paper

1. Euphytica 120: 301–307, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
301
Assessment of genetic variation in a collection of lentil using molecular
tools
Gabriella Sonnante∗ & Domenico Pignone
CNR, Istituto del Germoplasma, Via Amendola, 165/A, 70126 Bari, Italy; ( ∗author for correspondence)
Received 28 January 2000; accepted 23 September 2000
Key words: diversity, ISSR, Lens, lentil, RAPD
Summary
A collection of lentil landraces, mostly from Italy, but including Mediterranean and foreign germplasm as refer-
ence, was evaluated using RAPDs, microsatellite-primed PCR and ISSRs. A low level of useful polymorphic bands
was detected with the former two markers, whereas ISSRs revealed a higher degree of variation. The UPGMA trees,
based on Jaccard similarity index matrices, obtained with RAPD and ISSR respectively, did not produce similar
clusters. However, in both cases, the accession which differentiated the most from the others was from Ethiopia, a
country where also other cultivated species have shown a remarkable degree of variation. On the other hand, Italian
accessions showed a trend to group together. ISSR markers proved to be useful for distinguishing closely related
genotypes, and possibly for substantiating the genetic peculiarity of ecotypes applying for the obtainment of origin
and quality marks.
Introduction
Lentil (Lens culinaris Medik.) is a diploid (2n=2x=14),
autogamous species and is one of the oldest crops in
the world, which originated in the Near East (Zohary,
1972). Its ancestry is supposed to be the result of a
single domestication event (Zohary, 1999). Nowadays,
lentil is still traditionally cultivated in the Mediter-
ranean Basin, Asia and has a certain diffusion in the
Americas. Although not a major crop, it provides
a substantial portion of Dietary Energy Supply on
a regional basis (FAO, 1995). Two cultivated vari-
etal groups are distinguished in lentil (Barulina 1930),
based on seed shape and dimension: small seeded (less
than 6 mm in diameter) var. microsperma and large
seeded (over 6 mm) var. macrosperma. In Italy, most
cultivated types are actually local agro-ecotypes rather
than improved varieties, which usually take the name
of the area where they are cultivated (Foti, 1982).
Morphological variation within each varietal group
is quite appreciable. Nevertheless most genetic studies
do not find a parallel with variation for genetic mark-
ers. Genetic diversity in cultivated and wild lentil has
been explored using several methods. Isozyme ana-
lysis is a quite simple and inexpensivetechnique and in
Lens it has been employed for the formation of a core
collection (Erskine & Muehlbauer, 1991), to study
population genetic structure together with RAPD ana-
lysis (Ferguson et al., 1998), and to assess genetic
variation in cultivated and wild material (De la Rosa
& Jouve, 1992). However, isozymes show little vari-
ation and are not useful to distinguish among closely
related genotypes. Also DNA markers such as RFLPs
detected a low level of genetic variation in cultivated
lentil (Havey & Muehlbauer, 1989).
Polymerase chain reaction (PCR)-based tech-
niques provide several options for assessing genetic
variation and identifying genotypes. A relatively low
level of polymorphism was observed in a limited
number of cultivated lentil based on RAPD markers
(Abo-elwafa et al., 1995; Sharma et al., 1995; Ford et
al., 1997), whereas AFLPs detected a ten-fold higher
polymorphism as compared to RAPDs (Sharma et al.,
1996).
Microsatellite sequences can also be used as
primers to detect polymorphisms in plant species
(Weising et al., 1995). They do not require any se-
quence information, and they should provide more re-

2. 302
liable profiles than RAPDs do, since higher annealing
temperatures can be applied.
Inter-simple sequence repeat (ISSR) amplification
(Zietkiewicz et al., 1994) is a relatively recent tech-
nique which can differentiate closely related geno-
types. It is based on the amplification of a single
primer containing a microsatellite ‘core’ sequence
anchored at the 3 or 5 end by 2–4 selective, often de-
generate, nucleotides. The DNA fragments amplified
are flanked by inversely oriented, adjacent microsatel-
lites. Due to primer configuration and to separation
of DNA fragments on polyacrylamide gels, ISSR
amplification usually reveals a large number of DNA
fragments. This technique has been used to investig-
ate genomic origins (Salimath et al., 1995), to assess
genetic diversity in germplasm collections (Gilbert et
al., 1999), to identify cultivars (Prevost & Wilkinson,
1999).
In this article we report on the evaluation of genetic
variation in a collection of cultivated lentils, including
some Italian well-known ecotypes. Initially, RAPDs
and microsatellite-primed PCR separated on agarose
gel were used. Since both these markers produced
a low number of polymorphic bands, ISSR markers
were tested.
Material and methods
In total, 46 accessions of lentil (22 macrosperma and
24 microsperma types) were analysed, 31 of which
originated in Italy, while the remaining ones were
mostly from other Mediterranean countries (Table 1).
Among the Italian accessions, some well known eco-
types were comprised, i.e. Castelluccio, Altamura,
Ustica, Pantelleria.
Young leaves were collected and DNA extracted
according to Paz & Veilleux (1997). After measur-
ing DNA concentration, amplification was performed
with Operon RAPD primers (series OPO, OPP, OPQ
and the primer OPK15), microsatellites or primers
for ISSR, the latter being based on a ‘core’ sequence
of microsatellites, with 2 or 3 selective nucleotides.
The amplification protocol was as follows: 18µl of
reaction volume contained 20 ng DNA, 50 mM KCl,
10 mM Tris-HCl pH 9.0, 1.5 mM MgCl2, 0.1 mM for
each nucleotide (dATP, dCTP, dGTP, dTTP), 0.2 µM
primer and 0.8 unit of Taq DNA polymerase.
The amplification programme included the follow-
ing steps: one cycle at 94 ◦C for 2 , followed by 35
cycles at 94 ◦C for 45 , annealing temperature (AT)
Table 1. List of the material analysed and its origin. For Italian
materials, where available, region is specified
Number Accession number∗ Type∗∗∗ Origin
1 MG 103215 M ITALY (IT)
2 MG 106892 M IT-Sardinia
3 MG 106899 M IT-Sardinia
4 MG 110287 M IT-Apulia
5 MG 110288 M IT-Basilicata
6 MG 110438 M IT-Apulia
7 MG 110918 M IT-Apulia
8 MG 111863 M IT-Basilicata
9 MG 103653 m ITALY
10 MG 106665 m IT-Abruzzo
11 MG 110290 m IT-Apulia
12 MG 111106 m IT-Basilicata
13 MG 111849 m IT-Basilicata
14 MG 111854 m IT-Basilicata
15 MG 112253 m IT-Sicily
16 MG 113060 m IT-Sicily
17 MG 115108 m IT-Abruzzo
18 MG 115109 m IT-Abruzzo
19 MG 115496 m IT-Apulia
20 MG 115963 m IT-Basilicata
21 MG 116218 m IT-Pantelleria
22 MG 116219 m IT-Lampedusa
23 MG 116221 m IT-Linosa
24 ∗∗ m IT-Ustica
25 ∗∗ M IT-Apulia (Altamura)
26 ∗∗ M IT-Apulia (Gravina)
27 PI 298921 M IT-Apulia (Altamura)
28 PI 298922 m IT-Umbria (Castelluccio)
29 PI 298923 M ITALY
30 PI 298924 M ITALY
31 PI 320940 M ITALY
32 MG 116052 m ALBANIA
33 MG 106398 M ALGERIA
34 MG 112366 M CYPRUS
35 MG 110830 m EGYPT
36 MG 107574 M GREECE
37 MG 111911 M LYBIA
38 MG 112112 M MOROCCO
39 MG 107186 M SPAIN
40 MG 107187 M SPAIN
41 MG 106673 M TUNISIA
42 MG 106286 m IRAN
43 MG 109605 m NEPAL
44 MG 107385 m PAKISTAN
45 MG 103504 m ETHIOPIA
46 MG 115437 m SOUTH AFRICA
∗ MG entries are from CNR Istituto del Germoplasma, PI ones are
from UDSA, ∗∗ courtesy of Dr Gaetano Laghetti, CNR Istituto del
Germoplasma, Bari, Italy.
∗∗∗ M = macrosperma, m = microsperma.