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ISSR markers for gender identification and genetic diagnosis of Hippophae rhamnoides ssp. turkestanica growing at high altitudes in Ladakh region (Jammu and Kashmir)

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Abstract

Hippophae rhamnoides L. ssp. turkestanica (Elaeagnaceae) is a predominantly dioecious and wind-pollinated medicinal plant species. The mature fruits of the species possess antioxidative, anti-inflammatory, antimicrobial, anticancerous, and antistimulatory properties that are believed to improve the immune system. The identification of male and female plants in H. rhamnoides ssp. turkestanica is quite difficult until flowering which usually takes 3–4 years or more. A sex-linked marker can be helpful in establishing the orchards through identification of genders at an early stage of development. Therefore, we studied the genetic diversity of populations in Ladakh with the aim to identify a gender-specific marker using ISSR markers. Fifty-eight ISSR primers were used to characterize the genome of H. rhamnoides ssp. turkestanica, of which eight primers generated 12 sex-specific fragments specific to one or more populations. The ISSR primer (P-45) produced a fragment which faithfully segregates all the males from the female plants across all the three valleys surveyed. This male-specific locus was converted into a SCAR. Forward and reverse primers designed from this fragment amplified a 750-bp sequence in males only, thus specifying it as an informative male-specific sex-linked marker. This SCAR marker was further validated for its capability to differentiate gender on an additional collection of plants, representing three geographically isolated valleys (Nubra, Suru, and Indus) from Ladakh region of India. The results confirmed sex-linked specificity of the marker suggesting that this conserved sequence at the Y chromosome is well preserved through the populations in Ladakh region. At present, there are no reliable markers which can differentiate male from female plants across all the three valleys of Ladakh region at an early stage of plant development. It is therefore envisaged that the developed SCAR marker shall provide a reliable molecular tool for early identification of the sex in this commercial crop. The genetic diversity of populations as surveyed by ISSR primers revealed 85.71 % polymorphism at the population level. The dendrogram obtained divided the genotypes into three different clusters, and the distribution of male and female genotypes in all the clusters was random. The Nei’s genetic similarity index was in the range of 0.63–0.96.

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Acknowledgments

This work was supported by the Department of Biotechnology, Govt. of India (Project: BT/PR10800/NDB/51/172/2008); KD extends thanks to UGC and the Ministry of Social Justice and Empowerment for providing fellowship during the course of the work.

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    Authors and Affiliations

    1. Department of Botany, University of Delhi, Delhi, India

      Kamal Das, Showkat Hussain Ganie, Yash Mangla, Rajesh Tandon & Shailendra Goel

    2. Centre for Biodiversity Studies, School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, J&K, India

      Tanvir-Ul-Hassan Dar

    3. Department of Biotechnology, Amity University, Noida, Uttar Pradesh, India

      Manju Chaudhary, Rakesh Kumar Thakur & S. N. Raina

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    1. Kamal Das
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    2. Showkat Hussain Ganie
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    Correspondence to Shailendra Goel.

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    Handling Editor: Peter Nick

    Kamal Das and Showkat Hussain Ganie contributed equally to this work.

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    Suppl. Table 1

    Nei’s genetic similarity index based on 12 ISSR primers in different populations of H. rhamnoides ssp. turkestanica. (DOCX 29 kb)

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    Das, K., Ganie, S.H., Mangla, Y. et al. ISSR markers for gender identification and genetic diagnosis of Hippophae rhamnoides ssp. turkestanica growing at high altitudes in Ladakh region (Jammu and Kashmir). Protoplasma 254, 1063–1077 (2017). https://doi.org/10.1007/s00709-016-1013-8

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