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Identification of molecular markers associated with sex in kartoli ( Momordica dioica Roxb.)
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The sex of Carica papaya, an angiosperm and Cycas circinalis, a gymnosperm was studied using ISSR and RAPD techniques in pre-flowering stage. One female-specific band generated from ISSR profile using primer (GACA)4 was detected in papaya, which seems to have importance from agricultural point of view. Sequencing of a male-specific RAPD band (PCR with primer OPB 01) in C. circinalis revealed homology with putative retro elements of diverse plants, probably indicating its use in the detection of male C. circinalis in future.
The random amplified polymorphic DNA (RAPD) technique was used to determine the sex of a dioecious species, Carica papaya L., with three sex types, male, female and hermaphrodite. A 450 bp marker fragment, named PSDM(Papaya Sex Determination Marker),
exists in all male and hermaphrodite plants but not in the female plants so far analyzed. The DNA sequence of PSDM exhibited
no significant similarity to previously reported sequences. A sequence-characterized amplified region (SCAR) marker, SCARps,
was developed from PSDM to determine the sex of papaya. Southern hybridization, using PSDM as a probe, showed that PSDM exists
in the male and hermaphrodite genomes, but not in the female genome. This result strongly suggests that PSDM is located on
the chromosome region that is specific to the male and the hermaphrodite. SCARps is a suitable marker for the precise and
rapid diagnosis of sex in papaya.
Sexual dimorphism is the rule in most animals. In plant kingdom however, dioecy is found only in 4% of the angiosperms. Dioecism has originated independently in different families and genera [1] and several distinct genetic mechanisms regulating dioecy have been found in different plant species [2, 3]. Sex is the queen problem in evolutionary biology and tracing of molecular factor(s) for sex expression has potential importance in basic and applied research. Sex determination mechanism in plants is not well understood. Neither the genetic nor the physiological basis of gender has been completely resolved in any plant species, in spite of the striking progress made over the floral development [4]. The presence of sex chromosomes has been documented in some plants [1]. More often, the sex ratio in dioecious plant species is controlled by the expression of alleles at one to several loci [2]. Genetic marker system based on direct analysis of the genomic DNA have been used widely for genetic mapping, disease diagnostics and evolutionary studies and they could prove very useful in the study of sexual determination in dioecious plants such as pistachio [5], hemp [6] and basket willow [7]. There are three major sex strategies in angiosperms such as bisexual, monoecious and dioecious forms. Cucurbitaceae family exhibits similar sex spectrum. Momordica dioica, commonly known as spine gourd, a perennial cucurbitaceous rhizomatous distinctly dioecious climber found in the forests of southern India and Bengal. Tender fruits and tuberous roots are used as vegetable and ayurvedic medicine. Medicinal properties of this plant are sex specific and each sex has its own medicinal value [8]. Karyomorphological studies [9] reveal that male and female M. dioica exhibit no morphological markers and possess homomorphic chromosomes which enable sex screening. The sex ratio in natural distribution is 3:1 (Male:Female). In the present study, M. dioica plant was investigated for the molecular basis of genotypic differentiation between male and female plants using randomly amplified polymorphic DNA (RAPD) technique. Identification of sex through RAPD markers have been reported in Salix viminalis [10], Carica papaya and Cycas circinalis [11]. A method to determine the gender of the plant before flowering would facilitate breeding and selection by enabling screening for gender at the seedling stage simplifying the breeding of male and female plants for different objectives, thereby saving time and economic resources. Both male and female populations, with well defined sexual characters were collected from 20 different geographical locations. Total genomic DNA was isolated from young, fresh leaves of well differentiated (confirmed through flowers) male and female plants by modified CTAB method following the standard protocol [12]. Quantification and purity of isolated DNA was checked through uv-spectrophotometer. Purified genomic DNA was subjected to PCR amplification for RAPD analysis, using twenty decamer random primers. Each 20µl of the PCR reaction mixture consisted of 50ng genomic DNA, 1.5mM MgCl 2 , 200µM each dNTP, 15pM primer (Operon Tech., Alameda, USA) and 0.5 units of Taq DNA polymerase (Banglore Genei, Bangalore, India). Amplifications were carried out in a Gradient Palm
Simmondsia chinensis (Link) Schneider, a multipurpose dioecious shrub of arid zones, has emerged as a cash crop. It is being cultivated for its
seeds which store liquid wax whose properties are similar to spermaceti (Sperm whale oil), a substitute for petro products
and precious high-priced lubricants. Jojoba is a slow-growing desert shrub having a male biased (5:1; male:female ratio) population.
Since there is no method available to determine the sex at the seedling stage, current investigations have been carried out
to generate a sex-specific random amplified polymorphic DNA (RAPD) marker in jojoba which is based on the PCR amplification
of random locations in the genome of plant. Of the 72 primers tested, only one random decamer primer, OPG-5, produced a unique
∼1,400 base pairs fragment in male DNA. To validate this observation, this primer was re-tested with the individuals of male
and female samples of four cultivars. The unique ∼1,400bp fragment was present in male individuals of all the four cultivars
and completely absent in respective female individuals tested. To the best of our knowledge, this is the first report to ascertain
the sex of jojoba plants at an early stage of development of the taxon.
Commercial scale fingerprinting of potato cultivars is made difficult by the need for speed, reliability and the ability
to distinguish between large numbers of genotypes. There are also problems in extrapolating the results of small experimental
studies to predict the performance of techniques or primers for larger applications. The potential of ISSR-PCR for fingerprinting
purposes was evaluated using four primers on 34 potato cultivars. The complex band profiles generated were reproducible between
repeat PCRs, DNA extractions, electrophoreses and gel scorings. Two primers were each able to distinguish all cultivars. The
combined use of any two of the four primers also allowed complete diagnosis. It is concluded that ISSR-PCR provides a quick,
reliable and highly informative system for DNA fingerprinting that is amenable for routine applications. Two possible correlates
of the ability of primers to distinguish between genotypes were then examined. Marker Index failed to correlate significantly
with genotype diagnosis, but a strong and seemingly linear relationship was observed between Resolving Power of a primer and
its ability to distinguish genotypes (r2=0.98). Resolving Power of one or a pair of primers was found to provide a moderately accurate estimate of the number of genotypes
identified. Possible implications for future studies on DNA fingerprinting are discussed.
In many dioecious plants, gender influences economic value, breeding schemes, and/or opportunities for commercial use of genetically transformed materials. The objective of this study was to identify molecular markers linked to sex determination loci in the dioecious plant Salix viminalis L. A 4 x 4 factorial mating design was used to identify sex ratios in full-sibling progeny, to generate a working genetics model for segregating sex ratios, and to search for molecular markers linked to sex determination genes. Bulked segregant analysis, utilizing 380 arbitrary decamer primers to generate randomly amplified polymorphic DNA (RAPD) products was initially applied to progeny sets from 3 of the 16 full-sibling families. Of the 1080 RAPD bands examined, only a single 560 bp band was shown to be linked to a sex determination locus. The same 560 bp band occurred in three additional full sibling families and was present in one female parent and one male parent involved in the factorial mating design. This marker, UBC354 560 , is biparentally inherited, is associated with femaleness in certain genetic backgrounds, and is linked to allele A1 in the proposed two-locus epistatic genetic model of sex determination for S. viminalis . Southern blots confirmed marker homology among progeny and parents used in this study.
The dioecious Mercurialis annua L. was used as a model plant to study some aspects of the molecular basis of sex determination in plants. We report in this paper the characterization of a previously identified male specific DNA marker, OPB01-1562, from diploid dioecious M. annua. The marker co-segregated with male sex in the progeny of hormonally feminized males. Sequence analysis showed the presence of approximately 0.6 kb retrotransposon-like sequence at its 3' end. Homologous sequences were isolated from diploid female, hexaploid male and monoecious plants. These sequences contained RNaseH and integrase domains of reverse transcriptase and were most similar to pineapple retrotransposon dea1, hence were named M. annua retrotransposon-like sequences (MARL-1 to MARL-5). A 771 bp fragment isolated from a diploid female, named fem771, was homologous to the 5' end of OPB01-1562. Results from DNA blot hybridization suggested OPB01-1562 and fem771 to be from the same locus and MARL-1 from a different one. RNA blot hybridization with OPB01-1562 and MARL-1 detected an approximately 2.8 kb transcript which was expressed strongly in stems and flowers of females but not males. This transcript was named M. annua female expressed (Mafex). Sex linkage of OPB01-1562 and expression of Mafex detected by OPB01-1562 strongly suggested Mafex to be a candidate gene involved in sex determination in M. annua.
A high-density genetic map of papaya (Carica papaya L.) was constructed using 54 F(2) plants derived from cultivars Kapoho and SunUp with 1501 markers, including 1498 amplified fragment length polymorphism (AFLP) markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color. These markers were mapped into 12 linkage groups at a LOD score of 5.0 and recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. This map revealed severe suppression of recombination around the sex determination locus with a total of 225 markers cosegregating with sex types. The cytosine bases were highly methylated in this region on the basis of the distribution of methylation-sensitive and -insensitive markers. This high-density genetic map is essential for cloning of specific genes of interest such as the sex determination gene and for the integration of genetic and physical maps of papaya.
Fruit set in kiwifruit is strongly dependent on pollination, which is limited by the lack of efficient male pollen donors, among other factors. We searched for molecular markers that could be polymorphic in relation to flowering time in order to classify male kiwifruit plants to discard those that are not likely to perform as efficient pollen donors. Random amplified polymorphic DNA (RAPD) and modified amplified fragment length polymorphism (AFLP) markers were generated using 41 male kiwifruitplants in two flowering groups, early- and late-flowering males (with respect to the female cultivar ‘Hayward’). One RAPD and nine modified-AFLP markers polymorphic between male plants exhibiting different flowering time were identified, sequenced
and analysed in databases. Unweighted pair group method with arithmetic average (UPGMA) clustering and multidimensional scaling showed that these markers could be used to classify the male plants into flowering groups. Analysis of molecular variance (AMOVA) agreed with this classification, showing that most of the genetic variation is found between flowering groups. Sequence analysis based on a database search revealed that the polymorphism PolM contains a 7-nucleotide long element involved in the repression of the phytochrome A gene, that Pol4 is a partial sequence of a phytochrome B gene, and that sequences Pol3, Pol5, Pol7, and Pol9 show high identity with ESTs from kiwifruit buds treated with hydrogen cyanamide. Clustering analysis supported the previous classification of males into flowering groups, making it feasible to predict male plants’ flowering times with respect to the cultivar ‘Hayward’ based upon these molecular markers.
KeywordsKiwifruit-Flowering time-Molecular markers-RAPD-Modified-AFLP-Marker assisted selection
In the genus Momordica three species M. charantia, M. balsamina and M. dioica, have been cytologically investigated. M. dioica has a more asymmetrical karyotype than the other two species.
Meiosis in the three species is regular. The strictly monoecious M. charantia and M. balsamina show similarity in the range and frequency of bivalents and chiasmata, whereas M. dioica, a dioecious species, has fewer half chiasmata per chromosome. The evolutionary significance of perennial and annual habits along with allogamous and autogamous breeding systems is discussed. The incompatibility between 2n=22 and 2n=28 species in this genus is strongly indicated by the negative results of crossings between M. charantia and M. balsamina on one side and M. dioica on the other. The possible origin of M. dioica from M. charantia is discussed.
Decamer RAPD primers were tested on dioeceious and hermaphrodite plants of Commiphora wightii to identify sex-specific molecular markers. Sixty different random decamer primers were screened out of which only three
primers were found to be associated with sex expression. A ~1,280-bp fragment from the primer OPN06 was found to be present
in all the female individuals. Another primer OPN 16 produced a unique ~400-bp amplification product in only hermaphrodite
individuals. The third marker, OPA20 amplified a ~1,140-bp fragment from female and hermaphrodite DNAs, but failed to do so
from the male plant DNAs.
A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype
in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of
which, named OPA8400, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the
association of the OPA8400 marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female;
on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this
sex-specific association, the sequences corresponding to OPA8400 were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned
RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes
used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid
identification of male plants during breeding programs of dioecious and monoecious hemp.
Actinidia deliciosa var. deliciosa a dioecious plant species has become very popular for commercial cultivation in the low and mid-Himalayan regions of India. But dioecy represents an important constraint in kiwifruit breeding programmes and also requires identification of male and female genotypes before planting an orchard. The PCR-based RAPD technique can be used to detail the genetic control of dioecy in several species. In the present study, 34 random primers were studied to identify sex in kiwifruit and eight sex-linked markers were identified. Six were female-specific and two were male-specific in nature. This communication deals with the RAPD data generated by using 34 primers and a dendrogram has been developed using the UPGMA method.
Two sex-linked fragments were identified by RAPD analyses in the dioecious diploid shrub Aucuba japonica var. ovoidea and were converted into markers of male-specific sequence characterized amplified region (SCAR) markers. PCRs using the primers designed in this study correctly discriminated 24 flowering males and 24 flowering females at higher annealing temperatures (SCAR markers OPA10-424 at 55 degrees C and OPN11-1095 at 65 degrees C), although at relatively low annealing temperatures, the fragments were amplified in both males and females. These SCAR primers were also tested to see whether they were applicable to sex identification in the conspecific tetraploid Aucuba japonica var. japonica. One set pf SCAR primers could be used for sex identification even in this tetraploid variety, although the other failed. The SCAR markers developed in this study will provide a powerful tool in identifying the sex of immature plants of dioecious A. japonica, which is a commercially valuable shrub due to its conspicuous fruits.
Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms
(RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random
DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which
amplify from one parent but not the other, are inherited in a Mendellan fashion and can be used to construct genetic maps
in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.
Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis.
- Hormaza
Identification of sex in Carica papaya L. using RAPD markers
- Eliana