*Dirección para correspondencia

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Abstract

The arid Western Rajasthan, where the Thar Desert of India is immersed, is mostly covered by sand dunes, a common landscape. The region has confronted with fragilities of natural resources, low, erratic and ill-distributed rainfall, and is covered up with many perennial hardy shrubs. Calligonum polygonoides, the most common perennial shrub, is widely present in some localities of this Thar Desert. In this study, we evaluated the diversity present among 54 wild Calligonum polygonoides plants, sampled from eight different locations within the Thar Desert. Our methods included chemical/nutritional characteristics and random amplified polymorphic DNA (RAPD). Both chemical and molecular methods produced wider range of diversity, however, RAPD detected comparatively more diversity. A total of 163 band positions were produced by ten RAPD primers, of which 147 were found polymorphic with 90.18% polymorphism. RAPD-based Jaccard’s similarity coefficients ranged from 0.43-0.89. The analysis of various chemical and mineral constituents revealed that phog is an excellent source of calcium, potassium and phosphorous while relatively poor in zinc. Among minerals, average potassium content was found maximum (2 430mg/100g) with 0.14 CV. Zinc was observed comparably less in quantity while highest variable with CV 0.73. The chemical-based Manhattan dissimilarity coefficient values ranged from 0.01-0.22 with an average of 0.12. The comparison of the clusters obtained based on the chemical and mineral parameters with those of the RAPD data showed that the groups formed in both cases showed different patterns of relationships among the samples. Broader range of diversity might be due to the out breeding behavior of C. polygonoides and indicates the good adaptability of the plants in the region studied. However, low diversity observed in the Bikaner province is alarming and suggests that anthropogenic activities leading to heavy population disturbances can affect the genetic composition of the species in a considerable way.

Key words: chemical diversity, RAPD, nutritional parameters, Calligonum polygonoides.

Resumen

El árido Rajastán occidental, en donde está inmerso el desierto de Thar en la India, está cubierto principalmente por dunas de arena, un paisaje común. La región ha enfrentado la fragilidad de los recursos naturales, las lluvias escasas, irregulares y mala distribución, y está cubierta con muchos arbustos resistentes perennes. Calligonum polygonoides, el arbusto perenne más común, se encuentra ampliamente en algunas localidades del desierto de Thar. En este estudio, se evaluó la diversidad presente entre 54 plantas silvestres de Calligonum polygonoides, de ocho localidades diferentes del desierto de Thar. Nuestros métodos incluyen características químicas/nutricionales y ADN polimórfico amplificado (RAPD) al azar. Ambos métodos químicos y moleculares producen un amplio rango de la diversidad, sin embargo, RAPD detectó comparativamente mayor diversidad. Un total de 163 posiciones de la banda fueron producidos por diez cebadores RAPD, de los cuales 147 se encontraron polimórficos con un 90.18% de polimorfismo. El coeficiente de RAPD basado en la similitud de Jaccard varió desde 0.43 hasta 0.89. El análisis de varios constituyentes químicos y minerales reveló que Calligonum polygonoides es una excelente fuente de calcio, potasio y fósforo mientras que es relativamente pobre en zinc. Entre los minerales, el contenido de potasio promedio se encontró como máximo (2 430mg/100g), con 0.14 CV. El zinc se observó comparativamente menor en cantidad, pero presentó la mayor variabilidad con CV 0.73. El valor del coefficente de disimilitud de Manhattan varió en un rango de 0.01 hasta 0.22 con un promedio de 0.12. La comparación de los grupos obtenidos según los parámetros químicos y minerales con las de los datos de RAPD mostró que los grupos formados en ambos casos mostraron patrones diferentes de relaciones entre las muestras. Una gama más amplia de la diversidad podría ser debido al comportamiento reproductivo C polygonoides e indica la buena adaptabilidad de las plantas en la región estudiada. Sin embargo, la baja diversidad observada en la provincia de Bikaner es alarmante, y sugiere que las actividades antropogénicas que conducen a disturbios excesivos en la población pueden afectar la composición genética de la especie de una manera considerable.

Palabras clave: diversidad química, RAPD, parámetros nutricionales, Calligonum polygonoides.

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Phog ( Calligonum polygonoides L.), a xeric under-utilized shrub of Polygonaceae, dominantly found in arid and semi-arid region of Thar Desert and offer green advantage to desert in extreme adverse climatic conditions. The Calligonum genus includes more than 80 species with xeromorphic shrubby characteristics distributed throughout Southern Europe, North Africa and Western and Central Asia as its main biodiversity center (Brandbyge 1993). Of the 80 species of this genus, only C. polygonoides L. occurs in Indian arid ]]> et al. 1979). It has a massive network of underground root which works as ]]>

C. polygonoides has emerged one of the economically beneficial plants during last few years of continuous drought conditions in Western Rajasthan. The thick branching stem and roots are used as fuel for industries while buds are picked in food ]]> et al. 1996) and the aqueous paste of plant acts as an antidote against the heavy doses of opium and also against poisonous effects of certain harmful plants (Singh et al. 1996). The decoction of roots mixed with catechu is used as gargle for sour gums. It is consumed in dried form especially with curd based preparation (Goyal & Sharma 2008) and the only sources of food for desert animals in extreme drought conditions. Flower and small succulent ]]>

The plant has been quoted in Red Data Book of IUCN as endangered plant species due to its large scale exploitation (Singh 2004). Phog (C. polygonoides) and khejri (Prosopis ]]> ) from Thar Desert of Rajasthan, are the most popular fuel due to less smoke and good burning quality. These species are, therefore, over-exploited. A study published by CAZRI, Highlights of Research (2002) in sample villages of Bikaner district revealed an average fuel wood consumption of 15kg/day/family. The removal of Calligonum species from sandy terrain for growing dryland cash crops, e.g. Arachis hypogea is also a major threat to this species, which needs protection and ]]>

The plant has not yet drawn the attention for its systematic study and no efforts have been made except ]]> C. polygonoides using isozyme (Tao & Ren 2004) and random amplification of polymorphic DNA (RAPD) (Ren et al. 2002, Bewal et al. 2009). In order to develop a systematic strategy for collection of representative sample covering large part of the variability and re-establish them in the area needs information ]]> C. polygonoides. For genetic diversity estimation, RAPD was selected due to the unavailability of specific markers, e.g. simple sequence repeat markers (SSRs) for the species under study. Moreover, RAPD is simple and most widely used to analyze diversity and genetic structure of wild plant species (Xu et al. ]]> et al. 2005, Vyas et al. 2009, Bewal et al. 2009).

Materials and methods

Study species and site: Calligonum polygonoides L., a dominant species of Thar Desert of Rajasthan was selected for the study (Fig. 1). This desert forms the Eastern extremity of the great arid and semi-arid belt of the world. It is one of the smallest deserts of the world but exhibits a wide variety of habitats and a high biodiversity, due to the juxtaposition of Palaearctic, Oriental and Saharan elements (Blanford 1901, Prakash ]]> C. polygonoides plant samples were collected from eight locations of Thar Desert of Rajasthan. Most of the locations are located around 100km area from Bikaner except Churu zone of the study which was sampled from 165km apart of the Bikaner region. The ecological factors and soil characteristics for the collection sites are presented in table 1. The plants were tagged and their tender sprouts were collected in ice for DNA isolation while for chemical analysis, immature buds from two year old plants were collected. ]]>
RAPD analysis: Genomic DNA was isolated from tender twigs of each plant using the method of Doyle & Doyle (1990). After purification, DNA was quantified using Nano Drop spectrophotometer (ND-1000, Version 3.1.1, USA) and finally diluted to 25ng/μL for PCR analysis. A total of 10 RAPD primers of OPD and OPH series obtained from ‘OPERON TECHNOLOGIES’ (Inc. Alameda, California) were used for analysis. The PCR reactions were performed in ]]>

Analysis of chemical parameters: The following chemical parameters i.e. moisture, crude protein, proline, total carbohydrate, starch, soluble sugar, crude fat, crude ]]> et al. (1973), Dubois et al. (1951), respectively. Fat content, crude fiber, crude protein, total carbohydrate, calcium, potassium, phosphorous, and dietary fiber ]]>

The RAPD bands were scored as binary matrix for the presence (1) or absence (0). All calculations were done using computer program NTSYSpc v. 2.02 (Rohlf 1998). Similarity matrix was constructed using the Jaccard’s coefficients and ]]> et al. (1999). Polymorphism ]]> et al. (1993). Arithmetic mean, standard deviation and coefficient of variation (CV) were calculated for each trait using standard formula given in Chandel (1997).

Results
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RAPD analysis: A total 163 (16.3 bands per primer) RAPD markers were generated by 10 primers for 54 individual samples of C. polygonoides. Out of 163 RAPD markers, 147 were recorded as polymorphic with 90.18% polymorphism. Maximum number of polymorphic bands (66.26%) were recorded in samples collected from Dhantor region, while minimum (37.42%) from Bikaner region. Most of the scorable band positions ranged from 300bp-4000bp. Primer efficiency was ]]> et al. 1993) and discrimination power (Dj). The average PIC values for RAPD primers ranged from 0.26 (OPD-12) - 0.44 (OPH-19). Discriminatory power (Dj) for all the RAPD primers ranged from 0.93-1.0. The RAPD primer, OPH-13 and OPH-19 found maximum polymorphic (100%) and discriminatory (1.0) compared to other RAPD primers (Table 2). RAPD-based Jaccard’s similarity coefficients ranged from 0.43-0.89 with ]]> Fig. 2A). Average similarity coefficient (0.49) was used as cut-off value to classify the dendrogram. Most of the samples from Bikaner and Norangdesar regions were clustered into uppermost group of the dendrogram. However, samples of Bikaner province were sub-grouped with high similarity. Most of the clustering was found independent of the geographical region.
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Chemical analysis: All the chemical parameters with minimum, maximum, mean and CV values are presented in table 3. All the minerals were observed in the following order of decreasing quantity i.e. K>Ca>P>Mg>Fe>Zn. Average potassium content was found highest (2 430mg/100g DW) in C. polygonoides budswith 0.14 CV. Highest content variation (CV ]]> Table 3).

]]> The moisture content varied from 50.32-68.49 % with mean value 61.44±3.56%. The crude fiber was found to be 10.91±3.51%. The total carbohydrate was main constituent of the buds, ranged between 67.12-76.64%, averaged 72.39±2.18%. The NDF ranged ]]> ±5.26%. Starch and soluble sugar were present in approximately same quantity with mean value 19.11±3.51% and 17.91±4.39% respectively. The hemicellulose, cellulose and lignin ]]> ±2.62%, 10.64+±2.2% and 9.02±1.92%, respectively.

The crude fat in ]]> ±0.94%. The mean quantity of free proline was observed 8.06±1.49mg/100g. Crude proteins ranged from 15.26-21.95% with the mean value 18.66±1.64%. Most of the chemical ]]>

Chemical parameters based diversity ]]> Fig. 2B). The first group comprised all the samples from province of Bikaner while most of the samples from province of Churu were aggregated in group II. The remaining samples distributed throughout the dendrogram. Norangdesar and Gorabdesar samples were distributed randomly in the chemical-based dendrogram due to high dissimilarity coefficient.

]]> Discussion

In present study, an effort was made to find out current ecological behavior of C. polygonoides species in Thar Desert of Rajasthan, by analyzing its chemical/nutritional and genetic diversity. Using RAPD analysis a very great range of diversity ]]> C. polygonoides samples of eight different provinces of Rajasthan. In the study, 10 RAPD primers produced a very good range of discriminatory power and PIC values. All the RAPD primers used were found informative as the Dj value ranged from 0.93-1.0. A very high range of discriminatory power indicates that all the RAPD primers generated distinct patterns for all the phog samples with great diversity. A low mean genetic similarity (0.49) among C. polygonoides samples reflects diverse gene pool, able to withstand in harsh ]]>

At nutritional level, C. polygonoides is an excellent source of calcium, potassium and phosphorous while relatively poor in zinc. Nutritionally all the C. polygonoides samples were found rich in nutritional compounds. Plants with sufficient minerals are found to be more drought resistant (Bouis ]]> C. polygonoides plants was detected less (50.32-61.44%) in comparison to Capparis deciduas (74.2-84.6%), another wild plant from the same province (Vyas et al. 2009). The low moisture content indicates adaptability and hardiness of C. polygonoides plant in adverse conditions of the desert area and ability to survive in ]]>

The crude fiber, crude protein and total carbohydrate were found comparable with other plant species such as legumes (Olaofe et al. 1994), ker (Vyas et al. 2009) and specifically with C. polygonoides ]]> et al. 2005). There is evidence that crude fiber has a number of beneficial effects related to its indigestibility in the small intestine (Aremu et al. 2006). The mean proline content was found maximum in samples of Lunkaransar province followed by Churu province. The NDF contributed maximum to the total carbohydrate and hemicellulose, cellulose and lignin contributed to NDF in decreasing order with the mean value of 11.81±2.01%, ]]> ±1.76% and 9.02±1.47%, respectively which was corollary to the contents reported in other plant species by Agarwal & Chauhan (1988), Vyas et al. (2009). The co-similarity of chemical parameters in same as well different desert plant species might provide hardiness and adaptability to these plants in xeric conditions.

Comparative statements of chemical and RAPD-based diversity: In RAPD analysis, high level of genetic diversity was found among C. polygonoides samples, where near 90.18% RAPD markers were polymorphic. However, the RAPD patterns were not found systematic in respect to classification of samples accords to ]]> C. polygonoides samples were clustered into seven groups on the basis of Jaccard’s coefficient. The RAPD-based dendrogram generated poor groups because ]]> C. polygonoides samples. Krofta et al. (1998), Patzak et al. (2010) also found difference between molecular and chemical data of wild hops ( Humulus lupulus). High similarity in chemical parameters proposes least variable expression of the genes for these chemical parameters in extreme drought conditions.

Low level of diversity generated by chemical parameters compared to RAPD suggesting better efficiency of the RAPD profiles for diversity analysis that account unexpressed DNA ]]> C. polygonoides in the studied region. The evolution of wild C. polygonoides samples in distinct climatic zones demonstrates significant levels of variations in response to the selection pressure in their native environment (Singh et al. 1996). It is therefore not surprising to find significant levels of polymorphism among 54 C. polygonoides samples as revealed by RAPD markers and chemical parameters. High level of genetic diversity and no structure of RAPD-based dendrogram accords to locations were found comparable with previous study of Bewal et al. (2009). Diversification of C. polygonoides samples from same region into different clusters indicating frequent seed dispersal or other way of propagation from long distances as high velocity winds are characteristics of these regions. Samples from Bikaner region showed highest average genetic ]]>

]]> The C. polygonoides cross pollinates (anemophily) (Raju et al. 2001) which might lead to high variations at chemical as well as molecular levels, not with standing the fact that the populations of the species under study are located at tens to hundreds of kilometers of distance. Predominantly obligate out breeding behavior of C. polygonoides beside certain non-comprehensible reasons prompted these accessions to spread and occupy specific ]]> C. polygonoides, collected from different locations of Thar Desert of Rajasthan (India).

Despite having such ]]> C. polygonoides is included in Red Data Book of IUCN under near extinct plant category. Very low rain fall, high wind velocity, erosion of deserts, exploitation of shrubs for fuel purposes and consecutive grazing of plants by desert animals are some reasons might influence its proper growth and development. Plants have tremendous ability of growth and reproduction in adverse conditions of deserts but low and ]]> C. polygonoides, making it an important local forage plant. Samples from Norangdesar, Pugal and Churu were found very rich in phosphorus content while samples from the province of Bikaner were found rich in iron content. Nutritionally rich C. polygonoides plants might be added to fortify nutritionally ]]>

A study on genetic diversity of C. polygonoides in the Thar Desert is prerequisite to reveal the ecological and genetic consequences and to preserve the biodiversity, while an analysis for chemical parameters would help in selection of plants with higher nutritional values. Moreover, the combined information will reflect applicability of molecular markers in ]]> C. polygonoides and further help to plan an effective conservation strategy for the species.

Acknowledgment
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We are thankful to Department of Biotechnology, Government of India for providing the funds for this work under the project ‘‘Characterization of underutilized species of Thar Desert’’.

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*Correspondencia:
Govind K. Vyas: Plant Biotechnology Centre, SK Rajasthan Agricultural University, Bikaner 334006, India/ Author presently working in Department of Botany, University of Rajasthan, Jaipur, India. govindvyas111@yahoo.co.in.
Vinod Kumar: Seed Technology Research, Agriculture Research Station, SK Rajasthan Agricultural University, Durgapura, Jaipur 302018, India. veenu_yadav2003@rediffmail.com
R. Sharma: Plant Biotechnology Centre, SK Rajasthan Agricultural University, Bikaner 334006, India/ Author presently working in Department of Botany, University of Rajasthan, Jaipur, India.
R.A. Sharma: Department of Botany, University of Rajasthan, Jaipur. sharma_ra2007@rediffmail.com
S. Sharma: Department of Home Science, CBRG Govt. PG Girls College, Sriganganagar, Rajasthan, India. ravtarrau@yahoo.com
J.P. Singh: Central Arid Zone Research Institute, Regional Station, Bikaner, Rajasthan, India; singh_jpbkn@yahoo.com ]]> Sushil Kumar: NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110 012; sushil254386@yahoo.com
1. Plant Biotechnology Centre, SK Rajasthan Agricultural University, Bikaner 334006, India; govindvyas111@yahoo.co.in
2. Seed Technology Research, Agriculture Research Station, SK Rajasthan Agricultural University, Durgapura, Jaipur 302018, India; veenu_yadav2003@rediffmail.com
3. Department of Botany, University of Rajasthan, Jaipur; sharma_ra2007@rediffmail.com
4. Department of Home Science, CBRG Govt. PG Girls College, Sriganganagar, Rajasthan, India; ravtarrau@yahoo.com
5. Central Arid Zone Research Institute, Regional Station, Bikaner, Rajasthan, India; singh_jpbkn@yahoo.com
6. NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110 012; sushil254386@yahoo.com
7. Author presently working in Department of Botany, University of Rajasthan, Jaipur, India

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