Chromosomal sex determination and

Sex Plant Reprod ( 1996 ) 9 : 127 - 132
iD Springer-Verlag 1996
S. Si ljak-Ya kovIev • S. Ben m al ek . M. Cerbah
T. Co ba de la Pefia . N. Bo unaga • S.C . Brown
A. Sa r r
Chromosomal sex determination and heterochromatin structure
in date palm
Recei ved: 20 November 1995 I Revision acce pted: 26 Dece mber 1995
Abstrac t In tbe date palm. a dioecious mode (separate
male and female individuals ) and the late initial reproductive age (5-10 years ) are major practical constraints
for genetic imp rovement. Ear ly selection on young seedlings co uld enhan ce breeding prog ra mmes and generate
experimenta l male and fe ma le genetic stocks. but no cytoge net ic prot ocol exists for sex determination in an im matu re date palm. Here we describ e a cytu log ica l method based on chro rn o rn ycin staining which dem on strates
the occurrence of sexual chromosomes carrying di stinctive nucl eo lar heterochromatin and thu s offe rs, for the
first time . the po ssibili ty of identifyin g male and female
individuals by simple analysis of root mer istems . This
o bservat ion has been exte nde d by in sit u rD NA hybridization . co nfoca l microscopy and dual-label flow cy tornetry of nuclei.
Key words Sex chromosomes · Sex-di ffe rentiating
chro mocentres . Heterochro matin · In situ hybridization ·
Phoenix dactylifera
Only about 5 % of the ge nera of high er plants are recorded as wholly dioeciou s, Man y of the dio eciou s species
S. Srljak -Yakov lev ( ~) . M . Cerba h . A. Sa rr
Laboratoire d'Bvolution et Systcmatique vegerales.
CN RS URA 1492. U nivcr site Paris-Sud, Bfttimc nt 36 2.
F-9 I405 Orsay Ce de x. Fr ance ;
Te!. +33- 1-69 4 1 65 30. Fax +33-1-6941 7353
S. Bcnmalek . N. Bnunnga
Centre de Rech erch e Scic ntifiq ue et Technol ogiq uc
sur le, Regio ns Aridcs. Laboratoire de Botaniquc. BP 119.
1600 0 Algiers-G are. Algeria
T. Co ba de la Pefia . S.C. Bro wn
Institut des Sciences Veget ales, CNRS UPR 40 ,
F-9 1198 Gi f-sur-Y vette Ccd cx, Fra nce
T. Coba de la Pefia
Departamcnto de Rccurs os Naturai s e Media Ambicn tc.
Uui versida de de Vigo . vigo. Spain
do no t have disting uishab le heteromorphic ch ro mosomes
(Chattopadhyay and Sharma 1991 ; Negrutiu and Sala
199 I ; Parker and Clark 1991), while the cytogenetics of
most di oecious species rem ain unex plored (Wesrerg aard
1958; Durand and Durand 1984). S peci es of the Palrnae
fami ly are freq ue ntly dioecious. Data co nce rni ng their
sex determination are meagre (Sinoto 192 9; Sar kar
1956 ), but suggest one heteromorphic chromosome pai r
in the male. Date palm (Pho enix dactylifera L. , 2n=36) is
outstand ing for the econ omic value of its different part s
and its socio-economic significance in agrosystems of
arid zo nes. Annual world date production is about 3.7
milli on tons (Anonymo us 1992 ). Yet the success of
breeding programmes has been very low, due to the wide
gap bet ween pure scie nce (ge ne tics , physiology and
pl ant pat hology) and applie d science co nce rni ng this
species (To mlinson 1990).
There is a general lack of understand ing of the genetics of palm. One male is used to hand pollinate about
90-100 females (Bounag a 1993). and for centuries emphasis has bee n on clonal propagation of fema les. This
red uces the ge netic divers ity of the cultivars, acce lerating
vulnerability to biotic and ab iotic stresses. In particula r, a
severe fusariose wilt caused by Fusarium oxysporum f.
sp. albedinis (Killian & Maire ) Gordon and ca lled "B ayoud" is destroyi ng nob le palm s thro ughout Nort h Afri ca ; two-third s of the Moroccan stand has been eliminated
during this century (Brac de la Perric re and Be nkhalifa
199 I ). Tolerant or resistant cultivars have alrea dy been
identi fied (Lo uvet and Tou tain 1973; Bendiab et a!.
1993). G reater evaluation and ma nipulation of ge netic diversity within the Phoen ix ge ne pool is urgently needed
for sus tainable produ ction species conservalion and
env ironmental preservation. T he cyto logical marke rs of
sex in Phoenix , indicated herein, provide an ess ent ial tool
for any such genetic pro gr amme.
Materials and methods
Female individuals were fro m six cu llivars: Cherka from Belli
Abbes oasis (Algeria); and Degla Beid a, Deglet No ur, Ghares,
Fig. l A-D Metaphase and inte rphase plate s showing chromomycin fluore scent XX and X Y co mbinations in Phoenix dactyli/era.
Two homomorph ic chromosomes (A ) or cbr omocenrrcs (C) appeared on female plants. and a pair of heteromorphic chromoso mes (B ) or chromocentres ( 1) ) appeared on male plants. Bar
Fig. 2 Interp hase da te palm nuclei showing chro moce ntres of di ffcrent sizes according to sex (A fem ale. B male). Confoea l microscope (Biok ad (00) images of nucl ei reconstruc ted from about 40
op tical sections. Arrows indicate 90° rotations of spo ts. the larger
arro w correspondin g to the larger spot. Bar 10 urn
Fig. 3 The chrumoccn tres of interphase date palm nucle i have
eq ual intensifies in female s ( A ) and unequal iutens itics in males
(8). They sometime fuse (arrows) . For 49 female ce lls. cc rnpariso n of the areas of the two spots within each nucleus gave an average ratio of 0.85 (SD=O. I I). while for 45 male ce lls this ratio was
0.66 (50 =0. 14). Whcn comp aring the two spot intensitics, the ratio was 0.85 (SD=0. 14) for fe males and 0.5 2 (5 0=0. 14) for males.
Rar 10 p m
Tilem sou and Tinaseur from the collection of INRA. Adrar (Algcrial . Male individua ls were from spontaneous seed germin ation in
Belli Abbcs oasis. All the indi viduals (five per cultivar) were 15·
year-old adults. Root tips were prctreated accord ing 10 the usual
karyologica l methods tOsrcrgrcc n and Hcn ccn 1962) and submitted to all adapted chrumomycin fl uorochrome band ing techniqu e
to reveal the GC-rich DNA and heteroch romatin (Scbweiz cr 1976:
Marli n and Hesemann 191'8 ). Stainings wer e made in chromomycin A3 (Serva . Heidelberg, Germ any} 0.1 mglml in Mcl lvaines
pH 7 buffer. afte r a blockin g pretreatment by <I no n- fluorescent antibio tic di stamycin A tServa) 0. 1 mg/ml in the same buffer. Fading
was prevented by an addi tional staining in 0. 1q.. meth yl green solution (S OH. Lo ndon. UK ) in Mcl lvainc' s pH 5.5 buffe r.
For in situ hybridi zation. chromosome spreads of Cherka cultivar were obtained using the me tho d described by Geber and
Schweizer ( 1987). A subclonc of pTA7 1 containing a (U;·kb TaqI
fragment of wheat ribosomal DNA (Gerlach and Bedrock 1979)
which carries a part of the coding seque nce for the 185 gene was
used as the probe. Plasmid DN A was labelled by PC R with digoxigcni n- l l -dlj'IP (Boeh ringer Mannh eirn. Ma nnhe im. Germany). In situ hybrid ization. de tectio n and visualization was carried
out fo llowing the procedure of Heslo p-Harrison et a l. ( 1991) with
slight modific ation.
The epifluoresce nt Zeiss Axiophor micro scope was used with
filler set 07 (exc itation 436-460 nm. emission 5 10-520 nm lone
pass} for observation of chromomycin-stained slides. and with
FtTC tille r for in situ hybridization.
Flow cy tometry was do ne on an EPICS V (Coultronics France,
Margency. France ) using Spectra Physics 2025 lasers (argon at
45X nm for chromomycin and krypton at 346 nm for Hoechst ).
Leaves were chop ped in Marie' s nuclear iso lation buffer (Marie
nnd Brown 1993), alo ng with petunia leaf as internal standard. ATspec ific bisbe nzimidc Hocchsr 333 42 (Aldri ch) was added at
5 ug/ml and Gc-speciflc chromomyci n A3 (Serva) at 50 ~l g/ml
with 50 mM MgCI.'! . Genome size was assessed with ethidium bromide (Sigma) at 50 pg/ml after RNase treatment of the nuclear
suspe nsion. Each value is the mean of at least 2000 nucle i.
Chrom omycin-bande d nuclei (50 ) of principally Chcrka cultivar and a sample of abo ut 50 nucle i from a male accessio n were
analysed by confocal microscopy (Molecular Dynamics Sa rastro
2000 and BioRnd 600) using 457 nm excitation. em ission above
530 nm . a x 60 objective ( l A NA. Nikon) and a series of 20 sections (51 2x5 12 or 256x2 56 with Z increment 0 .2 pm) .
The Z series of images were filtered with 3x3 x3 matrices using
firstly a median. (hen a guussiau algo rithm. "Maximum intensity"
20 projec tions, retaining the highest intensity at each (x,y ) coo rdinare. were used to op timize contrast in nor mal im ages. On the o ther
hand. quantification of the fluo rescenc e assoc iated with heterochromatin was done in lWO ways using the Sarastro software. Either
"loo k through" 2D projections. integrating intenxities at each (x ,y )
coo rdinate. were used for 16 -hit image analysis, or. altern atively,
the vo lume so ftware was applied to filtered series using a threshold
chosen for the image most equatorial to a heterochromatin spot.
The latter procedure yie lded integral intensifies, average pixel in·
ten xlries. variance. and a vo lume estimate (J1m 3 per spo t).
Thresholding is an uncertain procedure in 3D; operatio ns were
more re produci ble from day to day or from person to perso n when
simply usi ng the "loo k through" 2 D projections.
120 , -- -- - - --
- --
- -- ,
• ~"'.1l'J
• •
90 -I---
c-- - - ' - - - - - - + - - - - j
Rel ative Int ensi ty Hoechst 33342
Results and discussion
Date palm is cytoge netically recalcitrant material, having
tiny and sticky chromosomes (Beal 1937; So liman and
AI-Mayah 1978; AI-Salih et al. 1987a,b). The potential
for sex diagnosis by baoding techniqu es or X-ray-induced fragmentation has been indicated (Chattopadhyay
and Sharma 1991) . Classical Giemsa C-banding (Marks
1975) has not allowed us to differenti ate male and female individuals. However, fluoroch rome banding of
root chromosomes with chromomyci n A3 (binding especially on GC- rich DNA sequences) did discri minate female and male individuals (Fig. I). The female has a pair
of GC-ric h homomorphic chromosomes (Fig. l A); the
analogo us pair is heteromorphic in the male (Fig. IB).
Therefo re, in our opinion, the XY sex-determining chromosome system cou ld be applicable to date palm . T his
heteromorphi c system was also visualize d on quiesce nt
nuclei by chromomyci n-binding spots indicating
chromocen tres. The nuclei of female root cell s show two
identical spots (Fig. IC), while the two spots on male nuclei have differen t sizes (Fig. ID ). The heteropycnosity
of the two sex-spec ific interphas e nuclear spots indicates
their heterochromatic constitution .
The se initial observations. readily discernible by epitluoresce nt microscopy, were confirmed by confocal microsco py (Figs. 2, 3). Because of considera ble variatio n
in staining between slides and even within slides, the essential co mpariso n invol ved calculating the ratio of sizes
and the ratio of summed fluorescent intensities for the
heterochromatin spots within each nucl eus. This ratio
should approach unity if the heterochrom atic spots are
identical, namely, on homomorphic chromosomes. In a
co mpariso n of the smaller and larger spots in a g iven
male ce ll, the ratio of project ed 20 areas was about 0.66
which, assuming spherical forms, would correspond to a
ratio of 3D volumes of about 0.54. In other words, one
Fig. 4 Simu ltaneous asses sment of fluorescence intcnsities due to
AT-spec ific bisbenzimide Hoechst 33342 (ab sci ssa) and Gc-specific chromomycin A3 (ordinate ). Each point is the mean of a
cytometric analysis of 2000--6000 nuclei. Each of the seven female cu ltivars (solid symbols) and four male individuals (ope n
symbols ) were measured at least twice
spot is about doub le the volume of the other. The ratio of
their heterochromatin content (integrated fluore scent intensities) was 0.52 for males. This ratio was 0.85 for
Cherka females, which is not significantly different from
1.0 given that the method of calculation (ratioesmaller/larger) allocated all dispersion to one side.
In some interphasic nuclei , fusion between sex-differentiating spots was occasionally observed (Fig. 3B, arrows).
Five other (female) c ullivars gave the same homomorphism, both for metaph ase plates and interphasic nuclei stained with chromomyc in. Four other male individuals showed the same heteromo rphis m.
By epitluorescence microscopy, it appeared that the
heterochromatin spots of the female were similar in both
size and intensity to the larger of the unequal spots in the
male. This observation was tes ted using a mixture of one
male and one female root meristern and assessing the
slides by confoca l microscopy to obtain the regres sion
between the spot size ratio (smaller/larger for each nucleus) and the summed intensity of heteroc hromatin
staining of this give n nucleus. These two parameters
were positively correlated; the heteromorph ism was associated with lower amo unts of heterochromat in. Apparently the evolution toward s differenti al localization of
sex-determining genes has been associated with the loss
of GC-rich heterochromatin in the Y chromosome or
gain of such heterochoma tin in the X chromosome.
Flow cyto metric analys is of dual-labelled nuclei using
chrornomycin A3 for GC content and bisbenzimide Hoechst 33342 for AT content also showed a slight increase
Fig.5 Two differentiating chromocentres (X.Y) associated with
the nucl eo lus in A mitotic interphase and B meiotic prophase. C
The se x chromos o me bivalent in me tap hase I. J) A naphase II: E
telophase 11 . F, F' Two op tical sectio ns of tetrad show ing spatial
segregation of spots. G Sex-differe ntiating spo ts in nu crosporc s:
general vie w of free rmcrosporcs. La rge and small lII"I"OIl'S indicate
X and Y sex-differen tiatin g spots. respec tive ly. Bar 10 ~Il1
Fi g. 6 In situ hybridization with an rDNA probe shows two chrorrmsoma l sites in me taphase plates (A) and two distinct spots in interphase nuclei (B) . These chromosomal (A') and intcrph asic (B')
spo ts are nega tive wi th DAPI staining (a rro ws ). Bar 10 pm
palm, as the nucleolus is always associated in interphase
wi th the two sex-differentiating spots (Fig. 5A). Th e
same feat ure is also ob serve d d uring meio sis propha se (Fig. 5B ). In add ition. in si tu hybridiza tion on
met aphase plates and interphase nuclei shows one pair
of rD NA sites (F ig. 6A . B). T hese sites were DAPI
(AT-dependent) negative and co nseq oenl1y are GC
rich (Fig . 6A'. B' ). T hey pro babl y cor res po nd to the nucleolar part of sex chro moso mes stained by chrom om y-
in the percen tage of AT in the male ge nome (Fig . 4) , sig nifi cant at P =O.Ol (Stude nt's r-rest with 27 (if!. Although
the ranges of fem ale and ma le values overlap in this scatter diagram, the slope of re lative intensities chrornomycinIHoechs t is higher for the female popu lati on than for
the male, which ag ree s wit h the concl usio ns from microscopy, Incide ntally, the geno me size calcu lated from
3 1 histogram s fo r Ph. dactyli/era was 2C=1.58 ±0.0l pg,
using ethidium bromide as DNA stain and Petunia hybrida (2C=2.85 pg, 41 .0% GC ) as internal reference. Th e
average GC compos ition was 42 .3±0.2 % as es tima ted
using Godellc 's simpli fied ca lculation (Godelle et a!'
1993 ) after flow cy tome try of isolated nuclei se parate ly
stained with Hoec hst and chromo myc in.
Sexual c hrom osom es can be nucleol ar (Chatto padhy ay and Shan n a 1991). T his is the ca se fo r da te
Figure 5B-G shows the behaviour and segregation of
sex-differentiating chromosome spots during meiosis divisio n fro m prophase to microspore stage . In some date
palm ge notypes the po llen exe rcises a direct effect on the
matern al tissues of the fruit, ind ucing differences in
terms of ripening or size and co lour. This phenomenon
(rnetaxenia) was first descri bed in 192 8 (Nixo n 1928)
and is curre ntly being studied (Lakho ua 1966; Osman et
al. 1974 ; Faki r and Carbonnier 1992 ). Altho ugh the
mechanism underlying metaxenia is unknown, given that
our method allow s sex chromosome identification on the
haploi d phase the incidence of X or Y chromosome s on
the ability of poll en to express this phenomenon can now
be studied. In particular, we can now test for any distortion in the frequ ency of X- and Y-bear ing pollen and determine the conformity betw een the sex ratios in such
pollen and the seedling population.
Date pa lm has been classified as dioecious without
recog nizabl e sex chromosomes (Chattopadhyay and
Sharma 199 1) and , until now, without karyological dis tinc tion between male and female plants. Alternative but
unsatisfac tory (Bennaceur and Bounaga, unp ublis hed da ta ) bio ch emical discriminators of sex have bee n studied.
A procedure based on peroxidase and/or esterase spec ificity for earl y distinction between male and femal e individuals has been patented (Su gan uma 1984) .
This is the first substantial evidence of sexual cytogenetic differentiation between ma le and female individuals in date palm. Mor eover, our methodology based on
specific DNA staining is reli able, easy to handle for
practical applications, and appli cab le to young seedlings.
Th is ana lysis should be performed on qu iescent nuclei or
0 11 met aphase plates. The heterochromatin spots containing nucleolus organizer regions tend to fragment as the
nucleus becomes ac tive, even in interphase. Therefore,
nuclei with more than two spots should be rejec ted, as
should those few nuclei with only on c spot resu lting
from fusion of the heteroc hromatin . In practi ce, image
analysis is not necessary. Simple microscopic observatio n of about 50 interphase nuclei (or some metaphase
plates) is adequate to discriminate male from female individ uals. We have seen cul tivars with additional GC rich heterochromatin, but these mino r band s do not hin der sexual diagnosis .
This fi nding will faci litate the urgentl y needed (Tomlinson 1990) genetic studies of thi s important crop. The
dioecism and the long time span until sex ual maturity
have led to selec tion programmes bas ed on clonal propagation of femal es from good date palm cuItivars. As stated hy Frankel (1989), this procedure promotes genet ic
unifor mity, accelerates the process of geneti c ero sion ,
and enhances the vulnerability of crops to environmental
stresse s. The high incidence and severity of fu sariose
disease in North African date palm orchards could be a
consequence of the wide diffus ion of genetically uniform
varieti es. Thu s, evaluation and proper use of the great
genetic diversity of Palmae (dat e palm and its wild re latives) is probably, as for any crop, the most appropriate
way to solve current and fut ure plant breeding problems
with respect to sustainable production and environment
The early sex determination on directed or chance
crosses (the latter being curre nt agronomic practice ) will
enable breeders to more efficiently create breeding male
and fem ale po pulations reflecting the dive rsity generated
by sex ual recombina tions .
The Pal mae fami ly also includes some monoecious
species (separate male and female inflo rescen ce s on the
sam e individual), while sex inversion and biased sex ratios have been repor ted (Tomlinson 1990; Oudejans
1974). The present protocol, which appears to be applicable to other Phoenix specie s (e.g. Ph. canariensis hort.
ex Chab aud), also opens new prospects for mor e fundamental studies on sex evo lution, parti cularly wi thin the
Palmae fam ily.
Acknowledgements Th e authors kind ly acknowled ge O . Robin
for cytolog ical ana lysis . Dr. M . Laure nt (Ors ay) and J. -R. Prat
(Gif-sur-Yvette ) for con focal micro scopy, D. Marie for initial cytometry an d Dr. R . Brac de la Perriere for indi catio ns concerning
cho ice of the material. Thi s work is support ed by a coop era tive
conve ntion bet ween the two fo llow ing bod ies: D irection du Developpement et de la Cooperation Sci cntifiq uc, Technique et Educative du Mini stere Frunc ais de s Affaires Etra nge res and Ministerc
Alge rien Dele gue aux Univ ers itcs et a la Rech erche Scicntiflque.
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