Hostname: page-component-78c5997874-lj6df Total loading time: 0 Render date: 2024-11-20T04:19:17.382Z Has data issue: false hasContentIssue false

Identification of morphological and variety diagnostic molecular markers for commercial varieties of nutmeg (Myristica fragrans Houtt.)

Published online by Cambridge University Press:  21 October 2024

Krishna Moothandassery Shaju
Affiliation:
Division of Crop Improvement and Biotechnology, ICAR-IISR, Kozhikode, Kerala, India
Vijesh Kumar Illathidath Payatatti
Affiliation:
Division of Crop Improvement and Biotechnology, ICAR-IISR, Kozhikode, Kerala, India
Saji Koryampallil Vijayan
Affiliation:
Division of Crop Improvement and Biotechnology, ICAR-IISR, Kozhikode, Kerala, India
Leela Neettiyath Kalathil
Affiliation:
Division of Crop Production and Post Harvest Technology, ICAR-IISR, Kozhikode, Kerala, India
Muhammed Nissar Vettathukattil Abdul Gafoor
Affiliation:
Division of Crop Improvement and Biotechnology, ICAR-IISR, Kozhikode, Kerala, India
Rema Jagadamma
Affiliation:
Division of Crop Improvement and Biotechnology, ICAR-IISR, Kozhikode, Kerala, India
Sheeja Thotten Elampilay*
Affiliation:
Division of Crop Improvement and Biotechnology, ICAR-IISR, Kozhikode, Kerala, India
*
Corresponding author: Sheeja Thotten Elampilay; Email: [email protected]
Rights & Permissions [Opens in a new window]

Abstract

Nutmeg (Myristica fragrans Houtt; family: Myristicaceae) is an important tree spice of great export value due to the presence of secondary metabolites with scope in health, wellness and cosmetic industries. Its dioecious in nature due to which it is propagated by budding and grafting which requires specialised skill and as a result authentic planting material production is very expensive. Based on the constant demand from farmers and stakeholders for a suitable, fast and easy technique for determining the authenticity of nutmeg varieties, an attempt was made to develop morphological and molecular markers for the identification of three commercial varieties that are in high demand in India; viz. IISR Vishwashree, IISR Keralashree, Sindhushree and a monoecious nutmeg (MN) accession, along with their biochemical profiles. Among the various morphological characters as per distinctiveness, uniformity and stability guidelines, the varieties showed distinctness mainly in leaf shape, shape of female flowers, fruit shape and seed shape. 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 capable of distinguishing the varieties. Among the varieties analysed, MN was found to be superior in terms of seed butter and essential oil in nut, mace and leaves. IISR Vishwashree was on par with MN in nut essential oil content. IISR Keralashree was superior in terms of oleoresin in nuts and mace. The morphological and molecular markers identified may be used for effective checking of authenticity of planting materials of commercially grown nutmeg varieties.

Type
Short Communication
Copyright
Copyright © The Author(s), 2024. Published by Cambridge University Press on behalf of National Institute of Agricultural Botany

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.

References

Archak, S (2000) Plant DNA fingerprinting: an overview. Ag Biotech Net 2, 16.Google Scholar
Gopalakrishnan, M (1992) Chemical composition of nutmeg and mace. Journal of Spices and Aromatic Crops 1, 4954.Google Scholar
Kumar, RS, Krishnamoorthy, B, Prasath, D, Venugopal, MN, Ankegowda, SJ and Biju, CN (2010) Variability in nutmeg (Myristica fragrans Houtt.) under high rainfall and high-altitude Kodagu region of Karnataka. Indian Journal of Plant Genetic Resources 23, 191194.Google Scholar
Nissar, VAM, Sasikumar, B, Aarthi, S and Rema, J (2019) Air layering in nutmeg (Myristica fragrans Houtt.). Journal of Spices and Aromatic Crops 28, 6669.Google Scholar
Sasikumar, B (2021) Nutmeg – origin, diversity, distribution and history. Journal of Spices and Aromatic Crops 30, 131141.CrossRefGoogle Scholar
Sheeja, TE, Johnson, GK, Jerome, J, Syamkumar, S, Krishnamoorthy, B and Parthasarathy, V (2008) Optimization of DNA isolation and PCR parameters in Myristica sp. and related gen era for RAPD and ISSR analysis. Journal of Spices and Aromatic Crops 17, 9197.Google Scholar
Figure 0

Table 1. Result of polymorphic bands in eight ISSR primers

Figure 1

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.

Supplementary material: File

Moothandassery Shaju et al. supplementary material

Moothandassery Shaju et al. supplementary material
Download Moothandassery Shaju et al. supplementary material(File)
File 780.3 KB