Introduction
Nutmeg (Myristica fragrans Houtt.) is an important tree spice belonging to the family Myristicaceae. Nutmeg is native to Moluccas; the spice island which is a part of Banda Island located East of Indonesia. It is an evergreen tree and grows mainly in tropical countries of the world. Important nutmeg-producing countries are Indonesia, India, Grenada, Sri Lanka, Malaysia, Mauritius, Zanzibar, Seychelles Reunion Island, Guatemala and the Solomon Islands (Sasikumar, Reference Sasikumar2021). An important problem in nutmeg cultivation is the segregation of seedlings into male and female plants resulting in about 50% unproductive male trees. In addition to this the seedling progenies show high variations in yield and quality. Hence the earlier practice of seedling planting has now completely been replaced by vegetative propagation through budding and grafting (Nissar et al., Reference Nissar, Sasikumar, Aarthi and Rema2019). In the present study we have utilised Inter-Simple Sequence Repeat (ISSR) markers for DNA fingerprinting of four varieties viz., IISR Vishwashree, IISR Keralashree, Sindhushree and monoecious nutmeg (MN) (IC 0645756), which are popular among nutmeg farmers.
Experimental
Morphological studies
The characters taken for morphological studies were as per the distinctiveness, uniformity and stability (DUS) guidelines for nutmeg (M. fragrans Houtt.) released by The Protection of Plant Varieties and Farmers' Rights Authority, New Delhi (http://plantauthority.gov.in/sites/default/files/dusguidelinesonnutmeg.pdf). The morphological characters of four nutmeg varieties based on 18 informative DUS traits were represented accordingly (online Supplementary Tables S2 and S4; Fig. S1).
Biochemical analysis
To estimate the content of essential oil, oleoresin and butter, fresh leaves, dried nut and mace samples of the four varieties were collected from ICAR-IISR Farm, Kozhikode and from a farmer's field in Pollachi, Tamil Nadu (see protocol in online Supplementary materials). Considerable differences were observed among the four varieties in terms of essential oil, oleoresin, seed butter and myristicin contents (online Supplementary Table S3; Figs S4 and S5).
DNA isolation and polymerase chain reaction
Good quality genomic DNA was isolated from young leaves of the nutmeg varieties as per the modified Cetyltrimethylammonium bromide (CTAB) method. The isolated DNA was polymerase chain reaction (PCR) amplified with the help of ISSR primers and a thermostable polymerase (Sheeja et al., Reference Sheeja, Johnson, Jerome, Syamkumar, Krishnamoorthy and Parthasarathy2008). A total of 35 ISSR primers developed in our institute were screened and six viz., IS 02, ISSR 12, ISSR 05, ISSR 14, ISSR 01 and UBC 834 generated clear, unique and reproducible polymorphic bands (Table 1, online Supplementary Tables S1 and S5; Fig. 1; Figs S2 and S3).
Table 1. Result of polymorphic bands in eight ISSR primers
Figure 1. Gel image depicting PCR amplification using primer IS 02 (A) and using primer ISSR 05 (B). L, ladder (1 kb plus); 1, IISR-Vishwashree; 2, IISR-Keralashree; 3, Sindhushree; 4, MN.
Discussion
Since the number of registered nutmeg varieties is increasing steadily, comprehensive and specific databases with precise descriptors and clearly distinguishable classes are necessary for authentication of varieties. Though traditional methods for varietal identification rely heavily on studying the morphological characters of plants (Gopalakrishnan, Reference Gopalakrishnan1992; Archak, Reference Archak2000), presently DUS test standards as well as DNA profiling/fingerprinting are gaining importance for identification of varieties. Three popular commercial nutmeg varieties and a monoecious accession were taken up in this study and out of the 18 morphological DUS test characters studied, the varieties could be distinguished mainly based on qualitative traits such as shape of fruits and seeds, leaves and female flowers. However, most of the quantitative characters described as per the DUS guidelines were inadequate to distinguish the varieties. Thus, it is evident that fine tuning of the DUS characters, incorporating more reliable and variable characters and eliminating less relevant ones may enhance the distinguishing ability of morphological markers. The results of biochemical profiling indicated that MN is superior in terms of essential oil in nuts (11%), mace (12%) and leaves (1.76%), and in seed butter content (31.2%). IISR Vishwashree was on par with MN in nut essential oil content. IISR Keralashree recorded the highest oleoresin in nuts (19.10%) and mace (24.67%). High myristicin (nut: 13.2%, mace: 15.9%, leaves: 10.5%) was observed in MN whereas the lowest myristicin and elemicin were recorded in IISR Keralashree and Sindhushree. Quantitative variations were observed among the varieties in essential oil, oleoresin, seed butter and major chemical constituents (online Supplementary Table S3). However, biochemical parameters are influenced by environment and hence can only be used as a supporting data for varietal authentication. Recording DUS traits as well as biochemical parameters is costly and labour intensive too (Kumar et al., Reference Kumar, Krishnamoorthy, Prasath, Venugopal, Ankegowda and Biju2010). Hence a highly efficient and stable strategy of DNA profiling by ISSR markers was opted in the study. Out of 35 ISSR primers screened, six primers viz., IS 02, ISSR 12, ISSR 05, ISSR 14, ISSR 01 and UBC 834 generated clear, unique reproducible polymorphic bands (IS-02800, ISSR-12700, ISSR-051200, ISSR-14500, ISSR-14900, ISSR-01500, UBC 834550), capable of distinguishing the four genotypes. However, we suggest that a concordance of DUS traits (online Supplementary Table S5) and DNA markers as mentioned above may be effectively deployed for varietal distinction in nutmeg. Distinguishing the varieties having high public demand, at the juvenile stages is highly significant as it will help the farmers and nurseries to sell the authentic planting material and will also aid in delegation of IPR and varietal registration.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S147926212400056X
Acknowledgement
The authors thank the Director, ICAR-IISR for the facilities provided for conducting the research work.
Competing interests
None.
