Int J Pharm Pharm Sci, Vol 8, Issue 11, 95-101Original Article


VALIDATION AND TRANSFERABILITY OF SIMPLE SEQUENCE REPEATS (SSR’S) FROM SOME SPECIES OF ACACIA GENUS TO ACACIA NILOTICA L.

AMITA YADAV, MANILA YADAV, SANDEEP KUMAR, DUSHYANT SHARMA, JAYA PARKASH YADAV*

Department of Genetics, Maharshi Dayanand University, Rohtak 124001, India
Email: yadav1964@rediffmail.com

Received: 18 Jun 2016 Revised and Accepted: 09 Sep 2016

ABSTRACT

Objective: In present study genetic transferability of SSR’s from related Acacia species to Acacia nilotica was evaluated along with its genetic diversity analysis from north Indian region.

Methods: A total of 30 primers selected from 5 different Acacia species were screened for amplification and polymorphism. Dendrogram and 2 D plot were constructed using NTSys PC version 2.02e. Different diversity parameters like Polymorphism information content (PIC), alleles per primer, number (no.) of amplicons were also calculated for each primer pair.

Results: SSRs from Acacia tortilis, A. senegal and A. koa were highly transferable in A. nilotica. Out of 30, only twenty-two primers showed amplification with an average of 1.36 alleles per locus. Polymorphic information content (PIC) values ranged from 0.5 to 0.96 with an average of 0.81. Jaccard similarity coefficient (J) values ranged from 0.04 to 0.67 showing a high level of diversity. Un-weighted pair group method with arithmetic mean (UPGMA), based cluster analysis, divided all accessions into three main clusters.

Conclusion: Geographical and climatic conditions showed a great impact on genetic diversity. The results indicated high transferability of genomic resources from related species and will facilitate more studies to characterize the relatively less studied Acacia nilotica genome.

Keywords: Acacia nilotica, SSR, Genetic diversity, Transferability, Geographical distance

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4. 0/)
DOI: http://dx.doi.org/10.22159/ijpps.2016v8i11.13550

INTRODUCTION

Acacia nilotica L. commonly known as Babool or kikar is a popular weed, native to Africa and Indian subcontinent. A. nilotica belongs to genus Acacia sensu lato (s. l.) which is the largest genus of subfamily Mimosoideae [1]. A. nilotica is well-known for fuelwood and forage tree naturalized in many countries [2, 3]. It grows in all types of environments except colder areas [4]. It is a single stemmed, medium sized tree having paired thorns at each node of the stem [5, 6]. It is an important ethnomedicinal plant having antibacterial [7], antioxidant [8], anti-mutagenic [9], antidiarrheal [10], antiviral and many more activities [11]. Various pharmacological compounds like gallates [12], flavonoids [13] and fatty acids [14] have been isolated from the various plant parts. It can be grown for soil reclamation [4], reforestation [15, 16], agro-forestry [17] and wasteland management [18]. It also provides tannins, gum and charcoal [19, 20].

Genetic diversity at molecular level reflects the difference in the DNA of individuals of same species [21]. An understanding of genetic relationship in germplasm is a valuable technique for biodiversity conservation and plant breeding programs [22]. Microsatellites or the SSRs are tandemly repeated sequences of 1-6 base pairs, found in almost all eukaryotic organisms [23]. Microsatellites are usually preferred due to their co-dominant nature, reproducibility, high allelic diversity and cross-species amplification [24, 25]. Microsatellites have been applied to many commercially important plant species and have been proven to be very useful [26, 27].

Genetic diversity of A. nilotica has been studied previously by using allozyme markers [28] and RAPD (Random amplified polymorphic DNA) molecular markers [29] and by SSR markers [30] for detecting the origin of A. nilotica in Australia. A. nilotica is widely distributed and cultivated in north India [18]. Unfortunately only very few molecular resources are available for A. nilotica. Genetic diversity studies in India using molecular markers are also limited. Keeping in view of that there is an urgent need to study genetic diversity of A. nilotica with new and emerging molecular markers like SSR. Genomic resources or the marker information from other economically important species might be exploited, but less studied in plant species like A. nilotica.

The present study focused on assessing the genetic diversity of A. nilotica from different accessions of north India collected from different geographical and climatic conditions using SSR molecular markers. The study also focused on providing information about transferability of SSR molecular markers from other well-studied species of genus Acacia to remarkably less exploited A. nilotica.

MATERIALS AND METHODS

Sample collection

A total of 14 accessions were collected from wild plants distributed in different states of north India in 2015 (fig. 1). Two collection sites were chosen from each selected state. Climatic and geographical conditions of all collection sites have been depicted in table 1. Healthy, fresh and young leaves of each plant material were harvested and brought to the laboratory in an ice box. Leaves were further stored at -80 °C until DNA extraction.

DNA extraction

Genomic DNA was extracted from each sample by using a genomic DNA extraction kit (Genetix biotech Asia Pvt. Ltd., India), following the manufacturer’s guidelines. Quality and quantity of isolated DNA were checked by 1% (w/v) agarose gel electrophoresis and nanodrop (myspec, Sigma-Svi, Germany). DNA samples were diluted to 20ng/µl and stored at -20 °C for further analysis of genetic diversity.

SSR amplification

Thirty polymorphic primer pairs reported from different species of genus Acacia were selected for amplification [30-35]. Out of these, only 22 primer pairs showed amplification and were selected for further study (table 2). PCR reactions were performed in a final volume of 15 µl; containing 9.35 µl water, 1.5 µl DNA polymerase buffer (10 X), 1.5 µl dNTP mix (2.5 mmol each), 0.15 µl Taq DNA polymerase at 0.5 U/µl, 1 µl primer (F+R at 1µg/µl) and 1.5 µl genomic DNA. The amplification reactions were performed in a thermocycler (Peqstar; Peqlab Biotechnologie GmbH, Germany) using the cycling profile: an initial denaturation of 10 min. at 95 ° C followed by 35 cycles of denaturation at 94 ° C (30 s), 1 min. at specific annealing temperature (Ta) of each primer (table 1) and extension at 72 ° C for 1 min. and finished by a final extension step at 72 ° C for 10 min. Amplicons were run over a 2.8 % agarose gel for allele size fractionation. Both 50 and 100 bp ladders were used to measure the size of the amplicon.

Fig. 1: Map of north India showing different collection sites


Table 1: Collection sites of A. nilotica leaves

S. No. Collection states Collection sites Latitude Longitude Altitude Climatic zone
1 Haryana

Jhajjar

Hisar

28.62 ° N

29.15 ° N

76.65 ° E

75.70 ° E

720 ft

705 ft

Semi-arid

Semi-arid
2 Punjab

Ludhiana

Amritsar

30.91 ° N

31.64 ° N

75.85 ° E

74.86 ° E

798 ft

768 ft.

Humid Sub-tropical

Humid Sub-tropical
3 HP

Una

Kullu (Bhuntar)

31.49 ° N

31.88 ° N

76.28 ° E

77.14 ° E

1211 ft

3573 ft

Humid Sub-tropical

Highland
4 UP

Agra

Varanasi

27.18 ° N

25.28 ° N

78.02 ° E

82.96 ° E

561 ft

264.8 ft

Semi-arid

Humid Sub-tropical
5 MP

Tikamgarh

Bhopal

24.74 ° N

23.25 ° N

78.83 ° E

77.41 ° E

807 ft

1729 ft

Humid Sub-tropical

Humid Sub-tropical
6 Rajasthan

Jaipur

Jodhpur

26.90 ° N

26.28 ° N

75.80 ° E

73.02 ° E

1417 ft

902 ft

Arid

Arid
7 Delhi Delhi 28.61 ° N 77.20 ° E 712 ft Semi-arid
8 Chandigarh Chandigarh 30.74 ° N 76.79 °E 1053 ft Humid Sub-tropical

Data analysis

Amplification data was scored in a binary matrix based on the presence (1) or absence (0) of bands based on their size. Only clear and reproducible bands were included in the study. The similarity between different genotypes was calculated using the SIMQUAL program of numerical taxonomy and multivariate data analysis NTSys-pc version 2.02e [36]. Jaccard similarity coefficient was calculated for different accessions. The dendrogram was constructed using SAHN module based on UPGMA to show a phenetic depiction of the genetic relationship between different accessions. PIC for each primer pair was calculated by using formula:

PIC = 1 − SHpi2,

Where p is the frequency of i-th allele [37].

Principal coordinate analysis was done using NTSys software. 2D plot of the similarity of different accessions was drawn with Eigen module of the ordinate option of NTSys execute the program. Eigen values were used to draw the 2 D plot along with two ordinates. Correlation between the geographical and genetic distances was calculated using MS Excel program. Relative geographical and genetic distances were calculated relative to Delhi.

Table 2: SSR primers used to detect diversity among A. nilotica genotypes

S. No. Primer Genbank accession no. Sequence Repeat motif Origin species

Annealing Temp.

(Ta in °C)
AN1 AN06 AY553916

F-AGCGTCATGGCATAACCT

R-CGAATTCATAATCTGTCTCGAT

 (GAA)10 A. nilotica 55
AN 2 AN11 AY553917

F-AGAAACCAAGGGGAGTAA

R-TCCTCTCTAATCATCTCAAC

 (TGC)6 A. nilotica 55
AN 3 AN12 AY553918

F-GGCCATAATTGGTAATGCATTC

R-CAATCCAGTGTTGCCAGTGT

 (CCA)6 A. nilotica 55
AN 4 AN17 AY553919

F-CATAATCCCTTTGATGTCCA

R-AAATTCTCCTGATTCTGCG

 (GCT)6. (GTT)2(GCT)2 A. nilotica 55
AN 5 AN18 AY553920

F-CAGGTAGCGGAGTAGGACGG

R-AAAGAGACAAAGCTAGAAGCGATT

 (CCCT)5 A. nilotica 55
AT 6 Acator_01934 -

F-CACATTTAACTCGCTTTGGCG

R-CACATATGGCTGCTGTGGAG

 (AG)13 A. tortilis 56
AT 7 Acator_15563 -

F-TCGTACTCATCGCCAAGACC

R-CGAAGAAGCTACAATGGCTG

 (TC)12 A. tortilis 58
AT 8 Acator_22993 -

F-CATCGGCCCAGTTATGAGTTG

R-TCCAAAGTTCTGATACTGGTAAGC

 (AC)21 A. tortilis 60
AT 9 Acator_24771 -

F-ACCGTTGAGCATATTTCAGTTG

R-AAGTACTGCTTAATCTGAGTGTG

 (AC)13 A. tortolis 55
AS 10 mAsCIRB09 FM883654

F-CCTTATGAACCAAAACCAGC

R-CAACGAGCATTAACACCAAC

 (TA)3 TG (TA)3 TG (TA)3 A. senegal 52
AS 11 mAsCIRE07 FM883644

F-GAAGCAGAAGCAGAAGCAGC

R-CCCCCCTCACACTCATCTC

 (GGA)2 (GAA) (GGA)4 A. senegal 52.3
AS 12 mAsCIRE10 FM883645

F-GAGAAACTGGAGAGGGGAAG

R-GCGACAAAAGTAGTAAAGGGC

 (GAT)6 A. senegal 54.5
AS13 mAsCIRF03 FM883651

F-CACTTTTACTTTGTGATCTCCC

R-CAGTCTTGTGTGCGTCTTAC

 (TA)4 (N)55 (AC)10 A. senegal 52.4
AS14 Ame03 DQ467673

F-GAACAATATCAGCAATCACT

R-CCTCATGCACACACAAGAT

 (AG)9 A. senegal 52.4
AS15 Ame07 DQ467658

F-ATAAAAACAAAAACCCAACTAAATG

R-GTCCAAAACTCTTCAATGTCAA

 ((GT)20 A. senegal 56.6
AS16 Ab26 AY843557

F-ATATTCTGCTTTAGTCTA

R-GGGGCATAAATATGAG

 (AG)8(AG)9 A. senegal 52.4
AM17 Am389 -

F-AATCCTTCCGAAAGTTATACATGG

R-GCACTTGTAAGTCGGAACTGC

 (AT)3(GT)9GC(GT)2 A. mangium 60
AK18 Ak08 -

F-ACAGTTCCACCTCACCGTTC

R-CGACCCTATCACCTTCTTGC

 (TACA)8 A. koa 51.3
AK19 Ak36 -

F-GCAGGACTTGACGAACCTTT

R-TTGGCTCCATCTTTTCCTTG

 (CA)9 A. koa 47.3
AK20 Ak99 -

F-GCTGGTCCAACGTAGAAGGA

R-CCAGCATGAGAACGAACAAT

 (CT)10 A. koa 45.7
AK21 Ak44 -

F-TTCTGTGTTCACCGTCGTTC

R-CTGCATCCAACCTTTGACCT

 (CT)13 A. koa 51.3
AK22 Ak284 -

F-ACAACCATCGGCAACTTAGC

R-CCCAACGAAGAAGCTAGACG

 (AG)3A(AG)11 A. koa 51.3

RESULTS

Genetic diversity by SSR

In present study SSR molecular markers were used to evaluate genetic diversity of 14 accessions of A. nilotica collected from north India. All 22 primer pairs showed the polymorphism. 30 alleles were generated by 22 primers with an average of 1.36 alleles per primer. Primer AK 22 generated maximum 4 alleles. Primer AK 18 showed 3 alleles while primer AN 5, AS 13 and AS 14 showed 2 alleles.

A total of 137 bands were observed ranging from 70 to 600 base pair in size. The average no. of amplicons per primer was 6.22. PIC was calculated for each SSR primer. PIC was higher than 0.5 in all cases. PIC values ranged from 0.5 to 0.96 with an average of 0.81. Lowest PIC was observed for primer no. AN1 and AS10. Table 3 enlists the PIC values, no. of alleles and amplicon size of each primer.

Genetic relationship

Genetic relationship between 14 A. nilotica accessions was measured by Jaccard similarity coefficient. Values of J ranged from 0.04 to 0.67. Maximum similarity (67 %) was observed between Jhajjar and Delhi accession while minimum similarity was observed between Jodhpur and Amritsar accession. Table 4 enlists the J values of all accessions with respect to each other.

Phylogenetic analysis

The dendrogram was constructed using UPGMA method based on values of Jaccard similarity coefficient (fig. 2). Cluster analysis divided all 14 accessions into three main clusters i.e. A, B and C. cluster A consisted of 8 accessions and was further divided into 3 sub-clusters A1, A2 and A3 based on their similarity. Sub-cluster A1 included Jhajjar, Hisar, Delhi and Chandigarh accessions. Sub-cluster A2 included accessions from Agra and Tikamgarh; sub-cluster A3 included Jaipur and Jodhpur accessions. Cluster 2 was further divided into 3 sub-clusters B1, B2 and B3; B1 comprising only one accession i.e. Kullu. Ludhiana, Varanasi and Bhopal, Una accessions were included in B2 and B3 sub-clusters respectively.

Amritsar was found separated from all other accessions and was included in Cluster C.

Table 3: Allele size, PIC, no. of alleles, no. of amplicons of each SSR locus used for A. nilotica diversity analysis

Locus Allele size (bp) PIC Alleles per locus No. of amplicons
AN1 150 0.50 1 10
AN 2 220 0.75 1 7
AN 3 150 0.68 1 8
AN 4 150 0.60 1 9
AN 5 600,200 0.69 2 7
AT 6 160 0.75 1 7
AT 7 90 0.96 1 3
AT 8 80 0.93 1 4
AT 9 70 0.83 1 6
AS 10 250 0.50 1 10
AS 11 250 0.93 1 4
AS 12 600 0.92 1 6
AS13 400,350 0.69 2 7
AS14 150,400 0.74 2 9
AS15 300 0.96 1 3
AS16 120 0.96 1 3
AM17 70 0.96 1 3
AK18 450,400,300 0.90 3 8
AK19 200 0.96 1 3
AK20 250 0.93 1 4
AK21 360 0.83 1 6
AK22 420, 400,210,120 0.88 4 10

Table 4: Jaccard similarity coefficient values for different A. nilotica accessions

Collec-tion sites C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14
C1 1
C2 0.56 1
C3 0.67 0.64 1
C4 0.41 0.42 0.58 1
C5 0.33 0.26 0.26 0.28 1
C6 0.23 0.36 0.19 0.20 0.43 1
C7 0.33 0.18 0.18 0.28 0.25 0.11 1
C8 0.11 0.20 0.20 0.28 0.15 0.07 0.15 1
C9 0.21 0.18 0.24 0.32 0.21 0.21 0.13 0.38 1
C10 0.08 0.06 0.05 0.06 0.05 0.04 0.17 0.05 0.08 1
C11 0.40 0.20 0.33 0.43 0.25 0.07 0.50 0.25 0.29 0.09 1
C12 0.15 0.18 0.24 0.39 0.31 0.13 0.13 0.30 0.20 0.08 0.22 1
C13 0.17 0.24 0.24 0.36 0.22 0.29 0.10 0.23 0.38 0.06 0.17 0.53 1
C14 0.12 0.16 0.23 0.25 0.20 0.09 0.09 0.31 0.19 0.08 0.21 0.27 0.09 1

C1-Jhajjar, C2-Hisar, C3-Delhi, C4-Chandigarh, C5-Jaipur, C6-Jodhpur, C7-Agra, C8-Banaras, C9-Ludhiana, C10-Amritsar, C11-Tikamgarh, C12-Bhopal, C13-Una, C14-Kullu


Fig. 2: Dendrogram generated using UPGMA cluster analysis for 14 A. nilotica accessions

Principle coordinate analysis (PCoA)

Two-dimensional plot of accessions was generated using principle coordinate analysis to know about the similarity among accessions. 2 D plot also segregated all accessions in 3 major clusters as clustered by cluster tree analysis. All three clusters on their respective coordinates have been shown in fig. 3.

Correlation analysis

Correlation between geographical and genetic distances was calculated to show the effect of geographical distances on diversity. Correlation analysis showed a positive correlation but the effect was not so pronounced. Correlation analysis resulted in correlation coefficient value of 0.464 (fig. 4).

Fig. 3: 2 D plot of 14 A. nitotica accessions generated by PCoA


Fig. 4: Correlation analysis between geographical and genetic distances between A. nilotica accessions collected from north India

DISCUSSION

A. nilotica is a relatively less admired weed occurring throughout India. A. nilotica is widely grown in saline soils [38], heavy metal contaminated soils [39] and drought conditions [40]. Various plant parts are known to have pharmacological effects [11]. Therefore it is very important to characterize the germplasm of plant from various locations in India. Arid and semi-arid regions of north India host populations of A. nilotica in varied climatic conditions. So different geographical locations were selected from north India; covering the four climatic zones i.e. arid, semi-arid, humid sub-tropical and highland.

Molecular techniques are very useful in characterization, domestication, and estimation of genetic relationship among different accessions. A. nilotica genotypes are not very well characterized by molecular markers, especially in India. A. nilotica genotypes have been characterized by RAPD and SSR markers previously [29, 30]. Some studies have focused on the interspecific genetic relatedness of A. nilotica but intra-specific genetic similarity has not been studied so much [41]. SSR molecular markers have been proven very useful in diversity analysis due to high polymorphism levels and their codominant nature [25]. SSRs have been used for diversity analyses in many medicinally and commercially important crops like Tinospora cordifolia [42], Aloe vera [43], Lagenaria siceraria [26], Ziziphus jujube [44], Prunus armeniaca [45], Eragrostis tef [46], Sphenostylis stenocarpa [47] etc.

The present study focused on genetic diversity analysis of 14 A. nilotica accessions collected from north India with the help of 22 SSR molecular primers. Out of these 22 SSR primers, 5 were developed for A. nilotica in Australia [30]. Rest 17 primers were selected from other species of genus Acacia. Transferability of genomic resources from one species to another depends on upon the phylogenetic relationship between different species [48]. Genus Acacia is presently divided into three main subgenera: Acacia, Aculiferum and Phyllodinae (syn. Heterophyllum) [49]. A. nilotica and A. tortilis belong to subgenus Acacia [49, 50]; A. mangium and A. koa belong to sub-genus Phyllodinae [51, 52]; A. senegal belongs to sub-genus Aculiferum [53]. SSR loci from Acacia tortilis, A. senegal and A. koa were highly transferable in A. nilotica. SSR primers from A. mangium were not so much transferable. Only one SSR loci showed transferability. The result of our study is in concordance with the finding of Butcher et al. (2000) that SSR loci developed for A. mangium are not transferable to A. nilotica [31].

In the present investigation, PIC values of 22 SSR loci ranged from 0.5 to 0.96 with an average of 0.81. Codominant markers usually have a PIC value from 0.5 to 1 [54]. It implies that selected SSR loci were highly informative. High polymorphism levels were detected among accessions. Jaccard similarity coefficient values ranged from 0.04 to 0.67. A positive correlation was observed between genetic and geographical distances of different accessions. The cluster tree analysis revealed that most of the accessions were clustered according to the geographical distance between them. Ge et al. (2005) also reported previously that a positive correlation was observed between geographical and genetic distances in populations of Ammopiptanthus mongolicus [55]. In the present study, A. nilotica populations of Jhajjar and Delhi were genetically most similar with a J value of 0.67. Climatic conditions also affect the genetic diversity. Kumar et al. (2015) showed that climatic conditions are one of the major factors affecting genetic diversity in Aloe vera [43]. Cluster analysis clustered accessions from arid and semi-arid regions in cluster A except for Tikamgarh which lies in the humid sub-tropical zone. Cluster B included accessions from the humid sub-tropical zone and one from the highland.

Genetic diversity is responsible for the existence of a species and its evaluation is helpful in conservation strategies. A. nilotica is a very important plant used for agroforestry and reclamation of soils in India [16, 18]. This study proves the potential of SSR as a tool to detect genetic diversity. The high value of detected diversity indicated the adaptability of species to various geographical and climatic conditions. It also provides information that genetic resources from other species of genus Acacia are highly transferable to Acacia nilotica.

CONCLUSION

SSR is a powerful tool for assessing genetic diversity in wild population of A. nilotica. Results of our finding indicate the rich genomic resources of A. nilotica in north India. Present study findings can be further imposed in germplasm management; marker assisted selection in breeding programs and agroforestry systems. Study also implies the high transferability of genetic resources from other related species of A. nilotica. Further research is required for developing more SSR molecular markers as well as exploiting genomic resources from related species to study the diversity of Indian germplasms of A. nilotica.

ACKNOWLEDGEMENT

The research was financially supported by UGC under UGC-SAP program (F.3-20/2012 (SAP-II) and UGC BSR fellowship (F.7-371/2012 (BSR).

CONFLICT OF INTERESTS

All authors declare that they have no conflict of interest

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