Page 8 - Molecular Plant Breeding

Molecular Plant Breeding 2014, Vol.5, No.3, 10-17
http://mpb.biopublisher.ca
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Table 2 Genotypes used for diversity analysis along with their pedigree
S.No. Genotypes Species Pedigree
1 RB- 55 Brassica juncea Local selection
2 RSPR- 01 Brassica juncea B.juncea×D.muralis
3 Pusa Bold Brassica juncea Varuna×BIC 1780
4 RSPR- 03 Brassica juncea Kranti×Pusa Bold
5 Varuna Brassica juncea Selection from Varanasi Local 786,02.021976
6 RL-1359 Brassica juncea RLM-514×Varuna
7 PusaAgrani Brassica juncea Early maturing Brassicajuncea×synthetic amphidiploids (B.campestris var. toria×B. nigra
8 PusaTarak Brassica juncea SEJ-8×PusaJagannath
9 Urvashi Brassica juncea Varuna×Kranti
10 Kranti Brassica juncea Selection from Varuna
11 RB- 24 Brassica juncea Spontaneous mutant of cultivar RH-30
12 RSPR- 69 Brassica juncea RLM 198×Varuna
13 NPJ- 112 Brassica juncea Agra Local×Purvi Raya
14 Nov-Gold Brassica juncea Bio- 902×BM- 185- 11
15 NRCDR- 2 Brassica juncea MDOC-43×NBPGR -36
16 RB- 50 Brassica juncea Laxmi×RH- 9617
17 BR- 24 Brassica juncea -
18 RH -30 Brassica juncea Selection from P26/3- 1
19 RH -0406 Brassica juncea RH 6908×RH 8812
20 RH -0749 Brassica juncea RH 781×RH 9617
21 RH -0119 Brassica juncea Pusa Bold×Rajat (PCR- 7)
22 GZ- 5 Brassica juncea -
23 CS- 56 Brassica juncea RH- 851×Pusa Bold
24 RSPT-1 Brassica campestris Mass selection
25 RSPN -28 Brassica napus Selection from Jupiter
26 DGS- 1 Brassica napus Selection from exotic collection
27 CCS- 08 Brassica oleracea Local selection
Genomic DNA Isolation and Quantification
The genomic DNA was isolated from 7-8 cm young
and actively growing fresh leaves using Doyle and
Doyle (1990) method with slight modifications. Leaf
material was grinded to fine powder in liquid N2,
transferred to 1ml of pre-warmed (at 65ºC) extraction
buffer and incubated for 35 minutes. An equal volume
of Chloroform: Isoamylalcohol (24:1) was added to
the tube, tilted for 10 minutes and centrifuged for 15
minutes at 8,000 rpm. Supernatant was collected to
which, 0.6 volume of ice-cold isopropanol was added
and stored at -200C for 3-4 hours. Centrifugation was
done at 10,000 rpm for 10 minutes at 40C and pellets
were purified with 0.01M ammonium acetate (200µl
-300µl). The pellet was washed twice with 70%
chilled ethanol, air dried, dissolved in 300µl TE (10
mM Tris-Cl, 1 mM EDTA pH 8.0) buffer, purified
with 3µl of RNase (10mg/ml) and finally stored at
-200C for further use. The amount and quality of DNA
was confirmed using Nanodrop (mySPEC, Wilmington,
USA). Finally the DNA was diluted to 25ng/µl
concentration for PCR amplification.
RAPD and SSR Assay
A set of 15 arbitrary random 10-mer primers and 3
EST-SSR primers (detailed in Table 3) were used in
the present investigation. The RAPD primers were
diluted to 5 pmol concentration and final concentration
of 25 pmol was used per reaction whereas the final
concentration of SSR primers were set to 15 pmol for
carrying out PCR reaction. DNA amplification was
carried out in PCR tubes containing 25 µl reaction
mixtures. Reaction mixture contained 2.5 µl of
template DNA (25 ng/µl), 2.5 µl of 10× PCR Buffer,
MgCl 2 (2 mM), 0.2mM of each dNTPs (dTTPs,
dGTPs, dCTPs, dATPs), primer (5pmol) concentration
with 1 U Taq polymerase per reaction. For SSR primers,