Molecular Plant Breeding 2016, Vol.7, No.23, 1
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These unique bands specific to different genotypes can serve as potential diagnostic markers in identification and
differentiation of genotypes and therefore may be of immense importance in Intellectual Property Right (IPR)
issues related to Plant Variety Protection and Farmer’s Right (PVP & FR) Act.
Efficiency of markers and their utility in terms of polymorphism and quantitative estimation could be expressed in
mean heterozygosity and marker index (Choudhury et al., 2007). The average Hav, (Hav)
p
and MI were found to
be 0.579, 0.124 and 0.640, respectively. The minimum (0.16) and maximum (0.88) PIC value were found with
markers UBC-861 and UBC-824, while the lowest (0.056) and highest (0.490) heterozygosity were found with
markers UBC- 815 and UBC-858 respectively. The average genetic distance coefficient value among the all
genotypes was 0.31 based on ISSR markers, which indicated a limited degree of genetic variation in the lentil
material. The genotype LL147 and L4147 were found to have maximum similarity (84%) which was closely
followed by L4147 and VL103 (82.9%). The lowest similarity was found between T-36 and Precoz (37%) which
was followed by IPL525 and T36 (40.9%) The average similarity was found to be 69.32 %.
The information on genetic diversity among these lentil cultivars will be helpful to lentil breeders in selection of
appropriate hybridizing parents in developing superior cultivars.
3 Materials and Methods
3.1 Plant materials
The experimental material comprised of 25 lentil genotypes from different lentil growing states of India and other
countries (Table 2).All seed material for this study was obtained from the germplasm unit of Indian Institute of
Pulses Research, Kanpur (India).
3.2 DNA Isolation and PCR amplification
Seeds of 25 lentil genotypes were germinated under etiolated conditions on paper towel soaked in sterilized water.
One-week-old seedlings were ground in preheated CTAB buffer and incubated at 60
℃
for 1 h. The aqueous
phase containing DNA was separated using chloroform: isoamyl alcohol (24: 1) (Abdelnoor et al., 1995). The
DNA was precipitated with chilled iso-propanol and the pellet was dissolved in 100 µl of T
10
E
1
buffer. The RNA
was eliminated by adding 0.5 U of RNAse. DNA concentrations were quantified by measuring absorbance using
Hoefer® Dyna Quant® 200 DNA fluorometer (Amersham Biosciences, Piscataway, NJ, USA) and stocks were
maintained at 25 ng/μl.
One hundred ISSR markers of the UBC series were used to study genetic similarity in 25 lentil genotypes. PCR
reaction mixture (20 μl) consisted of 20 ng of template DNA, 1X PCR buffer, dNTPs (Banglore Genei, Bangalore)
2.5 nM each, 10 pM primerand 0.6 U of
Taq
DNA polymerase (Banglore Genei, Bangalore). The thermal cycling
program was carried out in a PTC 200 thermal cycler (MJ Research, Biorad). The PCR program had an initial
denaturation step at 94
℃
for 2 min, followed by 41 cycles of denaturation at 94
℃
for 1 min, primer
annealing(depending upon the Tm of respective marker) for 1 min and DNA extension at 72
℃
for 3 min. A final
extension step was given at 72
℃
for 4 min. The amplified DNA fragments were resolved on ethidium bromide
stained agarose gel (2%) in 1X TAE buffer at 50 V. A 100 bp DNA ladder (MBI Fermentas) was used as a
molecular weight marker for determining the molecular weight of the amplified products.
3.3 Scoring and Data analysis
The amplification profiles of each marker in different genotypes were scored and recorded as presence [1] or
absence [0] of bands and binary quantitative data matrix was constructed. Unique alleles were defined as
thosedetected in only one genotype. The presence and absence of alleles for each marker was recorded for all
genotypes and then converted into genetic similarity matrix using Jaccard (1908) similarity coefficient in
NTSYS-PC 2.1 software. (Rohlf, 1998). The similarity coefficients were used to construct a dendrogram depicting
genetic relationship using unweighted pair group mean average (UPGMA) method (Sneath and Sokal, 1973). The
Polymorphism Information Content (PIC) values were calculated following the formula described by Botstein et
al. (1980):
