Sex determination in nutmeg (Myristica fragrans Houtt.) through molecular and biochemical markers

Abstract

Nutmeg (Myristica fragrans Houtt.) is an important tree spice of southern India, Malaysia and Indonesia yielding two products of commercial value, ‘nutmeg’ and ‘mace’. Nutmeg of commerce is the dried seed and mace is the dried aril. It is a spreading evergreen tree of the family Myristicaceae and is dioecious with long pre bearing period of 5 to 7 years. Presently, the dioecy in nutmeg is overcome by vegetative propagation or top working. Even though several vegetative methods have been reported, the large scale adoption of these methods is constrained due to insufficient number of orthotrops. So seedling continues to be the major propagating material. In the present study, an attempt was made to identify sex in nutmeg through molecular and biochemical markers so as to identify sex at seedling stage itself. Molecular marker used was RAPD and biochemical marker was isozymes of acid phosphatase and glutamate oxaloacetate transaminase (GOT). Based on bearing pattern and floral morphology, five typical male and female plants of age 10-15 years were selected for RAPD analysis. DNA isolation technique was standardised using CTAB method. Good quality DNA with UV absorbance ratio (A260/A280) 1.80- 1.84 was used for analysis. Sixty seven decamer primers were screened and four primers showing the polymorphism has been selected for further RAPD analysis. PCR amplification with selected primers viz. OPD 15, OPA 27, OPF 05 and OPK 01 was carried out with samples of bulk DNA from five male and female, DNA of individual male and female and negative control. Among them OPK 01 amplified reproducible female specific band (1.1 kb) in bulked and individual samples. The polymorphic female specific band amplified by OPK 01 primer was eluted and cloned in pDrive vector and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed white clones were sequenced at Bioserve, Hyderabad. The sequence obtained after VecScreen (Nut seq 816 bp) was analysed with various bioinformatic tools like Blastn, Blastp, GenScan, ORF Finder, Transeq, GOR and Protparam. Three open reading frames identified in the sequence Nut seq816 sing NCBI tool “ORF Finder”. The +2 ORF strand encodes two domains for amino asparate transaminase (GOT) and cystathionine beta- lyases with E value 3.6. The other ORFs didn’t encode any domain. GenScan predicted no gene in the Nut seq816. Based on sequence information two pairs (SP1 and SP2) of SCAR primers (24 bp) were designed using primer3 programme. The efficiency of SCAR primer to distinguish male and female plants was tested by PCR amplification of DNA from five male and females. The SCAR primers amplified around 300 bp single band in five females. SCAR primer was again checked with four occasional fruiting males and it amplified 300 bp band in one occasional fruiting male. Expression of SCAR primers was tested in ten seedlings and got amplified in four samples. Leaf samples from identified male and female trees were used for the biochemical marker analysis. Polyacrylamide gel electrophoresis (PAGE) for isozyme assay was standardized with standard protein and nutmeg leaf protein. Acid phosphatase assay recorded four zones of activity and all were monomorphic. Glutamate oxaloacetate transaminase showed polymorphism and need protocol refinement. Accuracy of SCAR primer SP1 to distinguish male, female and occasional fruiting male has to be done with more samples. Commercialization of the technique can be explored based on the accuracy and cost benefit analysis.