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Development of sex-associated SCAR markers in Piper longum L,

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Plant Genetic Resources Newsletter
published in Issue No.141, page 44 to 50

Development of sex-associated SCAR markers in Piper longum L.

P. Manoj N.S. Banerjee P. Ravichandran

Introduction

Piper longum L., a dioecious plant species, is well known in tropical India for the medicinal
value of its different plant parts For example, mature female spikes are highly valued for their
use in the treatment of upper respiratory tract diseases in traditional and ayurvedic medicine in
India (Viswanathan 1995).

Piper longum plants are functionally dioecious and have no distinguishing cytological or
vegetative features to identify the sex of plants. Both male and female plants remain
indistinguishable until the flowering stage, when male plants can be identified by their long
spikes and female plants by their short spikes. Where the plant is grown for its female spikes, the
requirement to grow plants to maturity before eliminating the unwanted males represents a waste
of resources. The plant is vegetatively propagated through stem cuttings and through tillers
arising from the base of mature plants because the seeds are very minute and embedded in the
spikes and are difficult to germinate eds. The plants maintain their sexual identity throughout
their successive generations.

In our experience, a 1:6 male:female population ratio is sufficient for cultivation. For a better
spike yield, a minimum number of male plants are necessary during cultivation. A method for
identifying the sex of plants at younger stage would therefore help in the elimination of
unwanted plants. The molecular basis of sex determination seems to affect the development and
accumulation of medicinal properties in the spikes of the female plant (Banerjee et al. 1999).
However, the chromosomal, genetic, or molecular factors determining the sexual phenotype in
this species are not known.

Dioecism is found only in a small number of plant species. Yampolsky and Yampolsky (1922)
noted that while only 5% of plant genera are wholly dioecious, 75% of flowering plant families
have some dioecious species. Dioecy (separate male and female individuals) is well established
in animals, but occurs sporadically in plants. Charlesworth (1985) extended the compilation of
Yampolsky and Yampolsky(1922) and identified 1303 genera and 170 families in which dioecy
is well established, understanding the genetic and epigenetic factors controlling sex
determination in dioecious plants is still at a preliminary stage. Chromosomal determination of
sexual phenotype has been detected in only five dioecious plant families (Parker 1990). As in
higher animals, strict genetic control of sex expression by heteromorphic X and Y chromosomes
has been established in the case of Silene latifolia and Silene dioica L. in the Caryophyllaceae
family (Westergaard 1946): in these species, the stamen development process in male flowers is
determined by the dominant influence of the Y chromosome. In contrast, in Rumex acetosa
(wood sorrel) the sex of an individual plant is influenced by a dosage compensation mechanism
based on thex chromosome to autosome ratio (Ainsworth et al. 1995). Yet, the influence of
epigenetic factors such as environmental conditions and phytohormones are known to control sex
determination in some other dioecious plant species such as Mercurialis annua (Louis 1989),
Cannabis sativa (Galoch 1978) and Arisaema triphyllum (Policanski 1981). Silene latifolia is the
only case where the molecular genetic control of sex determination is understood. Distinct Y
chromosomal regions and the genes essential for the suppression of pistil development and
promotion of stamen development in male plants have been identified from this species (Ye et al.
1991; Grant et al. 1994; Hardenack et al. 1994; Donnison et al. 1996; Matsunaga et al. 1996;
Barbacar et al. 1997; Robertson et al. 1997; Scutt et al. 1997; Farbos et al. 1999).

The RAPD technique (Welsh and McClelland 1990; Williams et al. 1990) has been used to
develop DNA markers linked to sexual phenotype in P. longum (Banerjee et al. 1999), Silene
latifolia (Zhang et al. 1998), Pistacia vera (Hormaza et al. 1994), Salix viminalis (Alstrom-
Rapaport et al. 1998), Myristica fragrans (nutmeg), Cannabis sativa (Mandolino et al. 1999) and
Actinidia species (Gill et al. 1998). It has been suggested that the RAPD markers should be
converted to sequence characterized amplified region SCAR markers based on their DNA
sequence, which could be detected through polymerase chain reaction (PCR) with longer
sequence-specific primers (Paran and Michelmore 1993). Therefore, several of the sex-linked
random amplified polymorphic DNA (RAPD) markers have been converted into SCAR markers
i.e., in Salix viminalis (Gunter et al. 2003), in Actinidia chinensis (Gill et al. 1998, Geoffrey et al.
1995) in Papaya (Urasaki et al. 2002) and several resistance RAPD markers converted to SCAR
(Paran and Michelmore 1993 , Arnedo et al. 2002).

Here, we report on the identification of two male-associated RAPD markers and the development
of one male sex-associated SCAR marker in P. longum.

Materials and methods

Plant material and DNA isolation

Various accessions of plants were obtained from the locations listed in Table 1. We analyzed
genetic differences in a germplasm collection consisting of 96 female and 40 male accessions of
P. longum L. bearing spikes. Total genomic DNA was isolated from young healthy leaves using
a standard protocol (Rogers and Bendich 1988).

RAPD and SCAR analysis

The purified genomic DNA was subjected to PCR for RAPD analysis using random decamer
oligonucleotide primers (OPA1-20, OPE1-20, OPD1-20 and OPAC1-20) from M/S Operon,
USA. The PCR reaction mixture consisted of 20 ng genomic DNA, 200 µM each of dNTPs
(dATP, dGTP, dCTP and dTTP), 15-picomole primer, 1x Taq DNA polymerase buffer and 0.5
units of Taq DNA polymerase (M/S Promega) in a final volume of 20 µl in sterile ultra-pure
water overlayed with 30 µl mineral oil. The PCR was performed in a Perkin Elmer PE480
thermal cycler for 40 cycles of denaturation at 94°C for 1 min, annealing at 40°C for 1 min and
extension at 72°C for 2 min followed by final extension at 72°C for 7 min. The PCR products
were visualized with 1.2% agarose gel electrophoresis in 1x Tris-borate–EDTA buffer
(Sambrook et al. 1989) and documented.

Cloning and sequencing

The PCR products were eluted and purified from agarose gels by GFX PCR DNA and a Gel
Band purification kit (Amersham, 27-9602-01) and cloned in pGEMT-Easy vector-II by using
the supplier’s protocol (Promega, Cat. No. A1380). Genomic DNA digested with EcoRI, HaeIII,
HindIII and BamHI (NEB) was transferred to Hybond N+ nylon membrane (Amersham) by
vacuum blotting (Model 785 vacuum blotter; Biorad) and hybridized for specificity. Automated
sequencing was performed through PCR (Big DyeTM terminator cycle sequencing kit, PE Bio
systems, Cat No. 4303152) and capillary electrophoresis on ABI Prism 310. Sequence analysis
for ORF and identity with other known sequences were done through the United States National
Center for Biotechnology Information (NCBI) BLAST search available at
http://www.ncbi.nlm.nih.gov/BLAST .

Results

Identification of male sex-associated RAPD bands

RAPD profile of 40 male and 96 female P. longum accessions were generated with 80 different
10-mer primers of arbitrary sequences and compared. RAPD profiles of the male samples
generated by OPA10 and OPA15 primers were distinctly different from those of the females
because of the presence of prominent male-associated bands OPA10827 and OPA15744 (see Figure
1A, C). When these marker bands were used as probes for hybridization to RAPD gel blots, only
the above bands were detected and no hybridization was found with any bands in the RAPD
profile of the females generated by the primers OPA10 and OPA15 (Figure 1B, D). OPA10827
and OPA15744 did not cross-hybridize among themselves. This indicates that the two amplified
bands are different and either the corresponding sequence is absent in the females or the primers
used did not amplify any band of similar sequence from the females.

Sequencing of the male-specific RAPD bands

The male-associated RAPD bands OPA10827 and OPA15744 were cloned in PGEM T-Easy vector.
Plasmids from the clones were amplified using vector-specific primers, T7 and SP6 (Figure 2A),
further confirmed by Southern hybridization using male-specific fragments OPA10827 and
OPA15744 as probe (Figure 2B, C). These are sequenced by ABI310 prism automated sequencer.
DNA sequences were compared with the available database by BLAST search. The sequences
were named MADP1 and MADP2 (male-associated DNA) from nomenclature proposed by
Sakamoto et al. (1995). MADP1 and MADP2 have a total length of 827 and 744bp, respectively,
and contained no identifiable open reading frame (ORF) in any orientation or frames. A BLAST
search did not reveal any significant similarity of MADP1 (Acc. No. AY046931, GenBank,
NCBI) and MADP2 (Acc. No. AF375880, GenBank, NCBI) with the other known DNA
sequences in the available database (e.g., EMBL, GenBank, DDBJ and PMB). A similar
comparison with male-associated DNA sequences from other plant species also did not have any
significant resemblance (Table 2).
RFLP analysis

Genomic DNA from 12 female and 22 male plants of 5 different cultivars was cut with 11
different restriction endonucleases, blotted and hybridized with [32P]-labelled MADP1 and
MADP2 DNA. Several different hybridization patterns were obtained; however, no
polymorphism pattern could be detected.

SCAR analysis

Four SCAR primers, MPS1A and MPS1B, MPS2A and MPS2B, were constructed based on the
DNA sequence of the 5’ terminal regions of the MADPI and MADP2 sequence. SCARs were
amplified in the presence of longer primers (containing the RAPD primer sequence followed by
the next 12–15 nucleotides of the cloned RAPD marker bands) obtained from M/S Gibco-BRL
and used for the PCR amplification. The sequences, annealing temperature and product size of
SCAR primers are listed in Table 3. The SCAR primers MPS1A and MPS1B amplified an
intense band of 827bp at an annealing temperature of 60°C in all the genomic DNA accessions of
male plants (Figure 2D). Primers MPS2A and MPS2B amplified an intense band of 744bp at
60°C annealing temperature in all male plants and a faint band of 747bp in few female plants’
genomic DNA when tested against a large population. When the annealing temperature was
raised to 68°C, these primers amplified a dominant band in males only. Increasing the stringency
by the use of Mg concentration gradient produced a faint amplification of a band in male plants
only. However, this result was highly unreliable. Digestion of the male and female amplification
products by common restriction enzymes failed to retrieve a polymorphism between the male
and female plants. Hence the SCAR primers MPS2A and MPS2B cannot be used for an early
screening of male and female plants.

SCAR primers MPS1A and MPS1B were tested in a population of 210 P. longum plants. When
210 plants were taken randomly to predict the sex using MPS1A and MPS1B primers, it was
found that 36 plants (17%) were male; the sex ratio was 1:6. The sex of the plants was confirmed
by spike morphology when they flowered 6 months later and the result was similar to the
prediction. The result confirms the 100% reliability of the primers. Hence, before cultivation,
individual plants can be checked for sex identity.

SCAR fragments, generated by MADP2 sequence specific primers from the female P. longum
plants, were cloned and sequenced (MADP2F). Comparison of the DNA sequence of MADP2F
with that of MADP2 revealed 98.10% identity between them (Figure 3). MADP2 was found to
have a deletion of three nucleotides (ATT) at positions corresponding to positions 457 – 459 on
MADP2F DNA. Additionally single nucleotide mismatches at five positions (as indicated in the
figures) were also noticed.

Discussion

We previously reported that the male sex-associated RAPD markers, OPA10827 and OPAC12,
could detect genetic differences between male and female plants of P. longum (Banerjee et al.
1999). In the present study, we have identified another two male sex-associated RAPD markers,
OPA10827 and OPA15744, that were amplified with OPA10 and OPA15, respectively, from the
genomic DNA of only male plants of P. longum by using Taq DNA polymerase from M/S
Promega and screening 80 RAPD primers. These male-associated RAPD markers were
reproducibly produced by the use of identical enzyme and reaction conditions at a high annealing
temperature of 42°C and, when used as probes, hybridized only with the same band in the RAPD
profile of male DNA in RAPD gel blot hybridization experiments, confirming their association
with male phenotype in this species. In the past, RAPD has been found to be a very effective
technique for the identification of sex-linked molecular markers in several dioecious plant
species, e.g., P. longum (Banerjee et al. 1999), Cannabis sativa, Silene latifolia, Actinidia
chinensis, Myristica fragrans (nutmeg) etc. However, the efficiency of the technique is
dependent on the size and complexity of genome structure as well as strategic application of the
technique, which needs to be optimized on a case-by-case basis. The efficiency of the present
study for the detection of male-associated markers through RAPD is comparable to those of
Sakamoto et al. (1995) and Mandolino et al. (1999), who reported two and one male sex-
associated RAPD markers, respectively, in Cannabis sativa after screening 20 primers. On the
other hand, it was necessary to screen a large number (400–900) of RAPD primers to identify
useful sex-linked markers in Pistachio vera and Humulus lupulus (Hormaza et al. 1994; Polley et
al. 1997).

In RFLP analysis, the male-associated DNA markers, OPA10827 and OPA15744, hybridized with
DNA from both male and female P. longum plants at identical positions, thus excluding any
possibility of their origin from male-exclusive chromosomal regions. Sex-linked markers from
other plant species (Cannabis sativa, Actinidia chinensis and Silene latifolia) also had similar
hybridization to both male and female plant DNA (Sakamoto et al. 1995; Gill et al. 1998; Zhang
et al. 1998; Mandolino et al. 1999). This could be explained by supposing that localized
differences in the corresponding genomic regions, including the sequences of the RAPD primers
used, had prevented amplification of such marker bands from the genomic DNA of the female
plants.

Interestingly, our earlier reported male sex-associated RAPD markers of P. longum, OPA10827
and OPAC12, (Banerjee et al. 1999), originally identified through PCR with Taq DNA
polymerase procured from M/S Bangalore Genei, India, could not be generated using Taq DNA
polymerase from M/S Promega, under identical reaction conditions. This lends support to the
existing apprehensions about the reliability of the RAPD markers for practical applications, thus
necessitating conversion of such markers into SCAR markers by designing suitable pairs of
primers based upon the nucleotide sequence of the original RAPD markers (Paran and
Michelmore 1993). SCAR markers, being sequence-specific, are not biased to minor variables in
experimental condition, however, they are highly reproducible, reliable, simple to use and
suitable for multiplex PCR analysis.

Two pairs of long sequence-specific primers were designed based on the 5’ sense and antisense
terminal regions of MADP1-Accn. No. AY046931, GenBank, NCBI (corresponding to
OPA10827) and MADP2-Accn. No. AF375880, GenBank, NCBI (corresponding to OPA15744).
These are the sequences for the conversion of OPA10827 and OPA15744 markers, respectively, to
sex-linked SCAR markers. We envisaged two possible outcomes: that the SCAR marker would
be male-associated, representing heterogametic mode of sex determination, or that the SCAR
markers would be amplified from all the males and some, or all, of the females. Amplification of
the sex-linked marker in females was expected by supposing the absence of polymorphism in the
nucleotide sequence of this genomic region from the males and females downstream of the
sequence of the original RAPD primer.

Homology search between MADP1, 827bp (GenBank Accession No. AY046931) and MADP2,
744bp (AF 375880) revealed only 46.6% homology. These sequences are originally from RAPD
using random primers OPA10 (GTGATCGCAC) for MADP1 and OPA15 (TTCCGAACCC) for
MADP2, hence not much homology was expected. Our experiment has proved that the pair of
primers (MPS1A and MPS1B) developed from MADP1 were found to be very effective in
amplifying a male-specific SCAR marker of the expected size This finding is comparable to
male-specific SCAR markers developed on the basis of respective RAPD marker sequences in
Humulus lupulus (Polley et al. 1997), Asparagus officialis, Actinidia chinensis (Gill et al. 1998)
and Cannabis sativus (Mandolino et al. 1999); one in each species and five in Silene latifolia
(Zhang et al. 1998). Gill et al. (1998) developed one female sex-specific SCAR marker in
Asparagus by following a similar strategy.

However, we found that the MADP2 sequence-based SCAR marker amplified a 744-bp band
from the males, and a 747-bp-sized fragment from some of the female plants. Non-specificity of
primary SCAR markers was noted earlier in Cannabis sativus (Mandolino et al. 1999) and in
Actinidia chinensis (Gill et al. 1998). Such markers could successfully be converted to male-
specific markers by incorporating nucleotide mismatches in the 3’ end of the redesigned internal
primers. Based on these results, we are now designing appropriate sets of primers by
incorporating nucleotide mismatches for the conversion of MADP2 SCAR into a male-specific
SCAR marker. We are also looking into the possibility of detecting a female-specific SCAR
marker by using the nucleotide deletion in the male MADP2 SCAR (Figure 3).

Previous studies have revealed that male sex-linked SCAR markers are either derived from the Y
chromosome, as in Silene latifolia (Zhang 1994), or their inheritance pattern suggested a
heterogametic (based on segregating X and Y chromosomes) mode of male phenotype
determination, as in Actinidia chinensis (Gill et al. 1998), Asparagus and Cannabis sativa
(Galoch 1978). Thus, strong male phenotype association of the MAPD1-SCAR marker provides
an indirect indication for a similar heterogametic mode of sex determination in P. longum. That
is, its hybridization to female genomic DNA might not constitute a serious deterrent to this
observation because considerable homology could be expected betweenx and Y chromosomes in
dioecious plants which are supposed to have more recent origin from some hermaphrodite
ancestors as in the case of Silene latifolia., compared with mammals. The characteristics of
MADP2-SCAR suggest two interesting possibilities: either an X-chromosome derived Y
homologue, or a polymorphic X chromosome locus. However, any definite proof in this direction
awaits analysis of the inheritance pattern of these markers in segregating male and female
progeny, which is yet to be developed. Such an effort will also help us in determining the sex
ratio in this species.

The above RAPD and SCAR markers proved to be extremely effective in identifying male P.
longum. As stated earlier in this paper, normally we have to wait for the male, or female, spikes
to appear for sex identification. These markers might be fruitfully used for an early screening of
Piper longum plants. Finding these markers has provided us with the necessary tools to examine
the mode of sex determination in addition to providing the possibility of identification of the
male phenotype with the MADP1-SCAR marker in P. Longum.

Acknowledgements

We thank Dr M.G. Purushothama, Dr E.V. Sonia and K. Seetha for their valuable help and
suggestions throughout. This work was supported by the Kerala State Science, Technology and
Environment Committee (KCSTE). We also acknowledge the late Dr M.R. Das, our former
Director, for all the support extended to us.

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