Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) markers.

Study of arbitrarily amplified (RAPD and ISSR) and gene targeted (SCoT and CBDP) markers for genetic diversity and population structure in Kalmegh [Andrographis paniculata (Burm. f.) Nees]

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.

/pdf/indirect-regeneration-and-assessment-of-genetic-fidelity-of-4p0z5riv0h.pdf

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations.

Flowering plants are known to exhibit vast diversity of sexual systems encompassing bisexual, monoecious and dioecious conditions. Dioecy offers opportunities to explore separately the male and female programmes giving an insight to the evolutionary, developmental and molecular processes leading to separate mechanisms for sex expression. Mechanisms controlling sex can either be genetic or epigenetic (physiological and environmental). Plant hormones too influence sex expression. An active Y sex determination system and an X to autosomes ratio systems are common amongst the flowering plants. Advances in our understanding of sex determination has been addressed both by conventional as well as molecular approaches. Using conventional techniques mainly cytogenetics, sex chromosomes in some dioecious plants have been identified and characterized. Surprisingly, the presence of well defined sex chromosomes was found in only few species. Some sex linked genes have also been identified and characterized using molecular approaches but none of these genes have a direct link to sex determination. Molecular markers have been employed to resolve the enigma associated with dioecism to a certain extent. Its application in plant breeding is immensely beneficial. Positively, it would be beneficial for validation of sex prior their sex expression at larger perspectives. The present review therefore emphasizes the mode of sex determination among dioecious plants vis-a-vis summarizes the works related to gender specific markers generated using male and female plants from agriculturally important dioecious crops.

Review on different mechanisms of sex determination and sex-linked molecular markers in dioecious crops: a current update

An efficient in vitro protocol has been established for clonal propagation of elite plant of Spilanthes calva DC., an important source of spilanthol, an antimalarial larvicidal compound. Nodal explants excised from field grown plant of S. calva DC. when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N6-Benzyladenine (BA), N6-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn), differentiated multiple shoots from the axils. BA at 10 μM proved optimum for elicitation of multiple shoots in 91.6 % cultures with an average of 7.12 shoots per explant within 6 weeks. The excised shoots rooted on half strength Murashige and Skoog’s medium supplemented with 0.1 μM IBA. Micropropagated plants were hardened and transferred to field for acclimatization, where 95 % plants survived and were phenotypically similar to the donor plant. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic fidelity amongst the regenerants. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 71 scorable bands, ranging in size from 100 bp to 1,100 bp were generated by a combination of the two markers in the aforesaid plants. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant. The similarity values amongst the aforesaid plants varied from 0.967 to 1.000. The dendrogram generated through UPGMA (Unweighted Pair Group Method with arithmetic mean) analysis revealed 98 % similarity amongst them, thus confirming the genetic fidelity of the in vitro clones.

/pdf/in-vitro-clonal-propagation-and-genetic-fidelity-of-the-3fkllo8xr1.pdf

In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker.

DNA fingerprinting studies have been carried out with the physiologically mature male and female plants of Jojoba using 80 ISSR primers with a view to generate sex-linked markers. After bulk segregant analysis, two unique ISSR markers, viz. ISSR8481500 and VIS111317 have been developed which can be used for determining the sex at the seedling stage. Of the eighty primers tested on the pooled male DNA and pooled female DNA samples, six ISSR primers were found to be associated with sex expression. Of the six, only two primers ISSR848 and VIS11 generated unique male sex specific bands of ~1,500 and ~1,300 bp which were consecutively present in all the male genotypes and absent in all the respective female genotypes. The remaining four primers when tried on individuals of different genotypes were confined to their sex specificity in only two female genotypes and absent in their male counterparts. One of the male-sex specific markers, VIS111317 has also been cloned and sequenced which showed homology with a sex linked gene, DD44 from dioecious Silene species. Furthermore, VIS111317 was converted into a male sex-specific sequence tagged sites (STS) marker of 584 bp. The male specific STS marker thus developed has been verified and validated on 100 populations of male and female individuals from ten different genotypes of Jojoba to endorse the diagnostic reliability of the STS marker. This can gainfully be employed for screening of sex at seedling stage which would be quite helpful for uprooting the undesired plants, thereby, saving resources like labor, water, fertilizers and space for highly desirable female plants.

Generation and validation of unique male sex-specific sequence tagged sites (STS) marker from diverse genotypes of dioecious Jojoba-Simmondsia chinensis (Link) Schneider

There is a recent surge in the marker-assisted selection of desired sex type among economically important dioecious crops. Simmondsia chinensis (Jojoba), a dioecious crop is of immense agricultural importance where only the female plants are preferred for commerce. DNA fingerprinting technology, ISSR analysis along with bulk segregant analysis (BSA), has been carried out on a diverse set of 17- to18-year-old mature male and female plants of Jojoba to validate a male sex-specific ISSR marker, UBC-8071200 on larger population that comprises 330 female and 255 male plants of Jojoba. This male sex-specific DNA fragment of ~1200 bp was cloned and sequenced. The sequence was found to be 1120 bp in length and based on the sequence information, a pair of sequence tagged sites primers was developed that amplified a single ~800 bp band, consistently only in all the male populations while no amplification was seen in their female counterparts. The marker was named as Jojoba Male Sex Marker which was further validated on 330 female plants from 22 genotypes and 255 male plants from 17 genotypes.

Validation of male sex-specific UBC-8071200 ISSR marker and its conversion into sequence tagged sites marker in Jojoba: a high precision oil yielding dioecious shrub

Monika Heikrujam

Papers

Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon targeted (SCoT) polymorphism and CAAT box-derived polymorphism (CBDP) markers.

Review on different mechanisms of sex determination and sex-linked molecular markers in dioecious crops: a current update

In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker.

Generation and validation of unique male sex-specific sequence tagged sites (STS) marker from diverse genotypes of dioecious Jojoba-Simmondsia chinensis (Link) Schneider

Validation of male sex-specific UBC-8071200 ISSR marker and its conversion into sequence tagged sites marker in Jojoba: a high precision oil yielding dioecious shrub