Title of Invention


Abstract This invention relates to a primers for developing genotype specific marker in Piper nigrum a) 5'-ACCTCCTCTTTGTCACTCACTTC-3', Pngfi 5'-CAGCCTTCCGGACTTCACCAC-S' Pngr1, b) 5'- GCAGCGACAGCCAGCACA-3' Pncf2, 5'- CCATCGTACCATTCAATCATC-3 Pncn, Wherein the designing based on the flanking sequence of the micro satellite region using primer premier, Tsp 5091 adapters are used to bind the restriction sites created by the Tsp enzyme in DNA fragments, the said oligos are labeled with bio tin and the beads are coated in streptavidin which binds with the biotin protein and a magnet is used to separate the streptavidin bound oligo DNA from unbound.
This mvenbori relates to a method of developing microsatellite marker for pper
Genetic polymorphisms are fundamental tool for studying the rules governing
inheritance as wel as tracing specific genes in braedng programmes. Genetic
diversity re a key factor in sustaining agricultural productivity. In order to preserve
and make trite diversity available lor crop improvement tremendous efforts have
been made in the cotoetion, maintenance and classical characterization of
germptesm. Plant breeding and plant identification have been carried out by man
already for a very long time The registration and identification of varieties was
till now based on morphological and physiological characteristics. However the
phenotype is only partialy determined by the genetic information. The impact of
the genotype can be masked by environmental factors. By using molecular
identification techniques it becomes possible to detect randomly on the genome
located fragments. AH geneticaly determined differences between individuate
are a reflection of changes in DMA sequences These DMA changes can be
demonstrated in oWerent ways: dhrectty on the DMA or imfractty via the gene
products or metabolites. The ability of researchers to assess genetic variation by
examining difference* at the DNA level has enhanced the evaluation of these
genetic resources and increased their utility.
Piper nigrum L(black pepper) is one of the oldest and worlds most important
spice originated in the humid forests of Western Ghats of Peninsular India from
where it has spread throughout the tropics . The flavour and fragrance of this
oriental spice had a magic spett in human cultivation and culture since very
ancient days, India contributes about 18% of the world eiport of pepper (2000-
01). More than 100 ouMvart and 12 Improved varieties of black pepper wHh
Intrinsic quaMes are available in the country. Western Ghats of India is the
center of origin and diversity of Piper nigrum L. the black pepper of commerce.
Use of molecular markers In genome analysis, the systematic mapping of
agronomteatty important traits and marker assisted selection have been greatly
advanced by the development of reliable PCR-based markers. Among many
PCR based markers, microsateMtes are of particular utttty because they are
typically both co-dominant and mutiallelic in nature.
MioroeateMite molecular identification of cuHivars or individuals and consequently for solving
wrong synorryrnec and homonyms. A microsateWte or simple sequence repeat
(SSR) consists of direct tandem repeats of short (2-6) nudeotide motifs.
Interpretation of microsatelltte bands was shown to be relatively easy, and the
date could b« stored conveniently in the form of ateto sizes in base pairs.
Furthermore, microsateffite allow to reach discrete genetic information such as
pedgree and thus to trace back to very ancient crossing events. For wide range
of genetic and population studtes SSR markers are a suitable choice based on
coat, labour and genetic informs&venest. In principle, the mtcrosateHto data
obtained from one individual represent the mkrosataite profile of that cuttivar.
The results of mtcrosatelte analysis can be reproduced and compared to
different laboratories or at different periods, provided standardization is carefully
performed. Piper nigrum is a major spice crop species for which several
germplasm coleclion exists and for which molecular marker data is less
available DevalopinQ genotype specific markers is very much importance as far
as the improvement of plant is concerned.
CuKaryotlc genomes have been shown to be densely mterspersed with a dass of
repetitive elements, termed microsateliites or simple sequence repeats (SSRs),
that consist of short In-tandem-arranged repeat motifs of one to a few nudeotides
in length (Hamada et al. 1082; Tautz and Rente 1084). The existence of repeated
simple sequence motifs (e.g. CACACA...) in plant nudear DMA was
demonstrated by Defeeny et at, (1083), It was subsequently shown that simple
sequence (also caHed microsateWte) repeats were ubiquitous in most organisms,
including plant and organele genomes (Lagercrantz et al., 1903; Wang et al.,
1994), and that these sequences represented a major source of genetic variation
(Tautz et al., 1986) suitable for plant genetics (Morgante and Ofeieri, 1993). In
mammalian genomes, where mterosateffitas were first analysed, the most
frequently occurring repeat sequence is (CA/TG), (Hamada et al. 1982). In plants
DNA sequence database searches indicated mterosatetttes to be less abundant
compared to mammals with (A), and (AT), the most prevalent repeat motif
followed by (GA/TC) n (Lagercrantz et al. 1993; Morgante and ONvieri 1993;
Wang et al. 1994), At many microsateflite loci, a high frequency of variation in the
number of repeat unite has been observed (Tautz et al. 1986). These length
variants can easily be analysed by polymerate chain reaction using primer pairs
specific to unique sequences flanking individual loci (Weber and May 1989; Utt
and Luty 1969), making microsatelites an abundant source of highly
polymorphic and co-dominant genetic markers. Me Couch and coworkerc (1997)
estimated that 5700 to 10,000 mkrosaAeNite repeat loci and present in the
relatively •mal rice genome. Many of these loci are incorporated into a high
density rnicrosatelet marker map for rice (Akagi et al., 1996; Chen et al) SSR
markers have been developed in a number of plant species and have been
shown to be useful in genetic mapping (Uu et al. 1996), variety identification
(Rongwen et al 1995) or the analysis of genetic variation (Raschke et al. 1995)
studying population genetics(Haymer, 1994;Tsumura et el., 1998; Thomas et al.,
1999) atoeMmsnt of parentage and dnpersalfDow et al,,1995), MiuuualsNils
markers were developed in many crop plants (hodgetts et al., 2001,Rudoph et al
1995, Liu et al., 2001, He et al.,2003). But in Piper ragrum no such efforts were
initiated and this is the first report where at least specific primers were designed
for the microsatelUte regions.
An object of this invention is to propose a primer for microsateMte region of piper
Another object of this invention is to propose a primer for mterosateffite region of
piper nigum for developing a marker.
Still another object of this invention is to propose a primer for rnicrosateHte
region of piper nigum which are detect high fevets of genetic dwersity and an
economically assayed by PCR.
Further object of this invention is to propose a primer for nmcrosataffita region of
piper nigum for the extent of genetic dmsrentiation within and between natural
Still further object of this invention is to propose a inethod for designing a primer
for microsateito region of piper nigum.
According to this invention there is provided a primer for microsatelto region of
piper nigum.
According to this invention there is also provided a method for designing the
process for microsateite region of piper nigum comprising:
isolating DMA from the plant;
subjecting the said DMA to the step of digestion,
adding an adapter to both ends of the DMA fragments;
amplifying the said adapter figated DMA using Tsp primers;
subjecting the amplified ONA to the step of hybridization,
enriching the hybridization DNA fragments for microsateWte repeats;
subjecting the enriched DNA fragments to the step of tailing to make the Kgation
of the fragments into pGEMT vector;
cloning into pGEMT vector to select transformed colonies;
sequencing the clones to design primers complementary to the flanking regions
of the microsatellrte repeats.
India is the land of spices. King of spice, the black pepper (Piper nigrum L) is the
oldest and best known spices in the world and it continues to be the most widely
used spice in the world today. The monsoon forests of the Malabar coast in the
Southwest India are home to this aromatic and pungent seasoning and the
region continues to produce the highest qualities of pepper for export.
Developing molecular signature is very much necessary for cataloguing and
identification of natural germptasm and wNI also help in legal matters concerning
the impending implications of Plant varietal Rights as per the convention on
Biological Diversity (CBT), QATT and WTO treaty.
Plant material used for the study is Piper nigrum cv. Karimunda which is grown in
the garden of Rajiv Gandhi center for Biotechnology. The detailed method of
developing microsateMite markers is described.
1. DNA isolation
DMA was isolated from the plant using Gen elutc from Sigma.
2. Digesting DNA
The first step in constructing a mferosatelte library is to use restriction enzymes
to cut up genomic DNA into fragments ranging in size from 200 to 600 base pairs
(bp). A rare cutter enzyme, Tsp5091 is used for this digestion.
1. Digestion was set up as follows:
water -36ul
10 x Buffer -5ul
GenomicDNA -7ul
Tsp 5091 -2ul
About 100ug of genomic DMA was Digested with Tsp 5091 (NEB) and separated
by eiectrophoresis in 2% agarose. Fragments between 200 bp and 600 bp were
excised and purified using GFX purification kit (Amersham).
2. Adapter ligation:
After digesting DMA, the next step is adding an adapter to both ends of the DMA
fragments. An adapter is just a piece of double stranded DMA that are designed
for tagging the fragments. Since the sequence of the fragments is unknown, by
adding a known sequence, we can use PCR to ampffy the fragment Here we
are using Tsp 5091 adapters, which w* be btaNng to the restriction sites created
by the Tsp enzyme. About 30ug of size fractionated DMA was fgated to 20 ug of
5' -end phosphorytoted Tsp 5091 adapter (consisting of 17-mer 5'-GGT
100 ul with 10 units of T4 ONA iigase.
The ligation was done at 16°C overnight for 16 hours.
3. Amplifying the adapter ttgated ONA using Tsp primers.
Water -35ul 94°C-4min
Tsp primer-2ul 94°C-30s
dNTP mix-2ul 55°C-30s
10xBuffer-5ul 72°C-120s
Adaptor ligated DNA-5ul 72°C-10min
Taq-1ul 35cydes.
The samples were again purified using GFX purification kit
4. Hybridisation with biotynyiated oKgo probes
During hybridization the btotynytoted otigo repeats bind to the complementary
sequence In the DMA Hybridisation was performed at 50°C overnight having a
total of 10Oul rxn as follows:
Denatured snap cooled per product-6ul
Biotylated oligo probe-2ul
0.5 M sodium phosphate-SOul
0.5% SDS-5ul
5. Enriching for mterosatdtte repeats
The oKgos are 3' labeled with btotin. The beads are coated in streptavkin which
strongly binds wNh the Wotin protein on our oNgo. Al DNA fragn^nts not attached
to a biotinylated ofigo are washed away a magnet is used to separata the
streptavidin bound ofigo DNA from the unbound DMA.
a. Race the tube in magnet and pipet off nybrkJrartwn sedition
b. Washed twice for Srmm with 200ul at room temperature 2Xssc 0.1 % SOS
c. Washed twice for Smin with 200ul at 45'C 1XSSC.0.1H SOS
d. Washed twice for Smin with 200ulat60°C IxSSC01% SOS
e. ElutedthegertomteDNAoourKitoOlgotyad
AmpMfy repeat enriched DMA. This makes the single stranded edition double
stranded and will increase the amount.
PCR were set up with hot start amplification.
6. Tailing of PCR products
The enriched DNA fragments were tailed with dATP inorder to make the Ugation
of the fragments into pGEMT vector. About 0 2mM dATP and 0.2 units of Taq
was used.
7. Cloning into pGEMT vector
The tailed products after purification was Kgated into pGEMT vector, later
transformed into JM108 competent cells. After blue white selection the
transformed colonies were selected.
8. Plasmid isolation
Plasmids were isolated from the transformed colonies using alkaline lysis method
9. Sequencing
The clones obtained were sequenced so as to design primers complementary to
the flanking regions of the microsatellite repeats.
1 0 . designing of primers
Primers were designed based on the flanking sequence of the microsateUrte
region using Primer premier.
The designed primers were tried in order to find out its working efficiency and
found that it is wording very well.

We Claim
1. Primers for developing genotype specific marker in Piper nigrum
Wherein the designing based on the flanking sequence of the micro satellite region using primer premier, Tsp 5091 adapters are used to bind the restriction sites created by the Tsp enzyme in DNA fragments, the said oligos are labeled with biotin and the beads are coated in streptavidin which binds with the biotin protein and a magnet is used to separate the streptavidin bound oligo DNA from unbound.
2. Primers for developing genotype specific marker in Piper nigrum as claimed in claim 1, wherein Tsp 5091 adapter consisting of 17-mer 5'-GGTCTACTGGACTCACT-3 and 21-mer 5'-pAATTAGTGAGTCCAGTA GACC-3').
3. Primers for developing genotype specific marker in Piper nigrum as claimed in claim 1, wherein during hybridization the biorynylated oligo repeats bind to the complementary sequence in the DNA.
4. Primers for developing genotype specific marker in Piper nigrum as claimed in claim 1, wherein the oligos are 3' labeled with biotin.
5. Primers for developing genotype specific marker in Piper nigrum as described and illustrated herein.








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Patent Number 237969
Indian Patent Application Number 3377/DEL/2005
PG Journal Number 4/2010
Publication Date 22-Jan-2010
Grant Date 15-Jan-2010
Date of Filing 15-Dec-2005
Applicant Address BLOCK-2, C.G.O. COMPLEX, LODI ROAD, NEW DELHI-110003
# Inventor's Name Inventor's Address
PCT International Classification Number C12N 15/09
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA