CARYOLOGIA
Vol. 64, no. 2: 189-196, 2011
Morphometric pattern of somatic chromosomes in three Romanian seabuckthorn genotypes
TRUTA1,* ELENA, GABRIELA CAPRARU1, CRAITA MARIA ROSU1, MARIA MAGDALENA ZAMFIRACHE2,
ZENOVIA OLTEANU2 and CIPRIAN MANZU2
1
Institute of Biological Research, Lascar Catargi, 47, 700107 Iasi, Romania.
University “Al. I. Cuza”, Faculty of Biology, Carol I 20A Bld., 700506 Iasi, Romania.
2
Abstract — The somatic chromosomes and karyotype traits of three Romanian genotypes (coded as CB-1,
S-16, L-4) of Hippophae rhamnoides L. have been studied. Diploid chromosome number is 2n=24. The karyomorphological data show that the complements have small chromosomes (1.33 ± 0.014 – 2.80±0.01 µm, for
CB-1 genotype; 1.13 ± 0.029 – 2.90 ± 0.029 µm, for S-16; 1.15 ± 0.099 – 2.88 ± 0.024 µm, for L-4 genotype) and
the length of haploid complements is 23.97 µm, for CB-1, 22.20 µm, for S-16, 21.73 µm, for L-4. In CB-1 and
S-16 male genotypes, the putative Y-chromosome has larger sizes than X-chromosome and it is of metacentric type (CI = 45.77, r = 1.18, for CB-1; CI = 46.22, r = 1.15, for S-16). The L-4 female karyotype is constituted
by twelve pairs of morphologically similar chromosomes. The karyotype formulae are 2n = 24 = 13m + 11sm
(putative male: 22 + XY), for CB-1 genotype, 2n = 24 = 18m + 6sm (putative male: 22 + XY), for S-16 genotype,
respectively 2n = 24 = 22m + 2sm (L-4 genotype, considered as female: 22 + XX). The small size of chromosomes
(< 3µm), the presence of only two morphometric chromosome types and preponderance of metacentrics confer
a relatively high degree of symmetry to the studied karyotypes. Our results show a low intraspecific karyotype
variation.
Key words: karyotype, seabuckthorn, sex chromosomes.
INTRODUCTION
Hippophaë rhamnoides L. has special characteristics, exploitable in biotechnological, nutritional, and pharmaceutical purposes or in cosmetics domain and in environment protection.
It can fix atmospheric nitrogen due to symbiotic
mycorrhizal Frankia fungus (KANAYAMA et al.
2008). The capacity to fix nitrogen is twice higher than that of soybean (LU 1992). The average
amount of nitrogen fixed in seabuckthorn forest is 30-60 kg/ha/year (ZIKE et al. 1999) or 180
kg/ha/year, according to STOBDAN et al. (2008).
More than 100 bioactive substances have been
evidenced in seabuckthorn leaves and berries:
*Corresponding author: e-mail: [email protected]
carbohydrates, organic acids, carotenoids, tocopherols, sterols and other lipids, tannins, phenols, amino acids, vitamins (C, E, B1, B2, F, K,
P, provitamin A), microelements (P, Ca, Mg, K,
Fe, Na) etc. (AHMAD and KAMAL 2002). Vitamin
C content largely ranges in berries, it exceeding
more times the content of dog rose hips, orange,
kiwi, hawthorn, tomato and other berries like
strawberry, raspberry and blackberry (YAO and
TIGERSTEDT 1993; LU 1996; LI and MCLOUGHLIN
1997). This complex composition results in utilization of seabuckthorn preparations in prevention and treatment of some diseases (YASUKAWA
et al. 2009; GREY et al. 2010). The pharmacological effects may be partly attributed to their high
content of phenolic compounds which show antioxidant, antimicrobial, antimutagenic and anticarcinogenic potential (ERCISLI et al. 2007; PANG
et al. 2008). More probably, the numerous health
benefits are the result of the synergy among many
different bioactive components in the plant parts
190
(YANG 2009).
The phenotypic and genotypic studies revealed a very large heterogeneity – morphological,
biochemical, cytogenetical – in all seabuckthorn
provenances, inclusively in Romanian varieties.
There are extremely numerous data concerning
seabuckthorn chemical composition, but a small
amount of information exists about chromosome pattern of this species. The knowledge of
genetic constitution of seabuckthorn – chromosome number, size and morphology – is of basic
importance in the deciphering of its populational
structure, of its polymorphism, geographical distribution, species systematics and evolution. In
spite of the thoroughgoing studies of molecular
genetics, the classical cytogenetic investigations
are still needed because they provide important proofs concerning numerical and structural
traits of chromosome set in view of karyotype
construction. To reach their full impact, it is essential that the new exciting molecular findings
to be fully integrated with the traditional cytogenetical data (STACE 2000). The highlighting of
numerical or structural chromosome variations
offers additional arguments in the explanation of
extremely large variability of phenotype traits in
seabuckthorn. Dioecious nature of seabuckthorn
represents a limitation for species breeding, the
sex determination mechanism in this species with
pronounced sexual dimorphism being not entirely deciphered. In dioecious plants, the genetic
basis of sex determination is diverse, including
heteromorphic sex chromosomes and also one or
several autosomal nuclear sex-determining loci,
possibly influenced by cytoplasmic genes and environmental cues (AINSWORTH 2000).
In Hippophae genus the most cytogenetic
data concern the chromosome number, rarely
their size and morphotypes. Although H. rhamnoides is recognized as a species having four different intraspecific cytotypes (2n = 12, 18, 20, 24)
(SINGHAL et al. 2008), the problem of its chromosome constitution is incompletely known
and the opinions are sometimes different, especially concerning the question of ploidy level.
SOBOLEWSKA (1926) (see work by COOPER 1932)
sustained the existence of n = 6 haploid number in Elaeagnus angustifolia, and n = 10 haploid number in H. rhamnoides species, whereas
DARMER (1947; 1952) (cf. ROUSI and AROHONKA
1980) claimed the existence of two morphologically close chromosome races – one diploid
(2n = 2x = 12), another tetraploid (2n = 4x = 24) –
in Hiddensee Baltic island. ROUSI (1971) sustains
as improbable this situation, by emphasizing in
TRUTA , CAPRARU , ROSU , ZAMFIRACHE , OLTEANU
and MANZU
this sense the existence, in Eleagnaceae family,
of range of variation between 11-14 of the basic
number, that makes more probable the existence
of x = 12 basic number in Hippophaë genus. It
concluded that 2n = 24 probably represents the
diploid chromosome number, while 2n = 12 Darmer’s count was based on an exceptional haploid
individual. An interesting point is represented by
the first record of n = 9 as meiotic chromosome
count (SINGHAL et al. 2008), established for Indian seabuckthorn populations grown in cold areas
situated at high altitudes of Himachal Pradesh
region. These data are in accordance with the earlier reports of SINGH (1982) and CHATHA and BIR
(1988) (cf. SINGHAL et al. 2008) on seabuckthorn
populations from Jammu and Kashmir regions of
India. Regarding the existence or characteristics
of heterosomes, only SHCHAPOV (1979) reported
the presence of male differentiated heterosomes,
but without detailed description of these. Based
on the results obtained in field crossing experiments, PERSSON (2001) sustains the genetic determination of sex in seabuckthorn individuals,
although the presence of sex chromosomes was
not substantiated nor in this case. LEBEDA et al.
(2003) speak about male and female forms based
on phenotype characters and not on genetic constitution of these ones. CAO and LU (1989) published data on chromosome size and morphology
in Chinese varieties, but they have not been identified heteromorphic sex chromosomes.
The present study was carried out to establish
the diploid chromosome number in three Romanian genotypes and to analyze in a comparative
manner the similarities and differences between
these, in order to identify some particularities
which permit us to presume the existence of morphologically distinguishable sex chromosomes.
MATERIAL AND METHODS
The cytogenetic investigations have been carried out on root tips of seedlings obtained by germination of seeds harvested from three genotypes
of seabuckthorn (H. rhamnoides ssp. carpatica
Rousi) with different geographical provenances
(Coteni-Buhoci, Sulina, and Letea areas), coded
as CB-1, S-16, and L-4. Seed germination was induced at 22°C, in dark, on moist filter paper, in
glass Petri dishes. The root tips having 10-15 mm
in length were pre-treated with 8-hydroxyquinoline (0.002 mol/L), for 4 h, then fixed in ethanol/
acetic acid (3:l), for 24 h, at room temperature.
The plant material was stored in refrigerator, at
191
CYTOGENETIC TRAITS IN SEABUCKTHORN GENOTYPES
4°C, in 70% alcohol. In view of analysis, the root
tips were hydrolyzed in 50% hydrochloric acid
for 8 minutes and then stained in modified carbol
fuchsin solution (GAMBORG and WETTER 1975).
The root tips were squashed in 45% acetic acid.
Microscopic investigation was carried out by
a Nikon Eclipse 600 microscope and the metaphases with well-spread chromosomes were photographed with Cool Pix Nikon digital camera,
1600x1200 dpi, 100x objective. The images were
processed by Adobe Photoshop programmer.
Our chromosome measurements included
absolute length of individual chromosomes
(CL), long arm length (L), short arm length
(S), arm ratio, r (r = L/S), centromeric index, CI
(CI = 100xS/CL), length of the haploid complement (LHC), and the relative length of each
chromosome (expressed as a percentage of the
absolute length of each chromosome pair out of
length of the haploid complement, namely CL/
LHCx100). These parameters helped us to establish the chromosome morphotypes of analysed
genotypes. According to LEVAN et al. (1964) terminology, the chromosome types were assigned
on the basis of centromere position (median,
submedian, subterminal, and terminal), respectively on CI and r values. Thus, the chromosomes
are metacentric when they have a mean arm ratio
of up to 1.7 and CI = 50.0-37.5, submetacentric
(r = 1.70 – 2.99, CI = 37.5 – 25.0), subtelocentric
(r = 3.00 – 6.99, CI = 25.0 – 12.5), and telocentric
(r = 7.00 and up, CI < 12.5). The chromosome
pairs, designed as I, II, III, …, XII, have been
grouped in descending order of their length.
RESULTS AND DISCUSSION
The literature on karyotypic features of different provenances of seabuckthorn is reduced and
mainly refers to the chromosome number found
in somatic cells. We think that some possible
explanations for the low number of cytogenetic
approaches are seed internal dormancy that seriously diminishes the germination rate, high chromosome stickiness that prevents well-spreading
of chromosomes in metaphase plates, and small
size of seabuckthorn chromosomes. The three
analysed genotypes are similar concerning the
somatic chromosome number (24 chromosomes)
(Fig. 1). In fact, 2n = 2x = 24 is the most reported number in H. rhamnoides (SHCHAPOV 1979;
CIREASA and DASCALU 1983-1984; CAO and LU
1989; CIMPEANU et al. 2004; RUAN and LI 2005)
and it is considered the true diploid number
of seabuckthorn (Rousi 1971). In ROUSI study
(1971), all the 2200 specimens from 33 herbaria,
34 samples from garden populations and 8 nature collections displayed 2n = 24 chromosomes.
In CB-1, S-16, and L-4 genotypes, relatively
similar patterns of karyotypes have been obtained (Fig. 2). Firstly, chromosomes are of small
dimensions – <3µm, except larger putative Ychromosome of CB-1 genotype, and the average
TABLE 1 — Morphometric characteristics of mitotic chromosomes in CB-1 genotype.
chromosome
morphotype
pair
I
m
CL
L
S
µm*
range
µm*
range
µm*
range
2.80±0.01
2.78-2.81
1.52±0.00
1.52-1.52
1.27±0.014
1.26-1.29
r*
CI
RL
(%)
1.19±0.006 45.61 11.66
II
m
2.68±0.004
2.67-2.68
1.54±0.00
1.54-1.54
1.13±0.004
1.13-1.14
1.37±0.005 42.28 11.16
III
sm
2.17±0.003
2.17-2.18
1.47±0.00
1.47-1.47
0.70±0.008
0.69-0.71
2.10±0.024 32.23 9.06
IV
m
1.99±0.004
1.99-2.00
1.20±0.003
1.20-1.21
0.79±0.00
0.79-0.79
1.52±0.10
39.64 8.31
V
sm
1.94±0.019
1.92-1.96
1.24±0.003
1.23-1.24
0.70±0.016
0.68-0.72
1.77±0.12
36.11 8.09
VI
m
1.79±0.008
1.78-1.80
1.11±0.00
1.11-1.11
0.68±0.004
0.67-0.68
1.65±0.004 37.79 7.47
VII
m
1.71±0.004
1.70-1.71
1.04±0.004
1.03-1.04
0.67±0.00
0.67-0.67
1.54±0.00
39.30 7.12
VIII
sm
1.65±0.004
1.64-1.65
1.05±0.00
1.05-1.05
0.59±0.004
0.59-0.60
1.78±.003
36.00 6.87
IX
sm
1.55±0.009
1.54-1.56
1.01±0.00
1.01-1.01
0.54±0.003
0.54-0.55
1.85±0.013 35.14 6.47
X
sm
1.53±0.004
1.52-1.53
1.05±0.003
1.04-1.05
0.48±0.00
0.48-0.48
2.19±0.05
1.32-1.35
0.79±0.008
0.78-0.80
0.54±0.004
0.54-0.55
1.45±0.019 40.81 5.57
XI
XII
31.37 6.37
m
1.33±0.014
X
sm
2.29
1.56
0.73
2.10
31.94 9.54
Y
m
3.40
1.84
1.56
1.18
45.77 14.18
* x ±SE = mean ± standard error; CL = absolute length of individual chromosomes; L = long arm length; S = short arm length;
r = arm ratio; CI = centromeric index; RL = relative length; m = metacentric; sm = submetacentric.
192
TRUTA , CAPRARU , ROSU , ZAMFIRACHE , OLTEANU
and MANZU
Fig. 1 — Metaphase plates (2n = 24) a CB-1 genotype; b S-16 genotype; c L-4 genotype.
of chromosome length calculated per genotype
is 1.99 ± 0.14, for CB-1; 1.85 ± 0.16, for S-16;
1.81 ± 0.16, for L-4. On the basis of measurements, the somatic chromosomes were grouped
in twelve pairs of morphologically similar chromosomes (I-XII) in L-4 genotype, respectively
eleven pairs of homomorphic chromosomes (IXI) and one pair constituted by two morphologically differentiated chromosomes, designed as X
and Y, in CB-1 and S-16 putative male genotypes.
The differences evidenced between the three
karyotype structures refer to the range of absolute chromosome length, relative length and the
length of haploid complement (Tables 1-4).
On the basis of arm ratio and centromeric
index values, all somatic chromosomes have
median or submedian constrictions, they being
exclusively of metacentric (m) and submetacen-
tric (sm) type. Some differences are present in
the proportion of these chromosome morphotypes. The metacentrics are more frequent. The
karyotype formulae are 2n = 24 = 13m + 11sm
(putative male: 22 + XY), for CB-1 genotype,
2n = 24 = 18m + 6sm (putative male: 22 + XY), for
S-16 genotype, respectively 2n = 24 = 22m + 2sm
(L-4 putative female genotype: 22 + XX). In the
studied material, the satellites have not been evidenced. These results showing the presence of
two chromosome morphotypes are in agreement
with the reports of CIREASA and DASCALU (19831984) (2n = 8m + 16sm), CAO and LU (1989),
CIMPEANU et al. (2004) (2n = 20m + 4sm). Karyotypes constructed by CAO and LU (1989) for five
H. rhamnoides subspecies (H. rhamnoides L.
ssp. yunnanensis, 2n = 14m + 10sm; H. rhamnoides L. ssp. mongolica, 2n = 16m + 8sm; H. rham-
TABLE 2 — Morphometric characteristics of mitotic chromosomes in S-16 genotype.
chromosome
morphotype
pair
CL
L
S
r*
CI
RL
(%)
µm*
range
µm*
range
µm*
range
m
2.90±0.029
2.87-2.93
1.57±0.024
1.55-1.60
1.33±0.004
1.33-1.34
1.19±0.019 45.71 13.08
II
m
2.85±0.024
2.82-2.87
1.46±0.023
1.44-1.49
1.38±0.003
1.37-1.38
1.06±0.019 48.54 12.83
III
sm
1.99±0.00
1.99-1.99
1.44±0.00
1.44-1.44
0.55±0.00
0.55-0.55
2.60±0.00
I
27.78 8.97
IV
m
1.96±0.003
1.93-1.99
1.02±0.003
0.99-1.05
0.94±0.002
0.93-0.94
1.09±0.029 47.89 8.84
V
sm
1.91±0.024
1.88-1.93
1.33±0.00
1.33-1.33
0.58±0.001
0.57-0.58
2.29±0.10
30.43 8.59
VI
sm
1.85±0.029
1.82-1.88
1.33±0.00
1.33-1.33
0.52±0.00
0.52-0.52
2.53±0.13
28.36 8.34
VII
m
1.69±0.024
1.66-1.71
1.05±0.00
1.05-1.05
0.64±0.00
0.64-0.64
1.66±0.07
37.70 7.60
VIII
m
1.52±0.003
1.49-1.55
0.83±0.00
0.83-0.83
0.69±0.029
0.66-0.72
1.20±0.049 45.45 6.85
IX
m
1.27±0.00
1.27-1.27
0.83±0.00
0.83-0.83
0.44±0.00
0.44-0.44
1.88±0.00
34.78 5.73
X
m
1.22±0.00
1.22-1.22
0.69±0.029
0.66-0.72
0.52±0.024
0.50-0.55
1.32±0.11
43.18 5.48
XI
XII
m
1.13±0.029
1.10-1.16
0.66±0.00
0.66-0.66
0.47±0.003
0.44-0.50
1.42±0.08
41.46 5.11
X
m
1.44
-
0.88
-
0.55
-
1.60
38.19 6.49
Y
m
2.38
-
1.27
-
1.10
-
1.15
46.22 10.73
* x ±SE = mean ± standard error; CL = absolute length of individual chromosomes; L = long arm length; S = short arm length;
r = arm ratio; CI = centromeric index; RL = relative length; m = metacentric; sm = submetacentric.
193
CYTOGENETIC TRAITS IN SEABUCKTHORN GENOTYPES
noides L. ssp. gyantsensis, 2n = 18m + 6sm; H.
rhamnoides L. ssp. sinensis, 2n = 18m + 6sm; H.
rhamnoides L. ssp. turkestanica, 2n = 20m + 4sm),
and for other three species of Hippophae genus (H. salicifolia, 2n = 10m + 14sm; H. thibetana, 2n = 14m(2sat) + 8sm; H. neurocarpa,
2n = 18m + 6sm) also showed only m and sm
chromosomes.
Generally, the karyotypes with preponderantly metacentric and submetacentric small sized
chromosomes (<4µm) are considered to be symmetrical, little evolved and relatively stable (STEBBINS 1971; ACOSTA et al. 2005; PASZKO 2006). If
CB-1 genotype has 54.17% m chromosomes and
45.83% sm chromosomes, in S-16 genotype the
ratio between metacentrics and submetacentrics
is 3:1, whereas in L-4 genotype only a small fraction (8.34%) is represented by sm chromosomes.
Sex chromosomes have evolved in a limited
number of dioecious angiosperms, and only in a
few of them the heterosomes have been certainly
demonstrated and characterized at cytological
and/or molecular levels (CHARLESWORTH 2002).
Like mammals, the dioecious angiosperms with
dimorphic sex chromosomes generally have XY
males and XX females (AINSWORTH 2000). According to the definitions of VYSKOT and HOBZA
(2004) and KEJNOVSKY et al. (2009), the heteromorphic chromosomes are microscopically sizeor shape-distinguishable chromosomes in the
two sexes, both from autosomes and from each
other.
In literature, H. rhamnoides is presented as
a species with heterogametic male and homogametic female, but the existence of heterosomes
never was irrefutably proved. In hemp (SAKAMOTO et al. 1998), white campion (NAKAO et al.
2002), sorrel (SHIBATA et al. 1999), Coccinia indica (SHARMA and SEN 2002) the Y chromosome
is lengthier than X chromosome and autosomes.
The chromosome analysis and estimation of nuclear DNA in both sexes of Coccinia indica evi-
TABLE 3 — Morphometric characteristics of mitotic chromosomes in L-4 genotype.
chromosome
morphotype
pair
CL
L
S
µm*
range
µm*
range
µm*
range
r*
CI
RL
(%)
I
m
2.88±0.024
2.85-2.90
1.48±0.024
1.45-1.50
1.40±0.00
1.40-1.40
1.05±0.014 48.70 13.23
II
m
2.83±0.024
2.80-2.85
1.63±0.024
1.60-1.65
1.20±0.00
1.20-1.20
1.35±0.024 42.48 13.00
III
m
2.13±0.16
1.95-2.30
1.18±0.12
1.05-1.30
0.95±0.049
0.90-1.00
1.24±0.06
IV
sm
2.05±0.049
2.00-2.10
1.33±0.024
1.30-1.35
0.73±0.024
0.70-0.75
1.83±0.028 35.37 9.43
44.71 9.78
V
m
1.78±0.024
1.75-1.80
1.10±0.00
1.10-1.30
0.68±0.024
0.65-0.70
1.63±0.05
VI
m
1.68±0.024
1.65-1.70
1.00±0.00
1.00-1.10
0.68±0.024
0.65-0.70
1.48±0.049 40.30 7.71
VII
m
1.63±0.024
1.60-1.65
0.90±0.00
0.90-0.90
0.73±0.02
0.70-0.75
1.24±0.042 44.62 7.48
VIII
m
1.50±0.00
1.50-1.50
0.90±0.00
0.90-0.90
0.60±0.00
0.60-0.60
1.50±0.00
IX
m
1.43±0.024
1.40-1.45
0.88±0.024
0.85-0.90
0.55±0.00
0.55-0.55
1.59±0.041 38.60 6.56
X
m
1.38±0.024
1.35-1.40
0.78±0.024
0.75-0.80
0.60±0.00
0.60-0.60
1.29±0.039 43.64 6.33
XI
m
1.33±0.024
1.30-1.35
0.70±0.00
0.70-0.70
0.63±0.024
0.60-0.65
1.12±0.041 47.17 6.10
XII
m
1.15±0.099
1.05-1.25
0.75±0.00
0.75-0.75
0.53±0.024
0.50-0.55
1.43±0.069 45.65 5.29
38.03 8.17
40.00 6.90
* x ±SE = mean ± standard error; CL = absolute length of individual chromosomes; L = long arm length; S = short arm length;
r = arm ratio; CI = centromeric index; RL = relative length; m = metacentric; sm = submetacentric.
TABLE 4 — Principal karyotypic features of three seabuckthorn genotypes.
genotype
somatic
chromosome number
karyotype formula
LHC (µm)
CL (µm)
x ± SE
putative sex
CB-1
2n=24
13m+11sm
23.97
1.99±0.14
male, 22+XY
S-16
2n=24
18m+6sm
22.20
1.85±0.16
male, 22+XY
L-4
2n=24
22m+2sm
21.73
1.81±0.16
female, 22+XX
LHC = length of haploid complement; CL = absolute length of individual chromosomes/genotype; x ±SE = mean ± standard
error.
194
TRUTA , CAPRARU , ROSU , ZAMFIRACHE , OLTEANU
denced a difference between 4C nuclear DNA
value of male (10.35 pg) and female (8.25 pg)
(SHARMA and CHATTOPADHYAY 1991), in relation
to the presence of longer Y chromosome in male
individuals. Similarly, the hemp genome size –
1683 Mbp for male, 1636 Mbp for female – additionally confirms larger dimensions of Y chromosome in this species (SAKAMOTO et al. 1998).
We specify that the following discussions are
issued with the reserve that in literature there is
no information about morphology and size of
sex chromosomes in seabuckthorn which allow
us to realize a comparative analysis about this
problem, although their existence is accepted in
this dioecious species. In the absence of specific
information, our considerations on heterosomes
in analysed genotypes are based on the fact that,
generally, in dioecious plants with sexual dimorphism, Y-chromosome is bigger than X-chromosome, these two chromosomes being heteromorphic. With respect to sex chromosomes in
analysed genotypes, their presence was searched
in this investigation by looking for morphologically different heterosomes. As we previously
showed, the literature data about seabuckthorn
sex chromosomes are based mainly on the results
obtained in field hybridization experiments, and
not on direct cytogenetic investigation. In present research, the CB-1 and S-16 genotypes have
eleven homologous chromosome pairs and one
heteromorphic pair of chromosomes differing by
size and morphology. Considering the previous
assertions on size of sex chromosomes in other
dioecious plants with sexual dimorphism, we
state that these chromosomes could be good candidates for the quality of sex chromosomes, and
and MANZU
the respective genotypes could be considered as
male genotypes. Consequently, L-4 genotype displaying twelve pairs of morphologically similar
chromosomes could be considered as being female genotype. Therefore, in CB-1 and S-16 genotypes, the pair XII puts together the putative
X-chromosome, with smaller size (2.29 µm in
CB-1, respectively 1.44 µm in S-16) and the putative Y-chromosome, of larger size (3.40 µm, in
CB-1, respectively 2.38 µm, in S-16) (Fig. 2). The
total chromosome length and the relative length
of Y-heterosome exceed X-chromosome size in
both analysed genotypes. The values of centromeric index and arm ratio (CI = 45.77, r = 1.18,
for CB-1; CI = 46.22, r = 1.15, for S-16) include
Y-chromosome in the category of metacentric
chromosomes, with median placed centromere.
The putative X-chromosome of male genotypes is of submetacentric type in CB-1 genotype
(CI = 31.94; r = 2.10), respectively of metacentric
type in S-16 genotype (CI = 38.19; r = 1.60). Value
of r parameter in S-16 putative X-chromosome is
very close to the limit beyond which the chromosome could be included in sm category, like Xheterosome from CB-1 male karyotype. Because
of the small and very small sizes of chromosomes,
it was somewhat difficult to make a very exact
determination of centromere position, especially
for the chromosomes smaller than 2 µm where
the details are few distinguishable.
In this species, the female genotypes are economically preferred over male genotypes and the
breeding efforts are mainly orientated towards
the development of valuable female phenotypes
and to removal of undesired males. In this moment there is no efficient method to discriminate
(a)
(b)
(c)
Fig. 2 - Seabuckthorn karyotypes. a CB-1 (male); b S-16 (male); c L-4 (female).
195
CYTOGENETIC TRAITS IN SEABUCKTHORN GENOTYPES
male and female individuals prior to sexual maturity in order to assure the commercially favourable sex ratio. A proportion of 7-12% of staminate trees is considered adequate for pollination,
namely a sex ratio of 1♂:6♀–1♂:8♀ (JANA et al.
2002; LI 2002), or even 1♂:9♀ (SINGH 1998). The
identification of specific molecular genetic markers in order to allow early discrimination and removal of superfluous male plants may be helpful
in seabuckthorn breeding programmes. In the
researches of PERSSON and NYBOM (1998) and
PERSSON (2001), although in the F1 descendance
of one cross, the RAPD marker was present both
in male parental and in all male descendants and
was absent in all female individuals, it can not be
considered as universal, because in F1 progeny
of another cross it was present in only one of the
male individuals. Instead, SHARMA et al. (2010)
have reported the detection of peroxidase isozyme markers associated with females and of one
RAPD marker (OPD-20911) certainly linked with
seabuckthorn males, fact extremely important in
the early assessment of gender in H. rhamnoides
L. before anthesis, as well as in the understanding the molecular basis of sex determination in
this dioecious species.
In conclusion, this cyogenetic study established the diploid chromosome number (2n = 24)
in three genotypes of Hippophae rhamnoides L.
Chromosomes are small sized (< 3µm), without
satellites, only of metacentric and submetacentric type. Based on their size and morphological characteristics, we presumed the existence
of heterosomes. In two of analysed genotypes,
presumed to be male, the karyotype formulae
have eleven homologous chromosome pairs
and one pair of heteromorphic chromosomes,
whereas one genotype, considered as female, is
different, showing twelve pairs of morphologically undifferentiated chromosomes. According
to obtained data, the three karyotypes display a
relatively high degree of symmetry and can be
considered as less evolved and less subjected to
significant genetic restructurations during evolution. Also, they show a low intraspecific variation
concerning metric and morphological traits of
chromosomes. Our results contribute to the enlargement of knowledge concerning the chromosome constitution of seabuckthorn, a dioecious
species with an impressive genotype and phenotype variability, little known in terms of mechanisms governing sex determinism. Undoubtedly,
these classical cytogenetic investigations must
be correlated with thoroughgoing molecular approaches in order to identify new unequivocal
markers linked to sex determination or to other
valuable phenotypic traits and to ensure a solid
support for efficient and modern breeding programmes of seabuckthorn.
Acknowledgements — This research was realized in the project CEEX-BIOTECH 109/2006-2008,
from National Research Program of Romania, with
the financial support of Ministry of Education and
Research. We thank Professor Ioan Viorel Rati and
Dr. Milian Gurau for providing the plant material for
cytogenetic investigations.
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Received November 18th 2010; accepted April 5th 2011