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