This document proposes using an omics approach to elucidate the mechanism of Pashmina fibre development. The objectives are to 1) characterize Pashmina goats and fibres, 2) generate proteomic maps of fibres and follicles, 3) identify markers for yield and quality traits, 4) generate transcriptome maps, and 5) analyze data to identify Pashmina-specific markers. Experiments will profile goats, fibres, and follicles using phenomics, proteomics, transcriptomics, and bioinformatics. Outputs include proteome maps, marker genes for yield/quality, and reference databases to differentiate Pashmina fibre.

1. 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

2. • 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

3. Shawl
IndustryWeavers
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

4. 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??

5. 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

6. Status of Pashmina Production

7. 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

8. 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

9. 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

10. Rationale
Unique
transcriptome
and Proteome
profiles

11. 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 …….

12. 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

13. 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

14. 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 mapof 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

15. Experimental Design

16. Objective 1: Phenotypic Characterization
of Pashmina goat vis-à-vis fibre
Cold
Arid
Changth
ung
Tempera
te
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

17. 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
Developmentofhighresolution
proteomicmapofpashminafibre
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

18. 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

19. 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

20. 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

21. 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

22. FIELD
Cold Arid
(Changthang)
Temperate
(Kashmir)EXPERIMENTAL
SETUP
• 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 preparation
TruSeq Illumina kit
RNA seq data analysis
WETLAB
EXPERIMENTS
OUT
SOURCING
BIOINFORMATICS
SUPERCOMPUTING
Filter Reads
Quality & adapter contamination
Goat TranscriptomeRead Mapping
Estimate expressionlevel
Bowtie2, samtools
eXpress
Differential Gene
Expression
EdgeR
RNA sequencing
Illumia HISEQplatform
Read quantification
Reads aligned
Marker development SNPs and microsatellites
Work Plan for
transcriptomic
analysis

23. Annotation: Pipeline shall take a single FASTA file as input
and proceed to assign each sequence to one of the six
annotation classes

24. 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

25. 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

26. 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