80%:susceptible
watering
0
235
30
150
No watering
190
Time
(minutes)
watering
Physiological evaluations
CI-340 Handheld
Photosynthesis System
Variables Evaluadas
Conductancia Estomática
Tasa Fotosintética
Tasa de transpiración
Fluorescencia de clorofila
Physiological evaluations
RNA extraction (Phenol protocol)
Sample ID
S1 L
S1R
S2L
S2R
S3L
ng/ul
4559.63
2853.82
3042.89
3527.75
3875.48
260/280
1.98
2.12
2.05
2.08
2.02
260/230
2.01
2.32
2.05
2.27
2.1
S3R
S4L
S4R
S4R
S5R
S6L
S6R
S7L
S7R
S8L
S8R
S9L
S9R
S10L
S10R
S11L
S11R
S12L
S12R
R1L
R1R
R2L
R2R
R3L
R3R
R4L
R4R
R5L
R5R
R6L
R6R
R7L
R7R
R8L
R8R
R9L
R9R
R10L
R10R
R11L
R11R
R12L
R12R
3042.53
3824.2
2571.61
2832.37
2442.19
4728.81
3885.68
4698.38
4289.25
4145.04
4442.63
3612.81
2881.96
4745.45
3734.45
5077.26
5312.15
4807.11
4535.37
4879.14
3570.38
4416.55
3030.63
3017.72
3973.27
3487.54
2109.99
3217.71
2886.41
3256.3
3301.64
5271.98
1588.56
3986
2157.42
3803.6
1734.23
4604.69
4771.53
4872.81
3420.75
4694.86
2799.66
2.13
2.04
2.16
2.06
2.13
1.9
2.05
1.96
2
1.98
1.93
2.02
2.12
1.85
2.07
1.71
1.51
1.98
1.97
1.81
2.09
1.91
2.12
1.99
2.02
1.82
2.15
1.98
2.14
2.02
2.1
1.1
2.1
1.9
2.12
2
2.12
1.84
1.82
1.76
2.09
2.06
2.13
2.32
2.08
2.35
2.04
2.32
2.03
2.23
1.98
2.15
2.02
2.09
2.09
2.3
1.93
2.29
1.82
1.64
1.99
2.22
1.83
2.27
1.98
2.31
2.02
2.16
1.82
2.36
1.96
2.33
2.04
2.27
1.3
2.31
1.92
2.34
2.07
2.34
1.94
2.03
1.77
2.42
1.98
2.45
RNAseq analysis
50bp SE, HiSeq Illumina (4 June, 2012)
analysis
•
•
•
•
QC of the raw reads
Mapping to the potato genome
Assembly and quantification
QC for transcript abundance
– Determine the highest expressed genes for each tissue
type
– Check the functional annotation of the highly expressed
genes
– Number of expressed genes (inconsistent in the literature)
– Expression patterns of known genes
– Correlation between replicates
DM LEAVES
PGSC0003DMG400019584
PGSC0003DMG400024182
PGSC0003DMG400022241
Ribulose bisphosphate carboxylase small chain 1,
chloroplastic
Ribulose bisphosphate carboxylase small chain C,
chloroplastic
PGSC0003DMG400011530
Photosystem II 10 kDa polypeptide, chloroplastic
Glyceraldehyde-3-phosphate dehydrogenase A,
chloroplastic
PGSC0003DMG400041620
Plastocyanin, chloroplastic
PGSC0003DMG400019257
PGSC0003DMG400019149
Chloroplast thiazole biosynthetic protein
Ribulose bisphosphate carboxylase/oxygenase activase,
chloroplastic
PGSC0003DMG400000926
Oxygen-evolving enhancer protein 2, chloroplastic
PGSC0003DMG400013460
Chlorophyll a-b binding protein 3C, chloroplastic
PGSC0003DMG400013751
Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
NATIVE LEAVES
PGSC0003DMG400024182
Ribulose bisphosphate carboxylase small chain 1,
chloroplastic
Ribulose bisphosphate carboxylase small chain C,
chloroplastic
PGSC0003DMG400041620
Plastocyanin, chloroplastic
PGSC0003DMG400031495
PGSC0003DMG400019149
Senescence-associated protein
Ribulose bisphosphate carboxylase/oxygenase activase,
chloroplastic
PGSC0003DMG400022241
Photosystem II 10 kDa polypeptide, chloroplastic
PGSC0003DMG400000493
PGSC0003DMG400011530
Carbonic anhydrase
Glyceraldehyde-3-phosphate dehydrogenase A,
chloroplastic
PGSC0003DMG400011816
Photosystem I reaction centre PSI-D subunit
PGSC0003DMG400005890
16kDa membrane protein
PGSC0003DMG400019584
What we would like to see
• Correlation between replicates
and qc metrics
• Expression level distribution
• Groups (genes, transcripts, tss
or cds) that differ between
conditions (volcano plots)
• Heatmaps & Clustering
• Public database with all the
data. (maybe integration into a
genome browser)
-Bioinformatics-
IDENTIFICACIÓN DE miRNAs ASOCIADOS
AL ESTRÉS EN SEQUÍA EN PAPA
Olga Ponce Trabajo de tesis de Maestria
Diversity studies using Sequence
tagged sites (STS)
Studies of Metabolic pathways