Gene 549 (2014) 171–178

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Diversity analysis in Indian genotypes of linseed (Linum usitatissimum L.)

using AFLP markers
Chandrawati a,b, Ramanuj Maurya a, P.K. Singh c, S.A. Ranade a,b, Hemant Kumar Yadav a,b,⁎
a
CSIR—National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India
b
Academy of Scientific and Innovative Research (AcSIR), New Delhi, India
c
AICRP on Linseed, CSAUA&T, Kanpur, India

a r t i c l e i n f o a b s t r a c t

Article history: AFLP fingerprinting of 45 Indian genotypes of linseed was carried out to determine the genetic relationship
Received 27 February 2014 among them. Sixteen primer combinations produced 1142 fragments with 1129 as polymorphic and 13 as mono-
Received in revised form 25 July 2014 morphic fragments. Polymorphic fragments varied from 44 (E-ACA/M-CTA) to 94 (E-AGC/M-CAC) with an aver-
Accepted 28 July 2014
age of 70.6 fragments per primer combination. The frequency of polymorphism varied from 93.7% to 100% with
Available online 30 July 2014
an average of 98.8% across all the genotypes. The PIC value ranged from 0.19 to 0.31 with an average of 0.23 per
Keywords:
primer combination. The primer pair E-AGC/M-CAC showed the maximum PIC value (0.31) followed by E-AGC/
AFLP M-CAG (0.29), E-AAC/M-CAG (0.26) and E-AGC/M-CTA (0.25). Resolving power (RP) and marker index (MI) var-
Flax ied from 13.73 to 43.50 and 8.81 to 28.91 respectively. The Jaccard's similarity coefficient varied from 0.16 to 0.57
Genetic diversity with an average of 0.26 ± 0.05. The maximum genetic similarities (57%) were detected between genotypes Him
Jaccard's coefficient Alsi-1 and Him Alsi-2, followed by Him Alsi-1 and GS41 and GS41 and LC-54. The genotypes R-552, Himani, RKY-
Polymorphism information content 14, Meera, Indira Alsi-32 and Suyog were found to be more divergent genotypes. The NJ clustering grouped all the
45 genotypes into three major clusters. In general the genotypes of cluster III had high oil content and those of
cluster I had low oil content. At the population level, within population variance was much higher than between
populations variance.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Linseed or flax (Linum usitatissimum) is a diploid (2n = 30, genome
size ~370 Mb), self pollinated annual plant (Ragupathy et al., 2011). It
has been under cultivation for its seed oil (linseed) or stem fibers
(flax) or both (dual purpose) for 1000 years (Dillman, 1953; Zohary,
1999). The center of origin of cultivated flax is believed to be the Middle
East, although secondary diversity centers were identified in the Medi-
terranean basin, Ethiopia, Central Asia, and India (Vavilov, 1926; Zohary
and Hopf, 2000). Globally, the important flaxseed–linseed cultivating
and producing countries include Canada, India, China, the United
Kingdom, Ethiopia, and the United States of America. In India, it is
mainly grown as an industrial oil seed crop in marginal soils and
rainfed conditions. Linseed is an important industrial crop plant as its
oil with high linolenic acid content (45–65%) is used for manufacturing
rapidly drying paints, stains, inks, varnishes and the polymer linoleum
Abbreviations: AFLP, amplified fragment length polymorphism; PIC, polymorphism in-
formation content; RP, resolving power; MI, marker index; EMR, effective multiple ratio;
NJ, neighbor joining.
⁎ Corresponding author at: CSIR—National Botanical Research Institute, Rana Pratap
Marg, Lucknow 226001, India.
E-mail address: h.yadav@nbri.res.in (H.K. Yadav).
(Rowland, 1998). Besides its oil, the linseed fibers (phloem fiber) are
used by industries for producing high quality linen fabrics, pulp, biofuel
(Diederichsen and Ulrich, 2009), raw materials of thermal insulations
(Kymalainen and Sjoberg, 2008) and bioplastics (Kwiatkowska et al.,
2009). Flax seeds also contain lignans which are reported to help in re-
ducing the risk of certain types of cancer (Westcott and Muir, 2003).
However, the high level of linolenic acid in the oil causes it to be unsuit-
able for use in edible products because of undesirable odors and flavor
that result from the auto-oxidation of this unsaturated fatty acid (Green,
1986). Efforts have been made to develop edible purpose linseed by re-
ducing the level of linolenic acid content to b5% and several varieties
with low linolenic acid have been developed and released for commercial
cultivation in Canada and Australia (Dribnenki et al., 1999, 2004; Green
and Marshall, 1984; Rowland, 1991). Recently, efforts are being made to
breed and develop dual purpose linseed varieties which could serve
both high quality fiber and oil. The availability and knowledge about the
extent of genetic diversity of genetic resource material play an important
role in identifying parental lines and developing new varieties with desir-
able traits.
Morphological trait-based diversity assessment has been widely used
in crop plants including linseed (Diederichsen, 2001; Diederichsen and
Raney, 2006; Saeidi, 2008); however the morphological characters are
not only sensitive to environmental factors but they also require labor

http://dx.doi.org/10.1016/j.gene.2014.07.067
0378-1119/© 2014 Elsevier B.V. All rights reserved.
172 Chandrawati et al. / Gene 549 (2014) 171–178

intensive field evaluation over long periods of time. On the other hand Table 1
DNA based molecular markers including RAPD, AFLP, SSRs and SNPs Details of genotypes used in the present investigation.

have several advantages like abundance, environment independent Sl. Variety Pedigree Oil content 1000 seed
early and rapid evaluation and non-tissue specific characteristics. As no. (%) weight (g)
in other plants, molecular markers have also been utilized by several re- 1. Shikha Hira × Crista 42.0 7.2
searchers to assess the genetic diversity in different sets of linseed 2 Neelum T-1 × NP (RR)-9 43.0 9.6
varieties/genotypes (Adugna et al., 2006; Braulio et al., 2012; Everaert 3. Rashmi Gaurav × Janki 41.4 6.6
4. Parvati [(EC41628 × EC77959) × 41.8 6.9
et al., 2001; Fu et al., 2003; Rajwade et al., 2010; Smykal et al., 2011;
(DPL 20 × Neelum)] ×
Uysal et al., 2010). Of all these studies only Rajwade et al. (2010) have [(216 × Hira) ×
assessed the diversity among 70 Indian genotypes of linseed by ISSR (BR1 × NP 440)]
method. More significantly, their study is confined to repetitive and 5. Sweta Mukta × T-1206 43.7 6.2
ISSR regions and is not truly reflective of the entire genomic coverage 6. Sheela Gaurav × Janki 40.6 6.6
7. Garima T-29 × Neelum 42.0 6.5
for the assessment of diversity. It is well known that, among the various 8. Shekhar Laxmi-27 × EC 1387 42.0 5.3
molecular marker systems AFLP offers a genome wide DNA profiling 9. Laxmi-27 [(Neelum × R1) × (Neelum × 45.0 8.2
and is thus recommended for molecular characterization and fingerprint- NPRR-9)] × [(Neelum × R10) ×
ing of germplasm lines (Azhaguvel et al., 2006) The present investigation (Neelum × Afg-8)]
10. Shubhra Mukta × K 2 44.4 8.1
was therefore undertaken to characterize and evaluate the level of genetic
11. Gaurav Sel. 3 × EC-1552 43.0 6.0
diversity among some of the prominent Indian genotypes of linseed 12. Sharda (Shubhra × J1) × (J1 × Kiran) 41.1 7.2
through AFLP analysis. 13. Azad Alsi-1 RL 904 × Kiran 39.9 4.8
14. Hira H 342 × NP (RR)-9 42.5 8.2
2. Materials and methods 15. C-429 No. 3 × IP-135 44.0 5.3
16. Pusa-3 K-2 × T-603 45.0 6.4
17. Jawahar-7 Selection of no. 55 37.7 9.2
2.1. Plant materials and DNA isolation 18. Jawahar-23 EC9832 × Hira 44.0 6.6
19. JLS-9 (RL-102 × R-7) × J 23 42.0 8.9
The plant material used for the present investigation consisted of 45 20. Suyog (Kiran × KL-168) × Kiran 41.4 8.0
21. Kartika Kiran × LCK-88062 42.9 5.7
genotypes obtained from All India Co-ordinated Research Program on
22. Indira Alsi-32 Kiran × RLC 29 39.2 6.0
Linseed (AICRP on Linseed), Chandra Shekhar Azad University of Agri- 23. Deepika Kiran × Ayogi 41.3 6.7
culture and Technology (CSAUA&T), Kanpur, UP, India. Majority of the 24. R-552 Sel. number 45 × B-67 43.0 6.0
entries are prominent and popular varieties which are under cultivation 25. RLU-6 Acc. 750 × RL 29-8 42.2 7.4
or widely used in various breeding programs. All the genotypes were 26. Meera (RL-75-6-2 × RL-29-8) × 42.2 7.6
LCK 8528
grown in field at CSIR-NBRI, Lucknow during crop season 2012–13.
27. Chambal Local × RR 45 40.1 5.2
The details of these genotypes are presented in Table 1. Total genomic 28. LC-185 NP (RR)-37 × Kangra local 45.7 4.6
DNA was extracted from fresh and young leaves following the CTAB 29. LC-54 K 2 × Kangra local 42.0 6.6
method (Doyle and Doyle, 1990) with some modifications. 30. LC-2063 1509 × LC 54 41.3 6.3
31. Neela SPS from indigenous collection 41.5 5.9
32. Him Alsi-1 – 38.7 6.2
2.2. AFLP analysis 33. Him Alsi-2 – 38.5 6.1
34. Janki – 41.5 9.0
The AFLP analysis was performed following the protocol described 35. Himani DPL 20 × KLS-1 36.4 6.1
by Vos et al. (1995) with minor modifications. In brief, genomic DNA 36. Nagarkot New River × LC 216 43.0 5.8
37 GS 41 – 41.5 8.1
(400–500 ng) was digested with 5 U EcoRI and 1 U MseI restriction en-
38. IC 15888 – 40.9 6.0
donucleases for 2.5 h at 37 °C and the digestion reaction was terminated 39. GS 234 – 38.6 6.8
at 65 °C for 15 min. The EcoRI and MseI adapter ligation was performed 40. JRF4 – 38.8 5.2
with digested DNA using 1 U T4 DNA ligase (New England Biolabs, USA). 41. BAU 616A – 39.2 5.7
42. EC-1392 – 41.1 5.7
The diluted restricted–ligated DNA was used for pre-selective amplifica-
43. T-397 T-491 × T 1103-1 44.0 5.8
tion with PCR profile of 20 cycles of denaturation at 94 °C for 20 s, an- 44. RKY-14 – 43.5 7.0
nealing at 56 °C for 30 s and extension at 72 °C for 2 min and a final 45. Padmini (EC41628 × EC77959) × 43.0 7.5
hold at 60 °C for 30 min. After confirming the PCR amplification by elec- (DPL20 × Neelum)
trophoresis of PCR products on 1.5% agarose gels, selective amplification Note: (-) pedigree information not available.
was performed with 10 fold diluted pre-selective amplified PCR prod-
uct. Selective amplification was perform in a 10 μl reaction volume
using selective primer combinations (labeled EcoRI and unlabelled primer combinations were combined with 8 μl of Hi-Di formamide con-
MseI primers) with amplification profile of denaturation at 94 °C for taining 0.20 μl GeneScan™ 500 ROX® as internal size standard. These
20 s, annealing at 66 °C for 30 s decrease by 1 °C/cycle up to 56 °C, ex- multiplexed PCR products were denatured for 5 min at 95 °C, quick
tension at 72 °C for 2 min for 10 cycles, followed by 94 °C for 20 s, chilled on ice for 5 min and loaded on an ABI 3730xl DNA Analyzer for
56 °C for 30 s, and 72 °C for 2 min for 33 cycles followed by 60 °C for electrophoresis. The fragment analysis was performed by GeneMapper
30 min on Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, v4.0 software (Applied Biosystems, Foster City, CA, USA).
USA). The sixteen selective primer combinations (PCs) used were:
E-ACA/M-CAC (PC1), E-ACA/M-CAG (PC2), E-ACA/M-CAT (PC3), E-ACA/ 2.3. Data acquisition and statistical analyses
M-CTA (PC4), E-AAC/M-CAA (PC5), E-AAC/M-CAG (PC6), E-AAC/M-CAT
(PC7), E-AAC/M-CTT (PC8), E-AGC/M-CAC (PC9), E-AGC/M-CAG (PC10), The AFLP data was scored as “1” (presence of fragment) and “0” (ab-
E-AGC/M-CAT (PC11), E-AGC/M-CTA (PC12), E-AGC/M-CTC (PC13), sence of fragment) manually by visualizing electropherogram with a
E-AGC/M-CTT (PC14), E-AAG/M-CAA (PC15), and E-AGG/M-CAA threshold value of 200 relative fluorescence units (rfu) for presence
(PC16). The selective PCR amplification was confirmed by electrophore- and less than that for absence of the fragment. The genotypic data
sis as mentioned above and then post PCR multiplex sets were prepared were used to calculate different parameters such as polymorphic infor-
based on fluorescence labeled primers. For post PCR multiplexing, 1.5 μl mation content (PIC), marker index (MI) and resolving power (RP). The
of each FAM, JOE and NED labeled PCR products, representing different PIC for each primer combinations was calculated according to Roldan-
 Chandrawati et al. / Gene 549 (2014) 171–178
Ruiz et al. (2000) formula: PICi = 2fi(1 − fi), where PICi is the polymor-
phic information content of marker i, fi is the frequency of the fragments
which were present and 1 − fi is the frequency of the fragments which
were absent. PIC was averaged over the fragments for each primer com-
bination. Marker index (MI) was calculated following Powell et al.
(1996) as: = PIC × EMR, where EMR (effective multiple ratio, EMR =
β × n) is defined as the product of the fraction of polymorphic loci (β)
and the number of polymorphic loci (n). The resolving power (RP) of
each primer combination was calculated according to Prevost and
Wilkinson (1999) as: Rp = ∑Ib, where Ib is the fragment informative-
ness and calculated as: Ib = 1–[2 × |0.5 − p|], where p is the proportion
of the genotypes containing the fragment. To understand the genetic re-
lationship pair-wise genetic dissimilarities among all 45 genotypes
were calculated according to the Jaccard's coefficient using DARwin
5.0.128 software (Perrier et al., 2003). The calculated dissimilarity
matrix was then used to construct a neighbor-joining (NJ) tree with a
1000 replicate bootstrap test. GenAlex 6.5 software was used to calculate
the number of different alleles (Na), number of effective alleles (Ne),
Shannon' information index (I) and molecular variance (AMOVA)
(Excoffier et al., 1992) within and among the populations based on
the NJ dendrogram.
3. Results
3.1. Marker polymorphism and AFLP features
To evaluate and characterize the 45 Indian genotypes of linseed a
total of sixteen AFLP primer combinations (PCs) were deployed to gen-
erate AFLP profiles. All the PCs have generated discrete AFLP profiles and
a representative snapshot from GeneMapper software showing poly-
morphic bands between two genotypes is presented in Fig. 1. Together,
the sixteen PCs produced 1142 fragments, of which 1129 (98.8%) were
found to be polymorphic and 13 (1.2%) monomorphic (Table 2). The
Fig. 1. Representative electropherogram (snapshot from GeneMapper) showing AFLP profiles with two genotypes GS41 (a, c) and Azad Alsi-1 (b, d) with primer combinations E-ACA/M-CAC
(a and b) and E-AAC/M-CAG (c and d). Arrows showing prominent polymorphic band.
173
total number of fragments of each PC varied from 45 (E-ACA/M-CTA)
to 95 (E-AGC/M-CAC) with an average of 71.37 fragments per PC. Poly-
morphic fragments ranged from 44 (E-ACA/M-CTA) to 94 (E-AGC/M-
CAC) with an average of 70.56. The percent polymorphism varied from
93.0% (E-AGC/M-CTT) to 100% (E-ACA/M-CAG; E-ACA/M-CAT; E-AAC/
M-CAA; E-AAC/M-CAG; E-AAG/M-CAT; E-AGC/M-CAT; E-AGC/M-CTC;
E-AAG/M-CAA; and E-AGG/M-CAA) with an average of 98.82%. The dis-
criminatory power of the informative AFLP profile mainly depends upon
three parameters namely polymorphic information content (PIC), marker
index (MI) and resolving power (RP) of a particular PC. The calculated PIC
value varied from 0.19 to 0.31 with an average of 0.23 per primer
combination (Table 2). The PC E-AGC/M-CAC showed the highest
PIC value (0.31) followed by E-AGC/M-CAG (0.29), E-AAC/M-CAG
(0.26) and E-AGC/M-CTA (0.25). The lowest PIC value (0.19) was no-
ticed for PC E-AAC/M-CTT. At the individual fragment level with each
PC, a majority of the polymorphic fragments (308) showed a lower
PIC value (b0.10) while 267 fragments showed higher PIC values be-
tween 0.41 and 0.50 (Fig. 2). The rest 554 fragments showed moderate
(in the range 0.11 and 0.40) PIC values. Resolving power (RP) varied
from 13.73 (E-ACA/M-CTA) to 43.50 (E-AGC/M-CAC) with an average
of 23.49. The marker index (MI) varied between 8.81 (E-ACA/M-CTA)
and 28.91 (E-AGC/M-CAC) with an average of 16.64. The number of
unique fragments and number of rare fragments were also calculated
across the genotypes and PCs. Unique fragments are genotype specific
and found only in a single genotype with a particular PC. The PCs pro-
duced at least 3 (with E-AAC/M-CAG) and at most 24 (with E-AAC/M-
CTT) unique fragments for a total of 183 fragments with an average of
11.4 per PC. The AFLP fragments which occur in less than 10% of geno-
types under study were considered as rare fragments. A total of 326
(28.5%) rare fragments were identified which varied from 7 (E-AGC/
M-CAC) to 36 (E-AAC/M-CAT) with an average of 20.4 rare fragments
per PC. The various marker features showed a strong and significant
correlation to each other. PIC and MI showed a correlation (r2) of 0.83,
174 Chandrawati et al. / Gene 549 (2014) 171–178

Table 2 similarity coefficient which varied from 0.16 to 0.57 with an average
Details of polymorphism and other informative content of 16 AFLP primer combinations of 0.26 ± 0.05. The maximum genetic similarity (57%) was noticed be-
used.
tween genotypes Him Alsi-1 and Him Alsi-2 followed by GS41 and Him
Primer TFa PFb PPc (%) PICd MIe RPf NUFg NRFh Alsi-1 (56%) and GS41 and LC-54 (54%). The genotype R-552 had the
combinations lowest range of pairwise genetic similarity coefficient with all other
E-ACA/M-CAC 66 65 98.5 0.22 14.38 19.60 4.0 29.0 genotypes varied from 0.16 to 0.29 with an average of 0.21 ± 0.03
E-ACA/M-CAG 72 72 100 0.25 18.49 25.40 5.0 24.0 (Table 3). Considering the genetic distance the genotypes R-552,
E-ACA/M-CAT 79 79 100 0.24 18.68 26.04 10.0 26.0
Himani, RKY-14, Meera, Indira Alsi-32 and Suyog were found to be the
E-ACA/M-CTA 45 44 97.8 0.20 8.81 13.73 9.0 12.0
E-AAC/M-CAA 68 68 100 0.21 14.18 19.29 13.0 24.0 most divergent among all the genotypes studied. The neighbor joining
E-AAC/M-CAG 55 55 100 0.26 14.11 19.24 3.0 18.0 clustering classified all the genotypes into three major clusters namely
E-AAC/M-CAT 78 78 100 0.21 16.40 22.20 9.0 36.0 I, II, and III (Fig. 3). Cluster II was found to be the largest with 27 geno-
E-AAC/M-CTT 85 81 95.3 0.19 15.72 22.00 24.0 34.0 types (60%) followed by cluster I with 13 genotypes (29%) and cluster
E-AGC/M-CAC 95 94 98.9 0.31 28.91 43.50 14.0 7.0
E-AGC/M-CAG 83 82 98.8 0.29 23.99 35.30 8.0 11.0
III with 5 genotypes (11%). Cluster II could be further subdivided into
E-AGC/M-CAT 76 76 100 0.22 16.91 23.50 20.0 19.0 three sub-clusters namely IIa, IIb, and IIc with 9, 5 and 13 genotypes re-
E-AGC/M-CTA 87 86 98.9 0.25 21.30 30.80 15.0 17.0 spectively. The other two clusters could not be subdivided further. Con-
E-AGC/M-CTC 68 68 100 0.24 16.36 23.00 14.0 16.0 sidering the oil content of the genotypes, it was noticed that all the
E-AGC/M-CTT 57 53 93.0 0.22 11.51 16.50 7.0 13.0
genotypes of cluster III showed oil content equal to or above the mean
E-AAG/M-CAA 56 56 100 0.20 10.98 14.98 14.0 18.0
E-AGG/M-CAA 72 72 100 0.21 15.46 20.76 14.0 22.0 oil content (41.7%) and at the same time 1000 seed weight was also no-
Total 1142 1129 – – – – – – ticed to be lower than the mean value (6.7 g) except for two genotypes
Minimum 45 44 93.00 0.19 8.81 13.73 3.0 7.0 (Table 1). In contrast, the genotypes of cluster I showed oil content
Maximum 95 94 100.00 0.31 28.91 43.50 24.0 36.0 below the average value except for two genotypes. Cluster II had geno-
Average 71.37 70.56 98.82 0.23 16.64 23.49 11.4 20.37
types having oil content that ranged from minimum (36.4%) to maxi-
a
Total number of fragments. mum (45.7%) and 1000 seed weight varied from 4.6 g (minimum) to
b
Number of polymorphic fragments.
c 9.6 g (maximum). The genotype LC-185 had minimum 1000 seed
Percent polymorphism.
d
Polymorphic information content. weight with maximum oil content among all the 45 genotypes. The
e
Marker index. partitioning of genetic variations within and between populations
f
Resolving power. (three populations based on dendrogram) using AMOVA showed that
g
Number of unique fragments. 13% of the total genetic variation exists among the populations
h
Number of rare fragments.
and 87% within each population (Table 4) with average pairwise
ΦPT (similar to FST) of 0.127. The genotypes used in the present in-
p b 0.005; MI and RP showed r2 = 0.99, p b 0.005 while r2 = 0.85, vestigation showed a low level of variation as indicated by the aver-
p b 0.005 was the correlation between RP and PIC. age Na (1.39 ± 0.015), Ne (1.24 ± 0.005), He (0.15 ± 0.003) and I
(0.25 ± 0.004) (Table 5).

3.2. Genetic diversity analyses

The AFLP profile data of 16 PCs with 45 genotypes was used to un-
derstand the level of genetic diversity and interrelationship (if any)
among linseed genotypes under investigation. A pair-wise genetic dis-
tance obtained from the Jaccard's coefficient was converted into a
4. Discussion
Genetic improvement of linseed is an important requirement for de-
velopment of high yielding varieties in India. In this context, it becomes
pertinent to understand the nature and extent of genetic diversity in the

200

180 185
176
160
Number of polymorphic fragmantes

140 144

132
120
117
100
100

80 86
82

60
54 53
40

20

PIC Value

Fig. 2. PIC distribution of 1129 polymorphic fragments across 45 genotypes of linseed.

 Chandrawati et al. / Gene 549 (2014) 171–178 175

Table 3 70.5 (98.8%) polymorphic fragments per PC. A high rate of polymor-
Minimum, maximum and mean of the Jaccard's similarity coefficient of 45 genotypes of phism was noticed indicating a considerable amount of genetic variabil-
linseed.
ity among the genotypes under investigation. The PIC value of the
Varieties Minimum Maximum Mean ± SD majority of polymorphic fragments was moderate but a considerable
Shikha 0.20 0.52 0.29 ± 0.06 number of fragments (16.4%) showed a higher PIC value. The PIC
Neelum 0.19 0.32 0.26 ± 0.03 value of most of the PCs was found to be low (average: 0.23). Similarly,
Rashmi 0.16 0.34 0.24 ± 0.05 a low PIC value (0.18) of ISSR markers was also reported by Rajwade
Parvati 0.17 0.35 0.24 ± 0.04
et al. (2010) in linseed. Out of 16 PCs used in the present investigation,
Sweta 0.19 0.35 0.24 ± 0.03
Sheela 0.19 0.41 0.29 ± 0.05 9 (56%) were found to produce 100% polymorphic fragments indicating
Garima 0.19 0.38 0.26 ± 0.04 that these PCs could be used to characterize other sets of germplasm
Shekhar 0.18 0.35 0.25 ± 0.04 materials of linseed. Considering the fragment distribution pattern
Laxmi-27 0.18 0.34 0.26 ± 0.04 among all the genotypes, the PC E-AAC/M-CTT generates the maximum
Shubhra 0.19 0.39 0.26 ± 0.04
Gaurav 0.16 0.37 0.26 ± 0.06
number of unique fragments (24) and second highest number of rare
Sharda 0.17 0.36 0.24 ± 0.04 fragments (34) though it had the minimum PIC value (0.19). The PCs
Azad Alsi-1 0.21 0.38 0.28 ± 0.04 which produced unique fragments in a specific genotype can be used
Hira 0.20 0.34 0.26 ± 0.04 to develop sequence tagged site (STS) markers to identify a particu-
C-429 0.19 0.36 0.24 ± 0.04
lar cultivar (Fernandez et al., 2002). Therefore, AFLP profiling of the
Pusa-3 0.16 0.36 0.24 ± 0.05
Jawahar-7 0.21 0.36 0.29 ± 0.04 PC E-AAC/M-CTT could be used to develop STS markers for variety
Jawahar-23 0.21 0.37 0.28 ± 0.04 identification in linseed. The PIC value has been primarily and most
JLS-9 0.17 0.41 0.27 ± 0.05 extensively used as a measure of discriminatory power or informative-
Suyog 0.16 0.33 0.23 ± 0.03 ness of markers in most of the diversity studies. Considering the PIC
Kartika 0.20 0.35 0.25 ± 0.03
value in the present investigation the PCs E-AGC/M-CAC (0.31), E-AGC/
Indira Alsi-32 0.18 0.36 0.23 ± 0.04
Deepika 0.18 0.40 0.26 ± 0.05 M-CAG (0.29), E-AAC/M-CAG (0.26), and E-AGC/M-CTA (0.25) were
R-552 0.16 0.29 0.21 ± 0.03 found to be highly informative and could be used in germplasm charac-
RLU-6 0.19 0.36 0.25 ± 0.04 terization and other marker based genetic studies in linseed in the future.
Meera 0.18 0.32 0.23 ± 0.04
The other two parameters, i.e. RP and MI provide complementary attri-
Chambal 0.18 0.33 0.25 ± 0.03
LC-185 0.18 0.31 0.25 ± 0.03
butes to the marker system and have been widely used in several genetic
LC-54 0.18 0.54 0.29 ± 0.09 diversity studies in different plants (Gupta et al., 2013; Laurentin and
LC-2063 0.17 0.42 0.27 ± 0.06 Karlovsky, 2007; Pecina-Quintero et al., 2013; Sehgal et al., 2009; Sharma
Neela 0.19 0.32 0.24 ± 0.03 et al., 2011; Stodart et al., 2005). Considering PIC, RP, and MI parameters
Him Alsi-1 0.17 0.57 0.29 ± 0.09
together, the PCs E-AGC/M-CAC, E-AGC/M-CAG and E-AGC/M-CTA could
Him Alsi-2 0.17 0.57 0.29 ± 0.09
Janki 0.20 0.41 0.30 ± 0.06 be considered as ideal PCs due to their higher values for these parameters.
Himani 0.18 0.26 0.22 ± 0.02 Therefore, these PCs could be highly useful for diversity and evolutionary
Nagarkot 0.19 0.38 0.28 ± 0.06 studies because of their high informativeness attributes. A strong associa-
GS 41 0.19 0.56 0.30 ± 0.10
tion between the ability of a primer to distinguish cultivars and resolving
IC 15888 0.19 0.41 0.30 ± 0.06
GS 234 0.19 0.51 0.29 ± 0.08
power has been reported and is thus considered as an important attribute
JRF 4 0.19 0.36 0.28 ± 0.04 for AFLP to determine its discriminatory power (Fernandez et al.,
BAU 616A 0.20 0.33 0.27 ± 0.03 2002; Prevost and Wilkinson, 1999). A strong correlation (r2 = 0.99,
EC-1392 0.17 0.42 0.29 ± 0.07 p b 0.005) between MI and RP was noticed here indicating that any of
T-397 0.16 0.40 0.27 ± 0.06
the two parameters could be used to assess the informativeness of a PC.
RKY-14 0.19 0.29 0.24 ± 0.02
Padmini 0.17 0.39 0.25 ± 0.04 Therefore, apart from PIC and RP, MI could also be used as a reliable attri-
bute to determine the discriminatory power of the PCs.
A pair-wise genetic similarity matrix based neighbor joining tree
available germplasm for devising appropriate breeding and selection was constructed to understand the extent of genetic diversity among
strategies. We have carried out AFLP based analysis of molecular diver- the Indian genotypes of linseed. In the present set of genetic material a
sity of varieties developed in India in order to identify and select diverse high level of genetic diversity was noticed (average similarity coefficient:
genotypes for use in further breeding programs. The AFLP analysis offers 0.26 ± 0.05). Six genotypes namely R-552, Himani, RKY-14, Meera, Indira
simultaneous screening of many different DNA regions distributed Alsi-32 and Suyog were highly diverse among all. The interesting point to
throughout the genome and has been extensively used for a wide note here is that out of these six diverse varieties five were grouped to-
range of taxa including plants, fungi, animals and bacteria (Mueller gether in one cluster (cluster II). The pedigree details of Suyog and Indira
and Wolfenbarger, 1999). AFLP has been widely used for studying ge- Alsi-32 revealed that they have one common parent i.e. Kiran which
netic variation, relatedness and phylogenetic relationships (Hill et al., might have contributed diverse alleles to the progenies. Among the six di-
1996; Kardolus et al., 1998), and characterization of natural populations verse genotypes, R-552 and Himani also showed differences in oil content
and breeding lines (De Riek et al., 2001; Dubey et al., 2010; Roldan-Ruiz and thus could be utilized as parental lines for developing mapping pop-
et al., 2000). In the case of linseed, limited studies have been carried out ulation and tagging oil content related QTLs. Likewise, Suyog and Himani
to characterize the genetic materials for diversity assessment through could be used for tagging test weight related QTLs through QTL mapping.
molecular markers. These include reports of RAPD (Fu et al., 2003), In comparison to other important oilseed crops only few linkage/QTL
ISSR (Rajwade et al., 2010; Uysal et al., 2010; Wiesnerova and maps have been reported in linseed (Cloutier et al., 2011, 2012; Oh
Wiesner, 2004), retrotransposon-based marker (Smykal et al., 2011), et al., 2000; Spielmeyer et al., 1998). To the best of our knowledge, only
AFLP (Adugna et al., 2006; Everaert et al., 2001) and SSR (Braulio et al., QTLs for Fusarium wilt (Spielmeyer et al., 1998), linolenic acid, linolenic
2012) analyses. However, the Indian genotypes have been analyzed acid, palmitic acid and seed color (Cloutier et al., 2011) have been report-
with only a limited number of markers (12 ISSR; 136 loci) by Rajwade ed till date. Therefore, it is important to carry out more mapping studies to
et al. (2010) and other than this study, no report of molecular assessment tag QTLs for other agronomically important traits in order to facilitate
of Indian linseed genetic diversity is known. marker assisted breeding for faster development of new varieties. The
High quality AFLP profiles were obtained with all the PCs deployed clear separation of the genotypes into three clusters in the NJ dendrogram
in the present investigation producing an average of 71.4 total and suggests that these were differentiated from each other. Considering each
176 Chandrawati et al. / Gene 549 (2014) 171–178

IIa

IIb

IIc

III

Fig. 3. Genetic relatedness among 45 genotypes of linseed based on neighbor-joining clustering. The scale at the bottom is for NJ distances.

cluster of genotypes as one population allowed the estimation of
partitioning of the genetic diversity as well as the Shannon index. The
within population variance was much higher (~6–7 fold) than the be-
tween population variance. This is consistent with the mostly similar
values of I and He in each population. These data trends also agree
well with those reported by Smykal et al. (2011) in the case of the
flax population using retrotransposon based markers for cultivated
flax germplasm accessions. They have reported that the corresponding
values were higher only in the case of the wild species of Linum. Thus
data in the present paper as well as that of Smykal et al. (2011) have
clearly shown that cultivated/pedigree accessions of the groups of
flax/linseed do not reveal a high degree of genetic variations.
The AFLP data after NJ analysis has revealed some interesting trends.
When the oil content of the genotypes as well as the 1000 seed weight
were considered in conjunction with the distribution of the genotypes
in the NJ tree, it was observed that all genotypes of cluster III exhibited
either equal to or higher than the mean oil content. In contrast, the ge-
notypes of the other clusters did not reveal any such correlation. Similar
Table 4
Analysis of molecular variance (AMOVA) for 45 linseed genotypes.
results with indirect correlation between specific traits and molecular/
PCR profiles have been reported in the case of papaya (De Jesus et al.,
2013; Saxena et al., 2005), mung bean (Lavanya and Ranade, 2013;
Lavanya et al., 2008), coconut (Perera et al., 1998) and Borago (De Lisi
et al., 2014) to cite a few examples. This result is useful for the selection
of higher oil content genotypes by AFLP profiles also and opens up the
distinct possibility of identifying one or more genes/QTLs for oil content
among these genotypes on the basis of suitable markers developed from
the AFLP profiles of these genotypes. Furthermore, these results are
promising for the possible correlation of AFLP profiles of the genotypes
with the fatty acid profiles of the genotypes so that molecular tagging of
specific fatty acid type genotypes could be facilitated.
The present investigation nevertheless helps in selecting diverse pa-
rental lines for developing mapping populations to carry out linkage/
QTL mapping studies. Apart from biparental QTL mapping, the diverse
accessions identified here could also be used in multi-parent breeding
Table 5
Different genetic diversity estimates for three populations (based on NJ dendrogram) of
linseed based on 1142 AFLP loci.
Population Na Ne I He

Pop1 (13) 1.43 ± 0.03 1.27 ± 0.009 0.27 ± 0.007 0.17 ± 0.005
Source Degrees of freedom Sum of squares Variance % variation
Pop2 (27) 1.77 ± 0.02 1.21 ± 0.007 0.25 ± 0.006 0.15 ± 0.004
Among population 2 711.33 18.61 13 Pop3 (5) 0.98 ± 0.03 1.23 ± 0.009 0.23 ± 0.008 0.16 ± 0.005
Within population 42 5365.15 127.74 87 Mean 1.39 ± 0.015 1.24 ± 0.005 0.25 ± 0.004 0.15 ± 0.003
Total 44 6076.48 146.35 100
Na—number of different alleles; Ne—effective number of alleles; He—Nei's (1973) gene
FST = 0.128 and p N 0.001. diversity; I—Shannon's information index.
 Chandrawati et al. / Gene 549 (2014) 171–178
programs for linseed genetic improvement and development of new
varieties having combinations of desirable traits. The biparental QTL
mapping approach has been reported to have problems related to low
resolution and limited genetic recombination (Cavanagh et al., 2008).
Therefore, a new approach of multiparent based population (Multiparent
Advance Generation Inter-Cross: MAGIC) is being widely used to over-
come demerits of biparental mapping population and to understand the
inheritance of various polygenic traits, identifying fine QTLs and marker
assisted breeding (Cavanagh et al., 2008). The diverse genotypes identi-
fied here will also be useful in developing MAGIC population and tagging
QTLs in linseed in the future. In conclusion, the present investigation char-
acterized and developed AFLP fingerprint profiles of prominent Indian
genotypes of linseed. Diverse sets of genotypes have been identified
which could be used in multi-parent breeding programs and developing
MAGIC population for QTL mapping and marker assisted breeding for de-
veloping new and elite varieties of linseed.
Conflict of interest
None.
Acknowledgments
Authors thank the Director, CSIR-NBRI, Lucknow for providing the
facilities to carry out the present investigation. Financial support in
form of DST-INSPIRE award to Chandrawati is gratefully acknowledged.
Authors also thank the reviewers of the manuscript for their suggestion
about population analysis.
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