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Assessment of the Genetic Relationship and Diversity of Mango and Its Relatives by cpISSR Marker
Available online at www,sciencedirect.com
Agricultural Sciences in China
2007. 6(2): 137- 142
ScienceDirect
Fcbruary 2007
Assessment of the Genetic Relationship and Diversity of Mango and Its Relatives by cplSSR Marker HE Xin-hua1-2,GUO Yong-ze2,LI Yang-mi1and OU Sh-jin2
’ ( h a n g x i Crop Genetic Improvement and Biotechnology
Laboratory, Guangxi Academy of Agriculturnl Sc.ience.s, Nulining 530007, P. R.
China College of Agriculture, Cuangxi University, Nanning 530004, P.R.China
Abstract Chloroplast inter-simple sequence repeat markers in mango were developed and used to analyze the genetic relationship and diversity of mango and its relatives. Thirty-six mango cultivars (Mungiferu indicu L.) and its relative species collected from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences, China, were examined by ISSR-PCR with chloroplast DNA (cpDNA). Eight better primers for chloroplast DNA that provided reproducible, polymorphic DNA amplification patterns were screened from 50 ISSR primers and used for UPGMA analysis. According to the band patterns with 8 primers for chloroplast DNA, all cultivars tested were distinguished from each other and these showed ample genetic diversity; the average percentage of polymorphism was 77.2%. The 36 samples could be clustered into four groups by UPGMA analysis at the coefficient 0.74. The results indicated that the cplSSR marker was a new powerful tool for the identification of mango cultivars or its relative species, and their genetic relationship analysis and diversity evaluation.
Key words: Mungiferu indicu, ISSR, cpDNA, genetic relationship, genetic diversity
INTRODUCTION Mango (Mangifera indica L.j is widely cultivated in tropical areas, and is a diploid fruit tree with 2n = 40 chromosomes (Mukherjee 1950). However, because of the wide sexual compatibility between mango and its relative species, the high frequency of bud mutations, long history of cultivation, and the occurrence of polyembryony in most of the species, the parentage and taxonomy of this genus are confusing. In the past, mango cultivars and relative species were often identified by the morphological and agronomic traits. With modern molecular biology development, most of the research with molecular markers based on the total DNA or the nuclear genome has been implemented in mango, and these include isozymes (Degani
et al. 1990), variable number tandem repeats (VNTR) (Adato et al. 1995), random amplified polymorphic DNA (RAPDs) (Schnell et al. 1995; Lopez-Valenzuela et al. 1997; Xu et al. 1998; Deng et ul. 1999; Karihaloo et al. 2003), inter simple sequence repeats (ISSRs) (Eiadthong et al. 1999; Gonzalez et al. 2002; He et al. 2 0 0 3 , and amplified fragment length polymorphism (AFLPsj (Eiadthong et al. 2000); however, there was no reports about chloroplast markers in mango. Since the chloroplast gene order is relatively conserved and its genome is small, non-recombinant, uniparentally inherited, and effectively haploid, which can overcome the disadvantages encountered in the analyses of nuclear DNA markers, it is the conservative nature of the chloroplast genome in higher plants that makes it more useful for parentage and taxonomic studies (Cheng et al. 2005). Chloroplast markers have been
Received 29 June, 2006 Accepted 27 September, 2006 HE Xin-hua, Tel: +86-771-3270184, Mobile: 01 3005921880. E-mail: [email protected]
02007, CAAS All rights reserved Publishedby Elsewer Ltd.
I38
HE X i n - h u a er al. ~
successfully used in the asscssment of relationship and genetic diversity of higher woody angiosperm plants, such as pine (Powell et al. 1995), Olea europuea L. (Besnard and Bervillk 2002), Coq1u.r avellana L. (Palmc and Vendramin 2002), Cnryocar brasiliense Camb. (Collevatti et al. 2003), Macarangu (Vogel et al. 2003), Sorbus urranensis Hedl. (Robertson et al. 2004), Fagus sylvatica L. (Vettori et al. 2004), and Citrus (Cheng et ul. 2005). In the present study, an attempt has been made to develop cplSSR markers for mango, thereby providing a powerful complementary method for studying the genetic relationship and diversity of mango and its relative species.
(Table 1) in thc present study were collected from the field h i t germplasm collection in thc Guangxi Academy of Agricultural Scienccs. Chloroplast DNA was extracted from fresh leaves according to the protocol (Zhu et al. 2003) with a slight modification. Mango chloroplast was obtained using the following methods: 10 g fresh leaves were pounded to pieces by a tissue triturator with buffer consisting of 0.2 mol L-' KC1, 0.8 mol L-' NaC1, and 8 mmol L' Na,S,O,, and then filtratcd with 9 layer gauzes. The filtrated homogenates were transferred to a tube of 50 mL, and equal volume of separated buffer was added, which consisted of 400 mmol L-' saccharose, 50 mmol L-' Tris-HC1,20 mmol L-I EDTA, 0.2% BSA, and 0.2% P-mercaptoethanol; pH 7.8, incubated at 0°C for 30 min and spun at 3 000 r/min for 5 min. The supernatant was transferred to another tube, and spun at 8 000 r/min for 8 min. The pellets were collected and purified with saccharose grads of 70, 60, 45, and 30%. DNA from the purified chloroplast was then extracted using the
MATERIALS AND METHODS Plant materials and chloroplast DNA extraction Leaves from 36 mango cultivars and relative species Table 1 Mango cultivars and the relative species used in the study Cultivar or species]) Red Ivory (seedling variation of Aroemwnis 26) Tianyangxiang Mang (seedling variation of Carabao) Alphonso Jin Mango Gaozhou Carabao Burma Mango Guandao Mang Indian Mango 15 Machew M . himnlis J. Y. Liarrg Okrong Pakistan Mango White Flower Mango Renmian Mang Liuzhou Lusong (variation of Philippine Mango) zhanjianp Carabao Aroemwnis Red Aroemwnis Zill Long Mang M. siamensis Warbg ex Craih Longjin Mango Jinqian Carabao Thailand Mango (unknown cultivar) Burma Ball Mango 3 Datou Mang (Yunnan) Bangalora Xianluo Mango Sri Lanka 8 I I Jinhuang Mane Xiamau Mane Neelum Dwarf Mango Yellow Aroernwnis M. persic$<)?-maWu & Ming Guike 5 -
Abbreviation ot the cultivar name') GxRI GxTY IAL YJM PhGZ BBM GxGD 11M 15 BMC GxMH ThOR PPM ThWF GdRM PhLZ Ph7J ThAS ThRA UZL YI M I1 4 GdLJ tidJQ ThTM BBB 3 YDT 1BG ThXL
SSL TJH GUM INL ThDM ThYA GxMW GxGG 5
Geographic origin Guangxi, China Guangxi, China India Yunnan, China Philippines Burma Guangxi, China India
Burma Guangxi, China Thailand Pakistan Thailand Guangdong, China Philippine3 Philippines Thailand Thailand USA Yunnan. China India Guangdong. China Guangdong, China Thailand Burma Yunnan. China Indian Thailand Sri I.ankn Taiwan Guanpdong, China India Thailand Thailand Guangxi. China Guangxi. China
Embryo type Pol yernbryonic Polyembryonic Monoembryonic Monoembryonic Polyembryonic Polyernbryonic Pol yembryonic Unknown Polyemhryonic Monoembryonic Polyenihryonic Unknown Polyembryonic Polyembryonic Polyembryonic Polyemhryonic Polyernbryonic Polyembryonic Monoembryonic Polyernbryonic Unknown Monoembryonic Polycinbryonic 1J nknow n Monocmbryonic Polyernbryonic Monoembryonic Polyembryonic Pnlyernbr~wnic Polyembryonic Polyemhryonic Monoembryonic Polyemhryonic Polyembrynnic Unknown Polyembryonic
' Except M . himalir J. Y. Liang, M . ,$iumcnaisWarhg ex Craib, and M. persiciforma Wu & Ming, and the others are the cultivars 0f.M. indlca L. Thrcc or four letters represent the abbreviation of thc cultivar's name or species.
02007,CAAS. All rights reserved. Publishedby Elsevierltd.
Assessmcnt of the Ccnetic Relationship and Diversity of Mango and its Relatives by cplSSR Marker~
hexadecyltrimethylammonium bromide (CTAB) method described by Doyle J J and Doyle J L (1990).
ISSR analysis Forty-four ISSR primers of the University of British Columbia, Canada for nuclear genome and six primers designed in the lab were used in the study, and these were synthesized by the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China. Each 20 pL amplification reaction consisted of 10 mM Tris-HC1 (pH 8.3), 50 mM KCI, 1.5 mM MgCl,, 0.2 mM mixed dNTP, 0.25 pM primer, 1 unit rTaq polymerase (Takara Biotechnology, Japan), and approximately 60 ng chloroplast DNA. Amplification was done in a thermocycler under the following conditions: 5 min at 94°C for 1 cycle, followed by 1 min at 92°C 1 min at 50"C, and 2 min at 72°C for 40 cycles, and 10 min at 72°C for the final extension. The amplification products were loaded onto a 2.0% agarose gel in a 1 x TBE buffer and were electrophoresed at 100 V for 1 h, and then visualized by staining the gel with ethidium bromide. The size of each fragment was estimated with reference to GeneRulerTM 100 bp DNA ladder plus (MBI).
Data analysis By electrophoresis, seventy-nine reproducible bands
_
_
139
from the selected primers were scored as 1 (presence) or 0 (absence) for 36 mango cultivars and the relative species were tested. Thcn, the unweighted pair-group method using the arithmetic average\ (UPGMA) cluster analysis was performed using the NTSYSpc version 2.1 e software.
RESULTS AND DISCUSSION Screening of primers and diversity analysis Four genotype sample\ (Carabao, Okrong, Zill, and M . persiciformis Wu & Ming) were used for the initial screening of the ISSR primers. Eight primers among the 50 ISSR primers that could amplify the chloropkdst DNA of 36 mango cultivars and their relatives and produce intense bands on agarose gels (Fig.1) revealed high polymorphism among the mango accessions as well as the relatives, generating 61 polymorphic bands (accounting for 77.2% of the total bands), and every primer among the 8 primers could amplify 5 to 11 cpDNA bands with length between 3 000 and 300 bp (Table 2). The cpISSR primers in the study were designed based on nuclear DNA ISSR primers, while the most optimal ISSR primers were different. The primers (GAA),, (GA),YC. and (GT),YG showed the highest level of polymorphism for the nuclear genome DNA ISSR of
Fig. 1 PCR amplified pattern by the UBC-826 primer in 24 mango cultivars. Lane M, GeneRulcrT" 100 bp DNA ladder plus; lanes I to 24 represent cultivars Red Ivory, Tianyangxiang Mang, Renmian Mang, Jin Mang, Macheso, Burma Mango. Dwarf Mango, Sri Lanka 81 1, Burma Ball Mango 3, Indian Mango 15, Jinhuang Mango, Guandao Mang, Ncelum, Okrong. Jinqian Carabao, Long Jin Mango, M. himalis J. Y. Liang, India Mango 4, Zill, M. siamensi.s Warbg ex Craib. Red Aroemwnis, Yellow Aroemwnis, Pakistan Mango. and Guike 5, respectively.
02007. CAAS All rights reserved Publishedby Elsevier Ltd
HE Xin-hua el ul.
140
mango (Eiadthong et al. 1999; He e t a l . 2005). However, the primers (AC),YG and (ACACACAT), showed higher level of polymorphism for the mango chloroplast DNA ISSR in the present study (Table 2). According to the banding patterns obtained by cpISSR with 8 selected primers, all cultivars tested in this study could be distinguished from each other and showed huge diversity, indicating that the cpISSR marker was effective for the identification of mango cultivars or the relative species and for evaluation of their genetic diversity.
Cluster analysis Based on the Jaccard coefficient of similarity obtained
from the chloroplast ISSR band sizes, a dendrogram was constructed using the UPGMA method. If the coefficient is 0.74, 36 mango cultivars and their relative species were divided into four major clusters (Fig. 2): (1) Red Ivory, Renmian Mang, Tianyangxiang Mang, Burma Mango, Macheso, Dwarf Mango, Thailand Mango, Jin Mango; (2) Zhanjiang Carabao, Sri Lanka 811, Burma Ball Mango 3, White Flower Mango, Indian Mango 15, Jinhuang Mang, Guandao Mang, Neelum, Okrong, Xiamao Mang, Longji'n Mango, Aroemwnis, Jinqian Carabao, M. himalis J. Y. Liang, India Mango 4, Alphonso, Bangalora, Zill, Guike 5 , Red Aroemwnis, Gaozhou Carabao, Datou Mang, Yellow Aroemwnis, M . siamerzsis Warbg ex Craib, Pakistan Mango; ( 3 ) Longmang, Liuzhou Carabao; and (4) M.
Table 2 Diversity analysis of ISSR-PCR with chloroplast DNA Primer'' ____
Sequence (S'-3')
UBC-8 11 UBC-835 UBC-840 UBC-841 UBC-851 UBC-XS7 GXU-1 UBC-826 Total
(GA),C (AG),YC (GA),YT (GA), YC
(GT), YG (AC),YG (ACACACAT), (ACLC
Total amplified bands
Number of polymorphic bands
~ - _ _
Percentage of polymorphic hands
7 10 8 I1 I1
5
71.4 80
X
5 9 8 9
10 11 11
10
19
61
62.5
81.8 72.7 90 90.9 63.6 71.2
7
"UBC represents primers designed by the Biotechnology Laboratory, University of British Columbia, Canada, GXU-I represent7 the primer designed in the laboratory.
I
. .. .. .
r IIXL
Fig. 2 A UPGMA dendrogram of relatonships among 36 mango cultivars and their relatives, bared on the Jaccard coefficient of similarity obtained from the chloroplast ISSR data. The abbreviations of the samples are shown in Table I .
02007,CAAS All rights reserved Pubilshedby Elsevter Ltd
Assessment of thc Genetic Relationship and Diversity of Mango and its Relatives by cpISSR Marker
persiciformis Wu & Ming. On the dendrogram, M . persicformis Wu & Ming showed the least similarity with all other mango cultivars tested in this study. The coefficients obtained from cpISSR between M. persicfirmis and Mangfera indica were less than 67%. Therefore, M . persicformis and Mangvera indica can be considered as two different species of genre mango at the molecular level. M . indica L. and M . siamenJis Warbg ex Craib were regarded as different mango species by traditional morphological taxonomy, but the result of the dendrograms of cpISSR Ahowed that these had considerably higher similarity, and these could be different cultivars of the same species. In the present study, M . himalis J.Y. Liang and Guandao Mang showed the highest similarity and these must be considered as the same species based on the two dendrograms, whereas, these are different species of genre mango by morphological classification. Based on the morphological characteristics and the result of the genome DNA ISSR (He et al. 2005) and cp ISSR, it is considered that M. h i m a h J. Y. Liang from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences could be Guandao Mang rather than M. h i m a h J . Y. Liang itself. In summary, chloroplast ISSR has proved to be an important complementaiy method in mango taxonomic studies and genetic diversity analysis. The high degree of chloroplast DNA polymorphism revealed by chloroplast ISSR verifies that this marker is a powerful tool for the study of mango parentage, as well as for the analysis of cytoplasm inheritance in mango.
141
and PCR polymorphisms. Theoreticd and Applied Genetics, 104, 1157-1163.
Cheng Y J, Carmen de Vicente M, Meng H J. Guo W W, Tao N
G, Deng X X. 2005. A set of primers for analyzing chloroplast DNA diversity in Citrus and related genera. Tree Physiology, 25, 661-672. Collevatti R G, Grattapaglia D, Hay J D. 2003. Evidences for multiple maternal lineages of Caryocar brasiliense populations in the Brazilian Cerrado based on the analysis of chloroplast DNA sequences and microsatellite haplotype variation. Molecular Ecology, 12, 105-1 15. Degani C, El-Batsri R, Gazit S. 1990. Enzyme polymorphism in mango. Journal of the American Suciew fur Horticultural Science, 115, 844-847. Deng J S, Lai Z C, Peng M Z. 1999. RAPD analysis of several mango cultivars. Journal uf Fruit Sciences, 16, 156-158. (in Chinese) Doyle J J, Doyle J L. 1990. Isolation of plant DNA from fresh tissue. Fucus, 12, 13-15. Eiadthong W, Yonemori K, Sugiura A, Utsunomiya N, Subhadrabandhu S . 1999. Identification of mango cultivars of Thailand and evaluation of their genetic variation using the amplified fragments by simple sequence repeat-(SSR-) anchored primers. Scientia Horticulturue, 82, 57-66. Eiadthong W, Yonemori K, Sugiura A, Utsunomiya N, Subhadrabandhu S. 2000. Amplified fragments length polymorphism analysis for studying genetic relationships among Mangifera species in Thailand. Journal ofthe American Society fur Horticultural Science, 125, 160-164. Gonzalez A, Coulson M, Brettell R. 2002. Development of DNA markers (ISSRs) in Mango. Actu Hurticulturae, 575, 139143. He X H, Li Y R, Guo Y Z, Tang Z P, Li R B. 2005. Inter-simple sequence repeat (ISSR) analysis of different native mango
Acknowledgements This research was supported by the National Natural Science Foundation of China (30560007),Natural Science Foundation of Guangxi Province of China (0542022) and Foundation of Guangxi Crop Genetic Improvement and Biotechnology Laboratory, China.
cultivars in Guangxi. Molecular Plant Breeding, 3, 829-834. Karihaloo J L, Dwivedi Y K, Archak S, Gaikwad A B. 2003. Analysis of genetic diversity of Indian mango cultivars using RAPD markers. Journal of Horticultural Science and Biutechnnlugy, 78,285-289. Lopez-Valenzuela J A, Martinez 0, Paredes-Lopez 0. 1997. Geographic differentiation and embryo type identification in Mangiferu indica L. cultivars using RAPD markers.
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Hurtscience, 32, 1105-1 108.
Adato AD, Sharon D, Lavi U?Hillel J, Gazit S. 1995. Application of DNA fingerprints for identification and genetic analyses
Mukherjee S K. 1950. Mango: its allopolyploid nature. Nature,
of mango (Mangtfera indica) genotypes. Journal of the
Palme A E, Vendramin G G. 2002. Chloroplast DNA variation,
American Society fur Hurticultural Science, 120, 259-264. Besnard G. Bervillk A. 2002. On chloroplast DNA variations in the olive ( O k a eurupaea L.) complex: comparison of RFLP
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postglacial recolonization and hybridization in hazel, Corylus avellana. Molecular Ecology, 9, 1769- 1779. Powell W, Morgante M, McDevitt R, Vendramin G G, Rafalski
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J A. 1995. Polymorphic simple sequence repeat regions in
Giannini R. 2004. Geographic distribution of chloroplast
chloroplast genomes: applications to the population genomes
variation in Italian populations of beech (FURUSsylvatica
of pines. Proceedings ofthe Natioml Academy of Sciences of
the USA, 92, 1759-1163. Robertson A, Newton A C , Ennos R A. 2004. Multiple hybrid origins, genetic diversity and population genetic Structure of two endemic Sorbus taxa on the Isle of Arran, Scotland.
Moleculur Ecology, 13, 123-134. Schnell R L. Running C M, Knight R 3. 1995. Identification of
L.). Theoretical and Applied Genetics, 109, 1-9. Vogel M, Banfer G, Moog U. Weising K. 2003. Development and characterization of chloroplast microsatellite markers in
Macaranga (Euphorbiaceae). Genome, 46. 845-851. Xu B Y, Jin Z Q, Peng S Q, Xu S P, Zhang X Q. 1998. RAPD analysis of genomic DNA in mango cultivars in Hainan Island.
Chinese Journal of Tropical Crop, 19, 33-37. (in Chinese)
cultivars and validation of genetic relationships in mangifera
Zhu X L, Zhang Y H, Zhou J: Lian Y W. 2003. Methods of
indica L. using RAPD markers. Theoretical and Applied
Castanopsis Kawaka mii chloroplast DNA extracting. Journal
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of Xiamen University, 42, 257-260. (in Chinese)
Vettori C, Vendramin G G, Anzidei M, Pastorelli R, Paffetti D, (Edited by ZHANG Yi-min)
02007,CAAS. All rights reserved.Published by Elsevier Lid.
Agricultural Sciences in China
2007. 6(2): 137- 142
ScienceDirect
Fcbruary 2007
Assessment of the Genetic Relationship and Diversity of Mango and Its Relatives by cplSSR Marker HE Xin-hua1-2,GUO Yong-ze2,LI Yang-mi1and OU Sh-jin2
’ ( h a n g x i Crop Genetic Improvement and Biotechnology
Laboratory, Guangxi Academy of Agriculturnl Sc.ience.s, Nulining 530007, P. R.
China College of Agriculture, Cuangxi University, Nanning 530004, P.R.China
Abstract Chloroplast inter-simple sequence repeat markers in mango were developed and used to analyze the genetic relationship and diversity of mango and its relatives. Thirty-six mango cultivars (Mungiferu indicu L.) and its relative species collected from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences, China, were examined by ISSR-PCR with chloroplast DNA (cpDNA). Eight better primers for chloroplast DNA that provided reproducible, polymorphic DNA amplification patterns were screened from 50 ISSR primers and used for UPGMA analysis. According to the band patterns with 8 primers for chloroplast DNA, all cultivars tested were distinguished from each other and these showed ample genetic diversity; the average percentage of polymorphism was 77.2%. The 36 samples could be clustered into four groups by UPGMA analysis at the coefficient 0.74. The results indicated that the cplSSR marker was a new powerful tool for the identification of mango cultivars or its relative species, and their genetic relationship analysis and diversity evaluation.
Key words: Mungiferu indicu, ISSR, cpDNA, genetic relationship, genetic diversity
INTRODUCTION Mango (Mangifera indica L.j is widely cultivated in tropical areas, and is a diploid fruit tree with 2n = 40 chromosomes (Mukherjee 1950). However, because of the wide sexual compatibility between mango and its relative species, the high frequency of bud mutations, long history of cultivation, and the occurrence of polyembryony in most of the species, the parentage and taxonomy of this genus are confusing. In the past, mango cultivars and relative species were often identified by the morphological and agronomic traits. With modern molecular biology development, most of the research with molecular markers based on the total DNA or the nuclear genome has been implemented in mango, and these include isozymes (Degani
et al. 1990), variable number tandem repeats (VNTR) (Adato et al. 1995), random amplified polymorphic DNA (RAPDs) (Schnell et al. 1995; Lopez-Valenzuela et al. 1997; Xu et al. 1998; Deng et ul. 1999; Karihaloo et al. 2003), inter simple sequence repeats (ISSRs) (Eiadthong et al. 1999; Gonzalez et al. 2002; He et al. 2 0 0 3 , and amplified fragment length polymorphism (AFLPsj (Eiadthong et al. 2000); however, there was no reports about chloroplast markers in mango. Since the chloroplast gene order is relatively conserved and its genome is small, non-recombinant, uniparentally inherited, and effectively haploid, which can overcome the disadvantages encountered in the analyses of nuclear DNA markers, it is the conservative nature of the chloroplast genome in higher plants that makes it more useful for parentage and taxonomic studies (Cheng et al. 2005). Chloroplast markers have been
Received 29 June, 2006 Accepted 27 September, 2006 HE Xin-hua, Tel: +86-771-3270184, Mobile: 01 3005921880. E-mail: [email protected]
02007, CAAS All rights reserved Publishedby Elsewer Ltd.
I38
HE X i n - h u a er al. ~
successfully used in the asscssment of relationship and genetic diversity of higher woody angiosperm plants, such as pine (Powell et al. 1995), Olea europuea L. (Besnard and Bervillk 2002), Coq1u.r avellana L. (Palmc and Vendramin 2002), Cnryocar brasiliense Camb. (Collevatti et al. 2003), Macarangu (Vogel et al. 2003), Sorbus urranensis Hedl. (Robertson et al. 2004), Fagus sylvatica L. (Vettori et al. 2004), and Citrus (Cheng et ul. 2005). In the present study, an attempt has been made to develop cplSSR markers for mango, thereby providing a powerful complementary method for studying the genetic relationship and diversity of mango and its relative species.
(Table 1) in thc present study were collected from the field h i t germplasm collection in thc Guangxi Academy of Agricultural Scienccs. Chloroplast DNA was extracted from fresh leaves according to the protocol (Zhu et al. 2003) with a slight modification. Mango chloroplast was obtained using the following methods: 10 g fresh leaves were pounded to pieces by a tissue triturator with buffer consisting of 0.2 mol L-' KC1, 0.8 mol L-' NaC1, and 8 mmol L' Na,S,O,, and then filtratcd with 9 layer gauzes. The filtrated homogenates were transferred to a tube of 50 mL, and equal volume of separated buffer was added, which consisted of 400 mmol L-' saccharose, 50 mmol L-' Tris-HC1,20 mmol L-I EDTA, 0.2% BSA, and 0.2% P-mercaptoethanol; pH 7.8, incubated at 0°C for 30 min and spun at 3 000 r/min for 5 min. The supernatant was transferred to another tube, and spun at 8 000 r/min for 8 min. The pellets were collected and purified with saccharose grads of 70, 60, 45, and 30%. DNA from the purified chloroplast was then extracted using the
MATERIALS AND METHODS Plant materials and chloroplast DNA extraction Leaves from 36 mango cultivars and relative species Table 1 Mango cultivars and the relative species used in the study Cultivar or species]) Red Ivory (seedling variation of Aroemwnis 26) Tianyangxiang Mang (seedling variation of Carabao) Alphonso Jin Mango Gaozhou Carabao Burma Mango Guandao Mang Indian Mango 15 Machew M . himnlis J. Y. Liarrg Okrong Pakistan Mango White Flower Mango Renmian Mang Liuzhou Lusong (variation of Philippine Mango) zhanjianp Carabao Aroemwnis Red Aroemwnis Zill Long Mang M. siamensis Warbg ex Craih Longjin Mango Jinqian Carabao Thailand Mango (unknown cultivar) Burma Ball Mango 3 Datou Mang (Yunnan) Bangalora Xianluo Mango Sri Lanka 8 I I Jinhuang Mane Xiamau Mane Neelum Dwarf Mango Yellow Aroernwnis M. persic$<)?-maWu & Ming Guike 5 -
Abbreviation ot the cultivar name') GxRI GxTY IAL YJM PhGZ BBM GxGD 11M 15 BMC GxMH ThOR PPM ThWF GdRM PhLZ Ph7J ThAS ThRA UZL YI M I1 4 GdLJ tidJQ ThTM BBB 3 YDT 1BG ThXL
SSL TJH GUM INL ThDM ThYA GxMW GxGG 5
Geographic origin Guangxi, China Guangxi, China India Yunnan, China Philippines Burma Guangxi, China India
Burma Guangxi, China Thailand Pakistan Thailand Guangdong, China Philippine3 Philippines Thailand Thailand USA Yunnan. China India Guangdong. China Guangdong, China Thailand Burma Yunnan. China Indian Thailand Sri I.ankn Taiwan Guanpdong, China India Thailand Thailand Guangxi. China Guangxi. China
Embryo type Pol yernbryonic Polyembryonic Monoembryonic Monoembryonic Polyembryonic Polyernbryonic Pol yembryonic Unknown Polyemhryonic Monoembryonic Polyenihryonic Unknown Polyembryonic Polyembryonic Polyembryonic Polyemhryonic Polyernbryonic Polyembryonic Monoembryonic Polyernbryonic Unknown Monoembryonic Polycinbryonic 1J nknow n Monocmbryonic Polyernbryonic Monoembryonic Polyembryonic Pnlyernbr~wnic Polyembryonic Polyemhryonic Monoembryonic Polyemhryonic Polyembrynnic Unknown Polyembryonic
' Except M . himalir J. Y. Liang, M . ,$iumcnaisWarhg ex Craib, and M. persiciforma Wu & Ming, and the others are the cultivars 0f.M. indlca L. Thrcc or four letters represent the abbreviation of thc cultivar's name or species.
02007,CAAS. All rights reserved. Publishedby Elsevierltd.
Assessmcnt of the Ccnetic Relationship and Diversity of Mango and its Relatives by cplSSR Marker~
hexadecyltrimethylammonium bromide (CTAB) method described by Doyle J J and Doyle J L (1990).
ISSR analysis Forty-four ISSR primers of the University of British Columbia, Canada for nuclear genome and six primers designed in the lab were used in the study, and these were synthesized by the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd, China. Each 20 pL amplification reaction consisted of 10 mM Tris-HC1 (pH 8.3), 50 mM KCI, 1.5 mM MgCl,, 0.2 mM mixed dNTP, 0.25 pM primer, 1 unit rTaq polymerase (Takara Biotechnology, Japan), and approximately 60 ng chloroplast DNA. Amplification was done in a thermocycler under the following conditions: 5 min at 94°C for 1 cycle, followed by 1 min at 92°C 1 min at 50"C, and 2 min at 72°C for 40 cycles, and 10 min at 72°C for the final extension. The amplification products were loaded onto a 2.0% agarose gel in a 1 x TBE buffer and were electrophoresed at 100 V for 1 h, and then visualized by staining the gel with ethidium bromide. The size of each fragment was estimated with reference to GeneRulerTM 100 bp DNA ladder plus (MBI).
Data analysis By electrophoresis, seventy-nine reproducible bands
_
_
139
from the selected primers were scored as 1 (presence) or 0 (absence) for 36 mango cultivars and the relative species were tested. Thcn, the unweighted pair-group method using the arithmetic average\ (UPGMA) cluster analysis was performed using the NTSYSpc version 2.1 e software.
RESULTS AND DISCUSSION Screening of primers and diversity analysis Four genotype sample\ (Carabao, Okrong, Zill, and M . persiciformis Wu & Ming) were used for the initial screening of the ISSR primers. Eight primers among the 50 ISSR primers that could amplify the chloropkdst DNA of 36 mango cultivars and their relatives and produce intense bands on agarose gels (Fig.1) revealed high polymorphism among the mango accessions as well as the relatives, generating 61 polymorphic bands (accounting for 77.2% of the total bands), and every primer among the 8 primers could amplify 5 to 11 cpDNA bands with length between 3 000 and 300 bp (Table 2). The cpISSR primers in the study were designed based on nuclear DNA ISSR primers, while the most optimal ISSR primers were different. The primers (GAA),, (GA),YC. and (GT),YG showed the highest level of polymorphism for the nuclear genome DNA ISSR of
Fig. 1 PCR amplified pattern by the UBC-826 primer in 24 mango cultivars. Lane M, GeneRulcrT" 100 bp DNA ladder plus; lanes I to 24 represent cultivars Red Ivory, Tianyangxiang Mang, Renmian Mang, Jin Mang, Macheso, Burma Mango. Dwarf Mango, Sri Lanka 81 1, Burma Ball Mango 3, Indian Mango 15, Jinhuang Mango, Guandao Mang, Ncelum, Okrong. Jinqian Carabao, Long Jin Mango, M. himalis J. Y. Liang, India Mango 4, Zill, M. siamensi.s Warbg ex Craib. Red Aroemwnis, Yellow Aroemwnis, Pakistan Mango. and Guike 5, respectively.
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HE Xin-hua el ul.
140
mango (Eiadthong et al. 1999; He e t a l . 2005). However, the primers (AC),YG and (ACACACAT), showed higher level of polymorphism for the mango chloroplast DNA ISSR in the present study (Table 2). According to the banding patterns obtained by cpISSR with 8 selected primers, all cultivars tested in this study could be distinguished from each other and showed huge diversity, indicating that the cpISSR marker was effective for the identification of mango cultivars or the relative species and for evaluation of their genetic diversity.
Cluster analysis Based on the Jaccard coefficient of similarity obtained
from the chloroplast ISSR band sizes, a dendrogram was constructed using the UPGMA method. If the coefficient is 0.74, 36 mango cultivars and their relative species were divided into four major clusters (Fig. 2): (1) Red Ivory, Renmian Mang, Tianyangxiang Mang, Burma Mango, Macheso, Dwarf Mango, Thailand Mango, Jin Mango; (2) Zhanjiang Carabao, Sri Lanka 811, Burma Ball Mango 3, White Flower Mango, Indian Mango 15, Jinhuang Mang, Guandao Mang, Neelum, Okrong, Xiamao Mang, Longji'n Mango, Aroemwnis, Jinqian Carabao, M. himalis J. Y. Liang, India Mango 4, Alphonso, Bangalora, Zill, Guike 5 , Red Aroemwnis, Gaozhou Carabao, Datou Mang, Yellow Aroemwnis, M . siamerzsis Warbg ex Craib, Pakistan Mango; ( 3 ) Longmang, Liuzhou Carabao; and (4) M.
Table 2 Diversity analysis of ISSR-PCR with chloroplast DNA Primer'' ____
Sequence (S'-3')
UBC-8 11 UBC-835 UBC-840 UBC-841 UBC-851 UBC-XS7 GXU-1 UBC-826 Total
(GA),C (AG),YC (GA),YT (GA), YC
(GT), YG (AC),YG (ACACACAT), (ACLC
Total amplified bands
Number of polymorphic bands
~ - _ _
Percentage of polymorphic hands
7 10 8 I1 I1
5
71.4 80
X
5 9 8 9
10 11 11
10
19
61
62.5
81.8 72.7 90 90.9 63.6 71.2
7
"UBC represents primers designed by the Biotechnology Laboratory, University of British Columbia, Canada, GXU-I represent7 the primer designed in the laboratory.
I
. .. .. .
r IIXL
Fig. 2 A UPGMA dendrogram of relatonships among 36 mango cultivars and their relatives, bared on the Jaccard coefficient of similarity obtained from the chloroplast ISSR data. The abbreviations of the samples are shown in Table I .
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Assessment of thc Genetic Relationship and Diversity of Mango and its Relatives by cpISSR Marker
persiciformis Wu & Ming. On the dendrogram, M . persicformis Wu & Ming showed the least similarity with all other mango cultivars tested in this study. The coefficients obtained from cpISSR between M. persicfirmis and Mangfera indica were less than 67%. Therefore, M . persicformis and Mangvera indica can be considered as two different species of genre mango at the molecular level. M . indica L. and M . siamenJis Warbg ex Craib were regarded as different mango species by traditional morphological taxonomy, but the result of the dendrograms of cpISSR Ahowed that these had considerably higher similarity, and these could be different cultivars of the same species. In the present study, M . himalis J.Y. Liang and Guandao Mang showed the highest similarity and these must be considered as the same species based on the two dendrograms, whereas, these are different species of genre mango by morphological classification. Based on the morphological characteristics and the result of the genome DNA ISSR (He et al. 2005) and cp ISSR, it is considered that M. h i m a h J. Y. Liang from the fruit germplasm collection in the Guangxi Academy of Agricultural Sciences could be Guandao Mang rather than M. h i m a h J . Y. Liang itself. In summary, chloroplast ISSR has proved to be an important complementaiy method in mango taxonomic studies and genetic diversity analysis. The high degree of chloroplast DNA polymorphism revealed by chloroplast ISSR verifies that this marker is a powerful tool for the study of mango parentage, as well as for the analysis of cytoplasm inheritance in mango.
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and PCR polymorphisms. Theoreticd and Applied Genetics, 104, 1157-1163.
Cheng Y J, Carmen de Vicente M, Meng H J. Guo W W, Tao N
G, Deng X X. 2005. A set of primers for analyzing chloroplast DNA diversity in Citrus and related genera. Tree Physiology, 25, 661-672. Collevatti R G, Grattapaglia D, Hay J D. 2003. Evidences for multiple maternal lineages of Caryocar brasiliense populations in the Brazilian Cerrado based on the analysis of chloroplast DNA sequences and microsatellite haplotype variation. Molecular Ecology, 12, 105-1 15. Degani C, El-Batsri R, Gazit S. 1990. Enzyme polymorphism in mango. Journal of the American Suciew fur Horticultural Science, 115, 844-847. Deng J S, Lai Z C, Peng M Z. 1999. RAPD analysis of several mango cultivars. Journal uf Fruit Sciences, 16, 156-158. (in Chinese) Doyle J J, Doyle J L. 1990. Isolation of plant DNA from fresh tissue. Fucus, 12, 13-15. Eiadthong W, Yonemori K, Sugiura A, Utsunomiya N, Subhadrabandhu S . 1999. Identification of mango cultivars of Thailand and evaluation of their genetic variation using the amplified fragments by simple sequence repeat-(SSR-) anchored primers. Scientia Horticulturue, 82, 57-66. Eiadthong W, Yonemori K, Sugiura A, Utsunomiya N, Subhadrabandhu S. 2000. Amplified fragments length polymorphism analysis for studying genetic relationships among Mangifera species in Thailand. Journal ofthe American Society fur Horticultural Science, 125, 160-164. Gonzalez A, Coulson M, Brettell R. 2002. Development of DNA markers (ISSRs) in Mango. Actu Hurticulturae, 575, 139143. He X H, Li Y R, Guo Y Z, Tang Z P, Li R B. 2005. Inter-simple sequence repeat (ISSR) analysis of different native mango
Acknowledgements This research was supported by the National Natural Science Foundation of China (30560007),Natural Science Foundation of Guangxi Province of China (0542022) and Foundation of Guangxi Crop Genetic Improvement and Biotechnology Laboratory, China.
cultivars in Guangxi. Molecular Plant Breeding, 3, 829-834. Karihaloo J L, Dwivedi Y K, Archak S, Gaikwad A B. 2003. Analysis of genetic diversity of Indian mango cultivars using RAPD markers. Journal of Horticultural Science and Biutechnnlugy, 78,285-289. Lopez-Valenzuela J A, Martinez 0, Paredes-Lopez 0. 1997. Geographic differentiation and embryo type identification in Mangiferu indica L. cultivars using RAPD markers.
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