Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers

Scientia Horticulturae 117 (2008) 297–301

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Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers Ishtiaq Ahmad Rajwana a, Nabila Tabbasam c, Aman Ullah Malik b, Saeed Ahmad Malik a, Mehboob-ur-Rahman c,*, Yusuf Zafar c a b c

University College of Agriculture, Bahauddin Zakariya University, Multan, Pakistan Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 January 2008 Received in revised form 21 April 2008 Accepted 23 April 2008

Knowledge about the extent of genetic diversity/relatedness in mango germplasm is vital for developing coherent strategies for future gains in productivity. The genetic diversity/relatedness among mango cultivars/genotypes developed in Pakistan has not been investigated previously. We have assessed the genetic diversity among 25 mango genotypes/cultivars using randomly amplified polymorphic DNA (RAPD). Sixty random ten-mer primers were surveyed, out of which 45 yielded amplicons in all the genotypes. Genetic similarity between genotypes/cultivars was in the range of 64–89% with an average of 74%. Similarly, the genetic relatedness among all variants derived from a mango cultivar Chaunsa was in the range of 81.18–88.63%. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The genotypes were grouped into three (A, B, C) clusters. Generally, genotypes originating from Pakistan were grouped in cluster ‘A’ while cluster ‘B’ primarily composed of southern India as well as Florida cultivars. Kensington Pride was the most distantly related genotype which grouped with Maya and Yakta, forming a distinct cluster ‘C’. ß 2008 Elsevier B.V. All rights reserved.

Keywords: Mangifera indica L. Cultivars RAPD Genetic diversity

1. Introduction Mango (Mangifera indica L.), one of the major fruit crops, is cultivated throughout the tropics. India is the leading mango producing country followed by China, Thailand and Pakistan. Pakistan produced 1753.9 thousand tons of mangoes in 2006 from 156.6 thousand hectares (Anon, 2006). It belongs to the genus Mangifera (Anacardiaceae) which comprises of 73 genera and about 830 species and has its origin in the northern foothills of Indian–Myanmar region (Yamanaka et al., 2006). It has been domesticated independently in the east (Myanmar, Bangladesh, Pakistan, Sri Lanka and North eastern India) by Buddhist monks between 400 and 500 B.C. Mango was introduced into the west in 16th century by Portuguese from Goa through East Africa to West Africa and the new world (Singh, 1976). Mango cultivars have been classified into two groups on the basis of embryo type: monoembryonic (one zygotic embryo per seed) and polyembryonic (one to six nucellar and one zygotic embryo per seed). The proposition of independent domestication of mango would account for the differences that exist between the

* Corresponding author. Tel.: +92 41 2554378; fax: +92 41 2651472. E-mail address: [email protected] (Mehboob-ur-Rahman). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.04.009

local polyembryonic cultivars of Southeast Asia (Myanmar, Thailand, Malaysia, China and Indonesia) and the monoembryonic cultivars of Southwest Asia (Indo–Pak sub-continent) (Iyer and Degani, 1997). Knowledge about the extent of genetic diversity is the major component in designing future breeding strategies for sustainability in mango production (Ravishankar et al., 2000). Earlier, morphological features were used to characterize fruit tree cultivars, rootstocks, and landraces; a time consuming process that is prone to environmental effects. DNA-based markers are useful tools for characterizing and studying genetic similarities among land races, varieties and cultivars (Duneman, 1994). Various DNA markers, including restriction fragment length polymorphism (RFLP) (Ravishankar et al., 2004), random amplified polymorphic DNA (RAPD) (Karihaloo et al., 2003; Ravishankar et al., 2004), amplified fragment length polymorphism (AFLP) (Yamanaka et al., 2006) and simple sequence repeats (SSRs) (Viruel et al., 2005; Schnell et al., 2006) have been utilized to determine taxonomic identity (Schnell et al., 2006), to estimate genetic diversity (Viruel et al., 2005) and draw evolutionary histories of mango (Yamanaka et al., 2006). The RAPD method is popular due to its simplicity and efficiency (Huff et al., 1993). Most of the mango cultivars grown in Pakistan have been developed by seedling selection from the population of ‘Chaunsa’ or hybridizing it as one of the parents, followed by selection of

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298 Table 1 Mango germplasm used for RAPD analysis S. no.

Variety

Origin

Region of cultivation in Pakistan

Average similarity

APL (cm)

NLC

AFW (g)

FS

Fiber

TSS (8Brix)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Faiz Kareem Kala Chaunsa Sufaid Chaunsa Late Ratole Yakta Sindhri Sobhay de Ting Haider Shah Anwar Ratole Chaunsa Taimooria Sanglakhi Dusehri Collector Lahootia Fajri Zardalu Bagan Palli Swarnarekha Keitt Tommy Atkins Sensation Kensington Pride Maya Pope

Multan Multan Multan Multan Sindh Tharparker Muzaffargarh Muzaffargarh Northern India Northern India Northern India Northern India Northern India Northern India Northern India North Eastern India Eastern India Southern India Southern India Florida Florida Florida Australia Israel Hawaii

Punjab Punjab Punjab Punjab Sindh Punjab, Punjab Punjab Punjab, Punjab, Punjab Punjab Punjab, Sindh Punjab, Punjab, Sindh Sindh Sindh Exotic Exotic Exotic Exotic Exotic Exotic

0.7727 0.7510 0.7470 0.7489 0.7314 0.7618 0.7417 0.7464 0.7476 0.7745 0.7194 0.7407 0.7650 0.7384 0.7726 0.7466 0.7142 0.7421 0.7660 0.7593 0.7216 0.7575 0.6820 0.7180 0.7389

22.07 26.20 29.40 28.70 29.60 25.10 26.50 26.60 20.37 23.14 26.60 35.10 26.80 27.50 30.00 28.50 21.20 25.30 32.90 32.00 30.90 31.00 27.90 28.90 26.50

Yellowish green Light brown Yellowish green Yellowish green Brownish green Light brown Yellowish green Yellowish green Yellowish green Yellowish green Yellowish green Light brown Yellowish green Yellowish green Yellowish green Yellowish green Greenish Light brown Greenish Greenish Light brown Brownish green Greenish Greenish Brownish

290 300 500 180 385 450 125 125 185 350 180 400 180 400 220 550 260 500 500 300 400 250 345 335 385

Ovate Oblong Oblong Oblong Ovate Round Ovate Oblong Ovate Oblong Ovate Ovate Oblong Ovate Round Ovate Oblong Oblong Round Ovate Oblong Round Ovate Oblong Ovate Oblong Round Ovate Round Ovate Round Oblong Round Ovate Round Ovate Round Ovate Round Ovate Round Ovate Oblong Ovate

Absent Much Scanty Scanty Scanty Absent Scanty Scanty Scanty Ovate Medium Scanty Absent Absent Medium Scanty Scanty Scanty Scanty Scanty Scanty Scanty Scanty Scanty Scanty Scanty

25–26 25–27 24–26 24–26 20–22 17–20 18–20 22–24 24–26 25–27 22–24 17–20 24–26 17–19 24–25 24–26 20–21 20–22 22–24 17–19 17–19 17–18 18–20 20–21 17–18

Sindh

Sindh Sindh

Sindh Sindh Sindh

APL = average panicle length; NLC = new leaf color; AFW = average fruit weight; FS = fruit shape; TSS = total soluble solids.

superior genotypes, thus creating a relatively small gene pool. The tendency to use similar parents extensively in a breeding program has led to concerns about the lack of genetic diversity (Fouilloux and Bannerot, 1988). Hence, the present study was conducted to assess genetic diversity and genetic relationships among mango cultivars/genotypes. The information will be useful for cultivar identification, genome mapping and initiating marker-assisted selection.

producing multibanded and scorable amplification products. Amplifications were performed in a Mastercycler Gradient thermal cycler (Eppendorf, Germany) using the following cycling program: one cycle of 94 8C for 5 min; 40 cycles of 94 8C for 1 min; 36 8C for 1 min; 72 8C for 2 min; followed by one cycle of 72 8C for 10 min and then held at 20 8C until the tubes were removed. Negative controls were setup without template DNA and cycled with other

2. Materials and methods 2.1. Plant material The experimental material used in the present study consisted of 25 mango genotypes. Leaves of 24 genotypes were collected from the Mango Research Station, Shujabad, (Punjab) Pakistan. Leaves of ‘Faiz Kareem’ were collected from Faiz Chaman Farm, Multan, (Punjab) Pakistan. The details of the genotypes are given in Table 1. 2.2. RAPD analysis Genomic DNA was extracted from the tender leaves of 10 individual plants of each genotype by the method of Doyle and Doyle (1987), quantified spectrophotometrically and by electrophoresis on 0.8% agarose gels. Total genomic DNA of each isolate was diluted in sterile distilled water to a concentration of 10 ng/ml for RAPD analysis. PCR was performed in a 50 ml reaction volume containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 100 mM each of dATP, dCTP, dGTP, dTTP, 30 ng of primer, 0.001% (w/v) gelatin, 20 ng of genomic DNA and 2 unit of Taq polymerase. Taq polymerase, together with buffer, MgCl2, dNTPs and gelatin were purchased from MBI Fermentas, USA. Out of 60 ten-mer random primers, 45 (OPB-1 through OPB-3, OPB-5, OPB-6, OPB-8, OPB-9, OPB-13 through OPB-15, OPF-1 through OPF-20, OPG-1 through OPG-9, OPG-16, OPH-1, OPM-7, OPQ-13, OPU-1 and OPW2) were selected for final RAPD-PCR analysis, based on their

Fig. 1. Amplification profile of 25 mango genotypes with primer OPF-12. M = kb ladder, 1 = Faiz Kareem, 2 = Anwar Ratole, 3 = Chaunsa, 4 = Taimooria, 5 = Kala Chaunsa, 6 = Sanglakhi, 7 = Sufaid Chaunsa, 8 = Late Ratole, 9 = Yakta, 10 = Dusehri, 11 = Sindhri, 12 = Fajri, 13 = Lahootia, 14 = Sobhay de Ting, 15 = Bagan Palli, 16 = Keitt, 17 = Tommy Atkins, 18 = Kensington Pride, 19 = Maya, 20 = Swarnarekha, 21 = Haider Shah, 22 = Collector, 23 = Zardalu, 24 = Pope, 25 = Sensation, 26 = positive control, 27 = negative control, M = kb ladder.

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PCR reactions. Amplification products were visualized on 1.2% (w/ v) agarose gels stained with ethidium bromide (10 ng/100 mL of agarose solution in TBE) for 30 min after electrophoresis. The gels were photographed under UV light with an electrophoresis documentation and analysis system 120 (Kodak Digital Science, Japan). 2.3. Statistical analysis All visible and unambiguously scorable fragments amplified by the primers were scored by visual observation. Amplification profiles (band in each position) were scored as present (1) or absent (0). The scored band data were used to estimate Euclidean distances. These distances were used to generate a dendrogram by

299

means of unweighted pair group method of arithmetic means (UPGMA). 3. Results and discussion Mango is a cross-pollinated fruit plant. The intra-varietal heterogeneity may arise due to cross pollination. In the present study, 10 individual plants of each genotype were studied for intravarietal genetic polymorphism by surveying with 10 decamerrandom primers. All the genotypes produced monomorphic amplification profiles (data not shown), reflecting intra-varietal genetic purity which is achieved by propagating the parent cultivars through asexual means (grafting) (Majumdar, 1989). Further analysis is needed to investigate the optimal number of

Table 2 Similarity matrix of 25 mango genotypes with 45 RAPD primers

Faiz Kareem Anwar Ratole Chaunsa Taimooria Kala Chaunsa Sanglakh Sufaid Chaunsa Late Ratole Yakta Dusehri Sindhri Fajri Lahootia Sobhay de Ting Bagan Palli Keitt Tommy Atkins Kensington Pride Maya Swarnarekha Haider Shah Collector Zardalu Pope Sensation

Faiz Kareem Anwar Ratole Chaunsa Taimooria Kala Chaunsa Sanglakh Sufaid Chaunsa Late Ratole Yakta Dusehri Sindhri Fajri Lahootia Sobhay de Ting Bagan Palli Keitt Tommy Atkins Kensington Pride Maya Swarnarekha Haider Shah Collector Zardalu Pope Sensation

Faiz Kareem

Anwar Ratole

1 0.8667 0.8863 0.7882 0.8078 0.7726 0.7843 0.7804 0.7373 0.7765 0.7922 0.8039 0.8078 0.7647 0.7686 0.7647 0.7451 0.6431 0.6980 0.7686 0.7529 0.7569 0.7412 0.7529 0.7843

1 0.8471 0.7490 0.7765 0.7726 0.7608 0.7804 0.7529 0.7765 0.7843 0.7726 0.7529 0.7647 0.7294 0.7333 0.6745 0.6745 0.6902 0.7294 0.7216 0.6863 0.7098 0.7059 0.7294

Fajri

Lahootia

1 0.8078 0.7333 0.7216 0.7647 0.6902 0.6431 0.6745 0.7608 0.7373 0.7255 0.6941 0.7529 0.7529

1 0.8157 0.7804 0.8000 0.7647 0.6628 0.6941 0.7961 0.7726 0.7608 0.7451 0.7726 0.8196

Chaunsa

Taimooria

1 0.7843 0.8196 0.7922 0.8118 0.7765 0.7647 0.7882 0.7882 0.8000 0.8118 0.7765 0.7647 0.7843 0.7255 0.6784 0.7098 0.7804 0.7333 0.7294 0.7373 0.7177 0.7804

1 0.7216 0.7412 0.7373 0.7020 0.7137 0.7608 0.6980 0.7098 0.7451 0.7333 0.7059 0.7020 0.7059 0.6431 0.6980 0.7059 0.6980 0.6941 0.7020 0.7137 0.7137

Sobhay de Ting

Bagan Palli

1 0.7529 0.7647 0.7373 0.6824 0.6902 0.7294 0.7373 0.6941 0.6941 0.7373 0.7765

1 0.7922 0.7490 0.6706 0.7098 0.7804 0.7255 0.7843 0.6667 0.7333 0.7333

Keitt

1 0.8314 0.6745 0.7373 0.8078 0.7529 0.7412 0.7255 0.7843 0.8235

Kala Chaunsa

1 0.8078 0.8118 0.7843 0.7020 0.7961 0.8196 0.7529 0.8118 0.7843 0.7569 0.7373 0.6784 0.6471 0.6549 0.7490 0.7412 0.6980 0.6824 0.7177 0.7647

Sanglakhi

1 0.7686 0.7804 0.7373 0.7765 0.7843 0.7412 0.7608 0.7490 0.7294 0.7255 0.6431 0.6745 0.7137 0.7529 0.7216 0.7255 0.6941 0.6902 0.7216

Tommy Atkins

Kensingt on Pride

1 0.6784 0.7177 0.7647 0.7333 0.7843 0.7216 0.7647 0.7726

1 0.7882 0.7098 0.6784 0.6667 0.6980 0.6471 0.6628

Sufaid Chaunsa

Late Ratole

1 0.7843 0.7333 0.7882 0.7804 0.7922 0.7726 0.7529 0.7555 0.7373 0.6706 0.6706 0.7098 0.7333 0.7412 0.7216 0.6902 0.6863 0.7333

1 0.7451 0.8078 0.8000 0.7961 0.7686 0.7333 0.7686 0.7569 0.6667 0.6980 0.7294 0.7451 0.7529 0.7098 0.6941 0.6902 0.7216

Maya

1 0.7569 0.7333 0.7294 0.7137 0.7020 0.7177

Yakta

Dusehri

Sindhri

1 0.7804 0.7333 0.6980 0.7333 0.6980 0.7098 0.7451 0.6863 0.7882 0.8196 0.7333 0.7098 0.7059 0.7294 0.6941 0.7020

1 0.8118 0.7608 0.7882 0.7686 0.7569 0.7686 0.7255 0.7333 0.7490 0.7647 0.7490 0.7216 0.7216 0.7412 0.7490

1 0.8314 0.7961 0.7294 0.7647 0.7686 0.6863 0.6549 0.6941 0.7804 0.7726 0.7608 0.7059 0.7726 0.7726

Swarnare kha

Haider Shah

1 0.8353 0.8157 0.7529 0.8039 0.8275

1 0.8000 0.7216 0.8118 0.7804

Collector

Zardalu

Pope

Sensation

1 0.7490 0.7843 0.7765

1 0.7216 0.7294

1 0.8353

1

300

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individuals and DNA markers that would be required to adequately represent allelic diversity in the genotypes. Out of 60 ten-mer random primers surveyed, 45 primers yielded amplicons in all the cultivar, of which 44 were polymorphic. However, none of the primers amplified a unique banding pattern which can distinguish all the genotypes. This is in agreement with earlier studies on mango (Ravishankar et al., 2000) and other field crops (Rahman et al., 2002). It reflects the occurrence of low genetic diversity among the mango genotypes. Moreover, very few efforts were undertaken to hybridize the material with the other mango genotypes or species which may help in broadening the genetic diversity (Mukherjee, 1997). Of the total 320 amplification products, 257 were polymorphic. The average number of fragments amplified was 6.95 per primer. The number of DNA bands ranged from 3 with primer OPB-14 to OPB-11 with primers OPB-2 and OPF-4. The genomic DNA of two cultivars, Kala Chaunsa and Taimooria, yielded the maximum number of DNA fragments each (276), while the fewest fragments (193) were amplified from the genomic DNA of Kensington Pride. The extent of polymorphism per primer ranged from 20% (OPB-6) to 100% (OPB-13, OPQ-13) with a mean of 80.59%. The approximate size of the largest fragment was in the range of 2.0–2.1 kb and the smallest scorable amplified fragment was approximately 200 bp (Fig. 1). The genetic similarity coefficients were in the range of 0.64– 0.89 with an average of 0.74. This is compatible with earlier studies that reported genetic similarities of 0.61–0.95 using RAPD analysis (Kumar et al., 2001). While in another study, relatively low estimates of similarity coefficients (0.32–0.72) were reported among 29 Indian cultivars (Karihaloo et al., 2003). The most genetically similar genotypes were Faiz Kareem and Chaunsa (88.63% similar) while Kensington Pride and Taimooria were the least genetically similar (64.31%) (Table 2). The most genetically diverse genotype was Kensington Pride, which was genetically 68.20% related to all the genotypes whilst Chaunsa was the most genetically similar (77.45%) (Table 2). There is a need to broaden

the genetic base of germplasm (Bally et al., 1996), which is an area of concern in modern agriculture. A single cultivar, Chaunsa, is the preferred cultivar in Pakistan and occupies around 25–35% of total mango acreage. Secondly, the remaining mango cultivars were developed through selection from the population of commercially important indigenous/introduced germplasm. Consequently, high genetic similarities (81.18–88.63%) are observed in Pakistani cultivars, bred using Chaunsa as a common parent. Kala Chaunsa and Sufaid Chaunsa are variants of Chaunsa, which were selected on the basis of yield and fruit quality. Faiz Kareem is a derivate of Chaunsa and Anwar Ratole (Ahmad et al., 2007). The similarity coefficients generated from the RAPD data were used to construct a dendrogram (Fig. 2). The dendrogram grouped the genotypes into three clusters, which correspond well with their pedigree relationship and area of cultivation (Ravishankar et al., 2000). In the present studies, the major cluster ‘A’ consisted of six genotypes developed in Pakistan and few northern Indian and north eastern Indian cultivars. This cluster is further subdivided into three sub-clusters. In first sub-cluster, the genotypes Faiz Kareem, Chaunsa, Anwar Ratole, Kala Chaunsa and Sufaid Chaunsa formed a sister group relationship. The mango cultivars originating from northern and eastern Indian regions are distinct from southern Indian cultivars emphasizing the need for further investigations (Fig. 2). The second sub-cluster consists of Late Ratole, Dusheri, Sindhri and Fajri. The sub-clustering of two cultivars, Dusehri and Late Ratole, may be attributed to commonalities in pedigree and morphological features, namely both are characterized by prolific and regular fruit bearing habit. Cultivars Sindhri and Fajri (north eastern Indian) also sub-clustered suggesting the origin of Sindhri as a chance seedling of either Fajri population or some northern (north eastern) Indian cultivars. Cluster ‘B’ comprises of two sub-clusters. In the first subcluster, Kiett forms a sister group with Tommy Atkins which confirms their evolution from a common ancestor (Schnell et al., 2006). In both sub-clusters, south Indian cultivars (Swarnarekha and Bagan Palli) grouped with the cultivars from Florida (Keitt, Tommy Atkins and

Fig. 2. Dendrogram of 25 mango genotypes/cultivars based on UPGMA analysis using Euclidean dissimilarity matrix.

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Sensation) which confirms their genetic inclination towards Indian cultivars (Schnell et al., 2006). Clustering of Haider Shah (Pakistan) and Collector (northern India) in cluster ‘B’ may be attributed to their close genetic similarity which needs further investigation. The cultivars Taimooria and Zardalu are genetically distinct from clusters ‘A’ and ‘B’ at a genetic distance of 0.27 and 0.29, respectively. Cluster ‘C’ contains the three most genetically diverse genotypes, Yakta, Maya and Kensington Pride, that have distinct pedigrees (Ian Bally, personal communication). Kensington Pride is the only polyembryonic genotype in the study, and is the least genetically similar (68.20%) with the other genotypes. Information about its parentage is unknown and it has been suggested that it was introduced in Australia from East Indies or India (Bally et al., 1996). Maya is the seedling of Haden (Viruel et al., 2005), while Yakta is an indigenous chance seedling of unknown parentage originating in Sindh province. The distribution of few indigenous (Pakistani) genotypes in the clusters (Fig. 2) suggests that both Indian and Pakistani cultivars were bred from a common gene pool as the two countries were united until 1947. Our results indicate that five cultivars (Taimooria, Yakta, Zardalu, Kensington Pride and Tommy Atkins) may be useful for widening the genetic base/genetic diversity of mango as a potential buffer against the spread of diseases (Zhu et al., 2000). The results obtained will be more useful for prospecting of additional material in Pakistan and conservation of genetic resources. Acknowledgements The study was supported by the Higher Education Commission, of Pakistan. We are grateful to Prof. Dr. Rob W. Briddon (employed at NIBGE under the Higher Education Commission [Pakistan] ‘‘Foreign Faculty Hiring’’ scheme) for critical reading of the manuscript. References Ahmad, I., Rahman, M., Tabbasam, N., Zafar, Y., Anwar, R., Malik, A.U., 2007. ‘Faiz Kareem’: a new mango cultivar. In: Malik, A.U., Pervaiz, M.A., Ziaf, K. (Eds.), Proceedings of the first International Conference on Mango and Date Palm,

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