national fund project on pashmina goat
TRANSCRIPT
Elucidating the mechanism of
Pashmina fibre development:
An OMICS approach
Lead Institute: SKUAST-Kashmir
Dr Nazir A Ganai
Cooperating Centre: NDRI, Karnal Dr Jai Kumar Kaushik
IASRI
Dr A R Rao
Concept Note 8467
• Background : How is this problem important to Indian Agriculture
– Role in Livelihood security and economy
– As a unique research Model
– Status of Pashmina Production
• Problem Statement – What is not known about the fibre
– Hypothesis
– Rationale / Long term Goal
• Objectives
• Experimental Set up
• Activities / output
• Outcome
• Budget
Shawl
Industry
Weavers
Changpas
Shawl Industry
• 50,000 shawls a year
• 125 million dollars a year
2 lac goats
50 tons raw pashmina
Women
Empowerment
15000 families
Livelihood Security
21,000 Changpas
Sustains Art
12,000 weavers
Cashmere
Swawl
GI
Scarce
vegetation
-40OC
Extreme
Cold
High
Altitude
• Altitude: 11 k-15 K ft
• Low O2 Tension
• Low Hb level ??
• Vegetation- rich in essential
elements ?
• Efficient Rumen micro-flora ?
• Evolved Metabolic System??
• Climate resillence
• Reduced Metabolic rate?
• Anti-freeze proteins
• Double Hair Coat?
• Outer – Guard fibre
• Inner - Pashmina
• Annual Cycling??
• Baldness??
Pashmina – a Speciality Fibre
– Produced globally , but Traded as Cashmere (GI)
– Costliest commercial animal fibre
• One Pashmina shawl Costs: Rs 30,000 to 1 lac
– Finest of all commercial natural fibres: • Pashmina Fibre: 12-14 μ m
• Cashmere Fibre: < 19 μ m
• Merino Wool : 17-21 μ m
– Texture: (Structural properties)
• Extremely soft, lightweight, and warm
• Very luxurious and
possesses excellent drape.
• Uniform fibre diameter • Cuticle:
• Unique scale pattern- • smooth, flat, regular,
ladder type • Cortex :
• ortho- and meso- cells more
• Para-cuticular cells less
Status of Pashmina Production
Status of Pashmina Production
India
Goat Breed
Region / State Pashmina Goat Population
Pashmina Production (Kg)
Yield / annum
Pashmina (Changthungi
Ladakh (J & K)
~ 1,60,000 45,000 240 gm/ animal/year
Chegu
Lahul & Spiti, Kinnur (HP)
Uttranchal
~ 12,000 1500 50-100 gm
What is not Known about the Pashmina Fibre
Question Mechanism What makes it so special?? Specific Protein profile not known
How does it cycle annually?? Pashmina grows and sheds naturally every year – mechanism is obscure.
Why Guard hairs donot shed?
Production in other climates? Horizontal spread of Pashmina Goat in other climatic zones
Ave. yield (~ 200 gms) is too low. How to breed this goat for sustainability ?
Genetic control of the yield and quality of fibre is not well known,
Why is Pashmina fibre finest of all Cashmere fibres ??
Is it hunger fineness or genetically determined??
How to test adulteration to avoid false trade??
Pashmina specific markers, required for
differentiating it from other fibres are not known.
“Cashmere Shawl” is a geographical indication of Kashmir (India).
Genetic profile need to be deciphered to protect this belonging
Hypothesis
The unique properties of the pashmina fibre
(fineness and texture, cyclicity)
are due to
specific molecular determinants
(relative composition and abundance
of fibre proteins like KPs, KAPs etc )
which need to be elucidated
through its proteome and transcriptome study
Rationale
Unique transcriptome and Proteome
profiles
Elucidation of the molecular determinants shall therefore help to:
• Gain insights into growth and typical annual cyclicity
of fibre.
• Molecular assisted breeding of Pashmina goat to break the fatigue in productivity;
• Help in differentiation of pashmina fibre from other fibres to avoid the false marketing practices,
• Help to protect the “Cashmere” – the geographical indication belonging to Kashmir, India
Rationale …….
Status of Current Research
• International Goat Genome Consortium- 2010 (www.goatgenome.org)
• First Goat Genome Sequence (~2.66-Gb genome sequence)- 2013 (http://goat.kiz.ac.cn/GGD/)
• 50K SNP Chip (http://snp.toulouse.inra.fr/~sigenae/50K_goat_snp_chip/)
• Differential expression studies:
– 6300 transcripts in the hair follicles -2013 – 1332 differentially expressed genes -2013
• Genes for Fibre Quality Prolactin receptor gene; KRT1.2 and KAP1.3, hemoglobin B allele 13 coat color genes in sheep
Proteomics of the fibre
• Proteins, which make up 90–95% -1997
keratin proteins (KPs)
kertin associated proteins (KAPs)
• Protein expression and quality traits of - 2011 Animal fibres, human hair and the wool
• Human hair - 343 proteins- 2006
• Wool- 8 major keratin Proteins- 2010
• Gene KAP5.1 - intrinsic strength
• Gene KAP6.1 - Load-bearing capacity/extensibility of wool
• Cashmere of Korean goats- 2004
Objectives 1. Phenotypic characterization of the pashmina goats for fibre
related traits under cold arid desert and temperate environments
2. Generate the proteomic map of Pashmina fibre and of follicles to
understand growth phases of the fibre development
3. Identify the markers for the yield and quality traits of fibre by
comparative proteome profiling under varying climatic conditions
4. Generate the transcriptome map of Pashmina hair follicles
to decipher the genetic control of fibre growth
5. Comparative analysis of the transcriptomic data for
identification of Pashmina fibre specific marker genes
Experimental Design
Objective 1: Phenotypic Characterization of Pashmina goat vis-à-vis fibre
Cold Arid Changthung
Temperate Kashmir
Growth and fibre related traits
Biochemical and hormone assays
Electron microscopy for ultra-structure of fibre & follicles
Histological assays of SKIN for S:P ratio
Possible phenotypic markers like • Biochemical • Hormonal • S:P ratio
linked to the yield / quality traits
Contrast Activities Out put
Objective 2 Generate the proteomic map of Pashmina fibre and follicles
to understand growth phases of the fibre development
Standardization of fibre sampling method,
protein extraction, solubilization and
sample preparation of pashmina fibre for
proteomic analysis
Generating the comprehensive proteomic
map of Pashmina fibres by combining gel
and non-gel proteomics approaches
Identification of novel isoforms and
variants of KPs and KAPs by combining the
protein sequence data (MS/MS) with
transcript sequence in Pashmina goat
1
Develo
pm
ent o
f hig
h re
solu
tion
pro
teo
mic
map
of p
ashm
ina fib
re
Activity Outputs
1
2
3
3
Protein Isolation
2D-gel electrophoresis
m/z spectra/peptide
mass fingerprints
Follicle proteins
identification
Proteins identification
Peptide fingerprints (m/z)
atching
with cDNA/RNA sequences
Deliverables
Activity Outputs
Comparative proteome analysis of pashmina fibre under cold arid and temperate environments
Putative protein markers of Pashmina fibre quality and yield. Protein networks/
interactome
regulating growth and
development of fibre The discovery of biomarkers would help in breeding program of pashmia goat for higher productivity (outcome)
Comparative proteomic analysis of fibre of flocks with varying fibre quality traits
Analysis of transcript and proteomic data for correlation with yield and quality traits
Objective 3: Identifying the markers for the yield and
quality traits of fibre by comparative
proteome profiling under varying climatic
conditions
1
1
2
2
3
Removal of various kind of fibres and follicles
Solubilization and extraction of fibre proteins
Sample cleanup and prefractionation of fibre/follicle proteins
Trypsin digestion of proteins
LC-ESI-MS/MS
Analysis of m/z spectra and identification of proteins using protein
and RNA/DNA sequence data
Comparative Proteome of pashmina fibre development
Anagen
Catagen
Telogen
Proteome Map of Pashmina Fibre
High Yield Low Yield
Sample cleanup and prefractionation of fibre/follicle proteins
Labelling of peptides Labelling of peptides
Pool the samples
Trypsin digestion of proteins
LC-ESI-MS/MS
Identification and quantitative analysis of differentially expressed proteins
Temp 1 Temp 2
Objective 4: Study transcriptomic profiles in Pashmina follicles
during cycling stages in low and high yielding animals
Activity Outputs
Tissue sampling (Skin / hair) from different experimental groups for RNA isolation and construction of cDNA libraries
• Annotated
Transcriptome map
SNP database,
Splice variants,
Novel transcripts
Generate transcript profiles from follicles
at different stages of growth in high and low
yielding animals under contrasting
environments
Annotation for Identification of novel transcripts, alternate splice variants, SNP and SSR markers associated with the fibre traits of experimental groups
1
1
2
2
3
Objective 5: Comparative analysis of the transcriptomic data for
identification of Pashmina fibre specific marker genes
Activity Outputs Differential Expression Analysis at three stages of
fibre growth
• Candidate gene
list influencing
yield and quality
traits
Annotation for Identification of novel transcripts,
alternate splice variants, SNP and SSR markers
associated with the fibre traits of experimental
groups
Validation of differential expression of candidate
genes through qRT-PCR analysis
Correlation of proteomic and transcriptomic
data to verify the marker genes
1
1
2
2
3
FIEL
D Cold Arid(Changthang)
Temperate(Kashmir)
EXP
ERIM
ENTA
LSE
TUP
• Cycling stages
• Yeild (High/Low)
• Length (Short/Long)
• Colour (White/Black)
•Genetic Groups(Pashmina , Non Pashmina, Xbred)
• Cycling stages
• Yeild (High/Low)
• Length (Short/Long)
FastQC, Trim Glore! FLASH
RNA Extraction TRIZOL/RNeasy cleanup Kit Qiagen
cDNA library preparationTruSeq Illumina kit
RNA seq data analysis
WET
LA
B
EXP
ERIM
ENTS
OU
T SO
UR
CIN
G
BIO
INFO
RM
ATI
CS
SUP
ER C
OM
PU
TIN
G
Filter ReadsQuality & adapter contamination
Goat TranscriptomeRead Mapping
Estimate expression levelBowtie2, samtoolseXpress
Differential GeneExpression
EdgeR
RNA sequencing Illumia HISEQ platform
Read quantification
Reads aligned
Marker development SNPs and microsatellites
Work Plan for transcriptomic
analysis
Annotation: Pipeline shall take a single FASTA file as input
and proceed to assign each sequence to one of the six
annotation classes
Work Distribution Phenomics Proteomics Transcriptomics Bioinformatics
Experimental animal groups Phenotyping Biochemical Hormonal Histology for S:P ration Micrography fibres
Proteome map of fibre Comparative proteome analysis Biomarker for differentiating fibres
cDNA libraries RNA sequencing qRT-PCR Validation of candidate genes
Transcriptome map DGE analysis for identification of candidate genes Annotations Correlation of transcriptome and proteome for marker identification
SKUAST-K NDRI Karnal SKUAST-K IASRI, SKUAST-K
Budget Head SKUAST-K NDRI IASRI
a) Non-Recurring
Civil works 15 5 0
Goods / equipments 77 25 11
Capacity building 21 7 10
b) Recurring
186 93 22
c) Institutional charges 15 5 1
Total 315 134 44
Expected Outputs • Structural differences between pashmina and non-pashmina fibres
• Proteome map of the Pashmina fibre and its dynamics through various follicular growth stages
• Relative composition of KPs and KAPs of Pashmina fibre which make the fibres qualitatively different.
• Unique peptide mass fingerprint – for differentiation from other fibres
• Reference data bases of pashmina fibre:
– Proteome
– Transcriptome
– SNP
• Marker genes for yield / quality
– Candidate genes,
– Novel transcripts
– alternate splice variants