Morphological, Molecular Characterization and Breeding for Biotic and Abiotic Stress in Mango (Mangifera indica L.) M.R. Dinesh, C. Vasugi and K.V. Ravishankar Division of Fruit Crops Indian Institute of Horticultural Research Bangalore-89 India Keywords: abiotic, biotic, breeding, characterization, mango, markers, morphological, molecular Abstract Mango (Mangifera indica L.) is the most important fruit crop in India having socio-economic significance. Morphological characterization involves the study of development of form, structure with attempts being made to interpret these on the basis of similarity and origin. Owing to the high heterozygosity and seedling propagation, nomenclature ambiguity is one of the main impediments in classification of mango. Under the morphological characterization the widely used keys are the ones developed by ‘Bioversity International’ which has 54 characteristics. Morphological characterization has assisted in the classification of cultivars but ambiguity in nomenclature persists due to its limitation by way of minor differences between the characteristics. Molecular markers are used to measure diversity at DNA level and are not much influenced by environmental conditions like morphological markers. Classification of cultivars and screening of cultivars for biotic and abiotic stress would help in selecting the parents for the hybridization program and development of recombinants resistant to various biotic and abiotic stresses. Breeding attempts for abiotic stress made in mango have centered on the rootstock improvement in few places where soil salinity is a problem. Screening of polyembryonic cultivars has shown the cultivars ‘Olour’ and ‘Bappakkai’ to be tolerant compared to others. INTRODUCTION Mango (Mangifera indica L.) is the most important fruit crop in India having socio-economic significance. It is known as king of fruits owing to the delicious quality of fruit rich in vitamins and minerals. The northeastern region is the centre of diversity for several important native fruits including mango. Mango is highly cross-pollinated and heterozygous, which has resulted in enormous variability over the years, which in turn has resulted in nomenclature ambiguity. A cultivar that is grown in a particular region is known by different names in other regions. In the case of mango, morphological keys have been developed by IPGRI (2006). One of the problems with morphological characterization is the expression of characters by a cultivar under a set of environment i.e., the genotype × environment interaction. This can mislead the results in the case of morphological characterization as a native to one region may not express in the same way as it expresses in its place of origin. However, to conclusively prove that two cultivars are synonyms, characterization at the genetic level would be more helpful. It is widely accepted now that molecular characterization is fairly accurate and would help in removing this ambiguity. MORPHOLOGICAL CHARACTERIZATION Morphological characterization involves the study of development of form, structure with attempts being made to interpret these on the basis of similarity and origin. It is likely that similar underlying causes of genetics, physiology, or response to the environment have led to this similarity in appearance. Scientific investigation into these causes can lead to one of two insights. One is ‘Homology’ – the structure is similar Proc. of the Global Conference on Augmenting Production and Utilization of Mango: Biotic and Abiotic Stresses Eds.: H. Ravishankar et al. Acta Hort. 1066, ISHS 2015 37 between the two species because of shared ancestry and common genetics and ‘Convergence’ – the structure is similar between the two species because of independent adaptation to common environmental pressures. Morphological, agronomical as well as biochemical parameters (Rick and Holle, 1990; Weber and Wricke, 1994; Kaemmer et al., 1995) have been widely used in the evaluation of various crops. Exploitation of such traits increases our knowledge on the genetic variability and strongly facilitates breeding for wider geographic adaptability. Mango has a long history of domestication, due to the high heterozygosity and seedling propagation, nomenclature ambiguity is one of the main impediments in classification. Several studies have been conducted on morphological descriptions of mango from time to time (Burns and Prayag, 1921; Mukherjee, 1948; Naik and Gangolly, 1950; Singh and Singh, 1956; Gangolly et al., 1957; Rajan et al., 2001; Yeshitela and Nessel, 2003; Desai and Dhander, 2000; Dinesh and Vasugi, 2002). Yeshitela and Nessel (2003) characterized 75 mango ecotypes based on the morphological traits. Under the morphological characterization the widely used keys are the ones developed by the ‘Bioversity International’ which has 54 characteristics. MOLECULAR CHARACTERIZATION Morphological characterization has assisted in the classification of cultivars in mango. However, ambiguity persists due to its limitation in identifying cultivars with little difference. Molecular markers are used to measure diversity at DNA level and are not much influenced by environmental conditions like morphological markers. The discovery of molecular markers has led to the detailed genetic analysis and approaches to improvement of crop plants. It is differentiated into protein markers, which were discovered first and DNA based markers. Recent advances made with the molecular characterization helped further in clearing this ambiguity. Biochemical markers have played an important role in the beginning. Many researchers have used biochemical markers for identifying cultivars in mango (Schnell and Knight, 1992; Degani et al., 1990; Gan et al., 1981; Truscott et al., 1994). Isozymes as genetic markers have been proved to be reliable, consistent and essentially unaffected by environmental conditions (Bailey, 1983; Torres and Bergh, 1980). However, isozymes can be affected by stages of development and tissue used for extraction (Feret and Bergmann, 1976). The DNA based markers like RFLP and RAPD provide excellent tools to study the genetic diversity, eliminate duplicates in germplasm, study relationships, gene tagging, genome mapping, PVR (Plant Varietal Rights) purposes, etc. The success of molecular markers in characterizing and analyzing genetic diversity in mango has been demonstrated by a number of studies (Schnell and Knight, 1993; Adato et al., 1995; Schnell et al., 1995; Lopez-Valenzuela et al., 1997; Ravishankar et al., 2000; Hemanthkumar et al., 2001). The RAPD technique was used by Schnell et al. (1995), Ravishankar et al. (2000) and Hemanthkumar et al. (2001) for cultivar identification and analysis of genetic relatedness in mango. Schnell et al. (1995), found the maternal halfsibs to cluster together indicating the usefulness of RAPD markers in studying the genetic relationships. In 1990, PCR based DNA marker called Random Amplification of Polymorphic DNA (RAPD) was developed (Williams et al., 1990; Welsh and McClelland, 1990). This RAPD technique was first used in mango by Schnell and Knight (1993). In their study, nine Mangifera species were analyzed and compared with traditional taxonomic relationships based on morphological characters. Later, Schnell et al. (1995) adopted RAPD analysis for identification of cultivars and to assess genetic relationship among cultivars. DNA from 25 cultivars was amplified using 80, 10 mer random primers. None of these primers amplified unique band pattern for each of the cultivars. However, 10 different combinations of 2 primers produced unique fingerprints of each of cultivars. Dendrogram analysis showed that maternal half sibs cluster together indicating usefulness of RAPD markers in genetic relatedness study. Later Lopez-Valenzuela et al. (1997) used 38 40 random primers, to assess genetic relatedness among 15 mango cultivar collected from Mexico, The Philippines, Florida and Reunion. Dendrogram analysis of RAPD markers showed 4 major groups, clearly classifying genotypes according to their geographic origin. This study also identified RAPD markers specific to polyembryonic and monoembryonic cultivars. Later in a study, RAPD markers were used to show diverse genetic base of Indian poly-embryonic and mono-embryonic mango cultivars (Ravishankar et al., 2004). RAPD analysis was done using 18 mango cultivars from different parts of India for RAPD analysis. The cluster analysis of RAPD markers generated showed that mango cultivars from a particular region group together indicating high degree of genetic relatedness among them (Ravishankar et al., 2000). Later there were several genetic diversity studies on Indian mango cultivars using RAPD markers and they demonstrated high degree of diversity in Indian mango germplasm (Karihaloo et al., 2003; Bajpai et al., 2008). Later a few studies employed another PCR based marker; inter-simple sequence repeats (ISSR) to study genetic diversity and genetic relatedness among mango genotypes (Pandit et al., 2007; Bajpai et al., 2008). Eiadthong et al. (1999) utilized anchored simplesequence repeat markers to analyze 22 mango cultivars. They were able to distinguish genotypes, but were unable to find markers unique to either mono-embryonic or polyembryonic types. Adato et al. (1995) used radioactively labelled Jeffery’s minisatellite probes and synthetic minisatellites for identification of mango cultivars. Jeffery’s minisatellite probe 33.6 was most useful, highly polymorphic and generated specific pattern for each of the 20 mango cultivars used. Kashkush et al. (2001) utilized amplified fragment-length polymorphisms (AFLP) to estimate genetic relationships between 16 cultivars and 7 rootstock cultivars. They also analyzed 29 progenies from the cross of ‘Tommy Atkins’ and ‘Keitt’ and produced a crude linkage map that identified 13 of the 20 linkage groups. Simple Sequence Repeats Markers have been used successfully in characterization of many crop species. SSR markers are highly accurate and amenable for large scale use in germplasm characterization. In recent years, there are a few efforts to develop SSR markers for mango. So far nearly 105 SSR markers are available for mango. However, this is a very small number for mango. Viruel et al.(2005) developed the first reported set of 16 microsatellite markers for mango, of which 14 produced the expected one or two amplification products per genotype. These 14 microsatellites were used to evaluate 28 mango genotypes. Discrimination of all 28 genotypes was possible and the average number of alleles per locus was 5.3. Schnell et al. (2005) developed a second set of 15 microsatellite markers and analyzed 59 Florida cultivars and 4 related species. Two of the microsatellites were monomorphic among the Florida cultivars: the other 13 had an average number of alleles per locus of 4.2 with PTC values varying from 0.21 to 0.63. Duval et al. (2005) developed 28 SSR primer pairs, they produced polymorphic amplification products for a diversity sample including 15 mango cultivars and two accessions from the related species Mangifera laurina and Mangifera applanata. 19 simple sequence repeat (SSR) loci with clear scorable patterns were chosen to study diversity in the mango germplasm bank of Guadalupe (FWI). The number of alleles ranged from 3 to 13 with observed levels of heterozygosity ranging from 0.059 to 0.857. Honsho et al. (2005) developed 6 microsatellite markers for mango using a microsatelliteenriched genomic library. They were characterized using 36 cultivars collected mainly in Thailand. The observed and expected heterozygosity ranged from 0 to 0.83 and from 0.29 to 0.73, respectively. The number of putative alleles was two to six. Recently at IIHR, we have developed 36 microsatellite markers and validated using 30 mango cultivars (Ravishankar et al., 2011). The number of alleles ranged from 3 to 19 with an average of 9.2 alleles per locus. Polymorphic information content values ranged from 0.185 to 0.920 with a mean of 0.687. The total value for the probability of identity was 2.42×10-31. We have also demonstrated cross species amplification in 5 related species, M. odorata, M. anadamanica, M. zeylanica, M. camptosperma, and M. griffithii, showing their 39 potential utility across species. Fourteen selected SSR markers (with high PIC) have been employed for molecular characterization of Indian mango cultivars (both at IIHR, Bangalore and CISH, Lucknow). So far nearly 400 mango cultivars have been characterized using 14 SSR markers. Genetic analysis showed that there are two major groups among Indian cultivars. In addition to molecular characterization, SSR markers have been used for pedigree analysis. Pedigree analysis of Florida mango cultivars showed that when compared by origin, the Florida cultivars were more closely related to Indian than to Southeast Asian cultivars (Schnell et al., 2006). Apart from molecular characterization of mango cultivars, DNA markers have been employed for phylogenetic studies of different Mangifera species. The phylogenetic relationship of 13 Mangifera species collected in Thailand was examined using cpDNARFLP technique. Based on the analysis of results, these species were classified into two groups. The first group consisted of M. indica and M. sylvatica, and the second group consisted of M. caloneura, M. cochinchinensis, M. collina, M. flava, M. foetida, M. gedebe, M. griffithii, M. macrocarpa, M. oblongifolia, M. odorata, and M. pentandra (Eiadthong et al., 1999). AFLP technique was employed to study phylogenetic relationship among 10 Mangifera species. Results showed that the common mango (Mangifera indica) was closely related to M. sylvatica, M. laurina and M. oblongifolia (Eiadthong et al., 2000). In our center, we have done analysis of chloroplast and its markers among M. indica, M. anandamanica, M. griffiti, and M. odorata. The results generated shows that M. anadamanica forms a different group indicating that it may not belong to the subgenus Mangifera. BREEDING FOR BIOTIC AND ABIOTIC STRESS Mango being perennial in nature, is subjected to several biotic stresses like pests and diseases and abiotic stresses like soil and environmental factors. The variability among the cultivars being very high, there is always scope for locating a source of resistance. The polyembryonic cultivars, for which variability is less and which are generally seen in regions of high rainfall should be screened for these stresses. There is also a need for creating variability in these cultivars, as these would help in improving the quality in these types. Identification of zygotic and nucellar embryony using markers would help in selecting new recombinants. However, as a prelude, there is an urgent need for evaluating the wide array of germplasm. It is of paramount importance to study the inheritance pattern of various quantitative traits in these cultivars. The breeding strategy that needs to be adopted is discussed hereunder. Genetical Studies – Basis for Combining Resistance and Quality The genetical studies to derive at the heritability of the different characteristics would help us in choosing the parents to develop recombinants which may be resistant to some of the biotic and abiotic stress as coupled with tolerance we need better quality. Hence, these studies act as a prelude to the breeding of cultivars. However, the fact remains that mango is highly heterozygous and progeny performance cannot be predicted. Lavi et al. (1989) observed in the distribution of different traits in seedlings derived from open-pollination (where the pollen parent is unknown) that there is no maternal effect on juvenile period and fertility, there is a slight effect of the female parent on fruit taste and size and there is a maternal parent effect on harvest season and fruit color. Varied reports are available on the inheritance of pulp color; that it is governed by additive gene action is one report by Sharma (1987), but Iyer (1991), observed yellow pulp color to be dominant over orange-yellow. The inheritance of skin color seems to be mediated by a number of loci as observed by gradation in the color (Sharma, 1987; Iyer and Subramanyam, 1987). Dinesh (2003) observed that heritability is low and the chances of hybrid vigour manifesting for the characters in the F1 generation are bright. Selection of progenies can be made based on fruit size i.e., medium sized fruits will have good TSS and selecting big sized fruits will bring down the TSS. It was also noticed that for the traits studied, phenotypic coefficient of variation was more than the genotypic coefficient emphasizing 40 the greater manifestation of characters and lesser influence of environment. The genetic studies in mango carried out earlier showed that red colored cultivars when crossed with green colored cultivars resulted in the progenies having gradation of colors, which indicated that it is controlled by a number of loci (Sharma, 1987; Iyer, 1991; Iyer and Subramanyam, 1987). The progeny population from the combination ‘Alphonso’ × ‘Banganapalli’, ‘Alphonso’ × ‘Janardhan Pasand’, ‘Raspuri’ × ‘Neelum’, ‘Alphonso’ × ‘Neelum’, ‘Alphonso’ × ‘Kerala Dwarf’, ‘Amrapali’ × ‘Arka Anmol’ was observed to have gradations in fruit skin color. The cultivars ‘Janardhan Pasand’ as male parent has imparted skin color to the progenies (Table 1). The role of heterozygosity and the number of loci controlling skin color can be gauged from the fact that even the progenies from the combination ‘Alphonso’ and ‘Neelum’ has resulted in attractive colored fruits, same is true for the progenies of the combination ‘Alphonso’ × ‘Banganapalli’. However, it is to be mentioned here that when cultivars like ‘Amrapali’ and ‘Arka Anmol’ were used, none of the progenies had attractive skin color, which shows that the expression of skin color in recombinants of heterozygous cultivars is highly unpredictable. It is also to be mentioned here that there is no repeatability in the recombinants. Brettell et al. (2004) inferred that in large number of mango hybrids important fruit characteristics such as fruit weight, fruit shape, skin color, fruit width and pulp thickness have high heritability and can therefore be readily selected in a breeding program, of particular interest is the observation that a high frequency of hybrids with red or burgundy blush can be recovered from crosses where one parent has an intense red blush color. Similarly, while the unique flavor compounds associated with ‘Kensington Pride’ are also found in nearly 50% of the hybrids involving ‘Kensington Pride’, leaf fragrance was not found to be a reliable predictor of fruit flavor in this case. Resistance Breeding Mango is subjected to biotic stresses during the various stages of growth. Mango breeding started in 1911 (Burns and Prayag, 1921) has undergone a lot of changes in the selection of parents as well as in the methodology with the objectives becoming more focused. Hybridization programs carried out at different centers have resulted in many hybrids, which have excellent skin color and good sugar acid blend, which may be suitable for export. Several insect pests are known to attack the mango crop. The most important ones are hopper (Idiocerus spp.), fruit fly (Dacus dorsalis) and stone weevil (Sternochetus mangiferae). Varying degree of susceptibility by different cultivars has been reported (Iyer, 1991). We need to carry out screening of germplasm for pests like fruit fly and stone weevil and for diseases like anthracnose extensively to determine the degree of tolerance/resistance. The screening of cultivars for fruit fly at the Indian Institute of Horticultural Research showed that the cultivars ‘Amrapali’ and ‘Vellaikolamban’ did not record any infestation under artificial conditions. Rami Reddy et al. (2005) observed that ‘LA Resource-1’ can be one of the options for hopper resistance source. The cultivar ‘Bhadauran’ is resistant to malformation and has been used in hybridization. However, all the F1 hybrids were susceptible when it was used as one of the parents in the breeding program (Sharma and Majumder, 1988a). The screening of 102 cultivars by Ram Nath et al. (1987) showed that the cultivars ‘Bhyadayam Dula’, ‘Samar Bahisht Rampur’ and ‘Mian Sahib’ were found to be free from malformation. These cultivars also can be tried in a breeding program. Gupta (1976) has reported the cultivars ‘Neelum’, ‘Zardalu’, ‘Janardhan Pasand’ to be tolerant to powdery mildew (Oidium mangiferae Berthat). Bacterial canker is another serious problem, Om Prakash and ‘Srivastava (1987) reported that the cultivar ‘Bombay Green’ is resistant to canker. However, it is to be mentioned here that screening of cultivars needs to be carried out at least for two to three years, as it is our experience that the cultivar ‘Pulihora’, which was found tolerant to anthracnose during one year was observed to be susceptible during another year. 41 Breeding for Off Season Types – a Strategy to Combat Biotic and Abiotic Stress? Utilizing the off season cultivars in the hybridization program is one of the major challenges in mango breeding. In the Kanyakumari region of India, a number of cultivars has been reported to be off-season bearers. Since, there is no synchronization in the flowering there is need to conserve the pollen and use them in the hybridization program. A large number of recombinants thus derived would help in the selection of progenies. It is imperative that we have to use indigenous cultivars viz., ‘Kalkand’, ‘Chengavarikkai’, which basically belong to the Kanyakumari region and not the other introduced types, as the indigenous types would have the inbuilt genes in them, which may probably express in other environments also. For utilizing off season cultivars it is imperative that pollen storage will have to be taken up. Vasugi et al. (2009), observed that the species M. odorata and M. zeylanica are cross compatible with M. indica. The percent fruit set was observed to be 33.64 and 24.78% for M. odorata and M. zeylanica, respectively using stored pollen with ‘Arka Anmol’ as the female parent, among the M. indica cultivars, the fruit set ranged from 12.4 to 58.06% by the use of cryopreserved pollen. Use of Wild Species Wild species of mango can be of great use in crop improvement programmes. Iyer (1991) suggested that the species could be useful in crop improvement in two ways; 1. species having edible fruit with other desirable characters; 2. species that could act as gene donors for specific improvement like resistance to pest and disease. The potential use of wild species in breeding has also been enumerated (Bompard, 1993). The wild species viz., Mangifera rufocostata and M. swintonioides have the peculiarity of flowering and fruiting out of the main season. In East Kalimantan their fruiting time usually coincides with the famine season hence called as famine food. Very little work has been done on the evaluation of wild species. Hence, there is an urgent need to conserve, screen and use them in breeding program. Mukherjee (1963) felt that different Mangifera species could be inter-crossed easily based on the success obtained from the crossing of M. zeylanica and M. odorata. Similar studies on the crossability of wild species, using M. odorata and M. zeylanica indicated that the percent fruit set was 33.64% in M. odorata and 24.78% for M. zeylanica using stored pollen with ‘Arka Anmol’ as the female parent (Vasugi et al., 2009). Iyer (2001) has listed the species that are useful as parents in the breeding program; he has mentioned that wild species, Mangifera zeylanica can be used as rootstock for saline soils and M. minor as resistant to anthracnose. Use of Tolerant Rootstock to Abiotic Stress The use of rootstocks can help in two ways, one is by development of new rootstocks tolerant to problematic soils and another is by way of screening existing polyembryonic cultivars. Screening of mango rootstocks to salinity has shown that the polyembryonic cultivars ‘Color’ and ‘Bappakkai’ could withstand higher level of salinity (Palaniappan, 2001). Mango cultivar ‘13-1’ was selected as a polyembryonic (3-6 embryos) rootstock for calcareous soils and/or for irrigation with saline water. Mango trees on ‘13-1’ rootstocks showed excellent performance on soil containing 20% lime, three other cultivars on ‘13-1’ rootstock showed good development on sandy soil with 10-20% lime and irrigation water containing 250 ppm. The rootstock, Cl growth was best in soil with good drainage (Gazit and Kadman, 1980). Creating Variability in Polyembryonic Cultivars In India and elsewhere, the variability for poly-embryonic cultivars is less. Although there are several good cultivars like ‘Prior’, which is one of the main commercial cultivars of Kerala; compared to mono-embryonic cultivars the variability is low. Lavi et al. (2004) observed that mother trees should not be chosen entirely on the basis of their phenotypes, and trees with inferior performance could also be included when necessary since in mango progeny performance is quite unpredictable. Since they 42 observed that most of the variance components of the agriculturally interesting traits are non-additive (Lavi et al., 1998) and since most of these traits result from dominant and epistatic interactions the approach in the Israel mango breeding programme is to rely on open pollination. Many of the cultivars like ‘Osteen’ are selections from open pollinated progenies. Many of the poly-embryonic cultivars find their origin in the Western Ghats or in the north-eastern region of the country, which are heavy rainfall areas. Hence, there are chances of making selections for anthracnose tolerant or resistant types and further these can be used as parents in the breeding program. Some of the poly-embryonic cultivars like ‘Peach’ and ‘Nekkare’ also have attractive skin color. Literature Cited Adato, A., Sharon, D. and Lavi, U. 1995. Application of DNA fingerprints for identification and genetic analyses of mango (Mangifera indica) genotypes. J. Amer. Soc. Hort. Sci. 120:259-264. Bailey, D.C. 1983. Isozymic variation and plant breeders’ rights. p.425-440. In: S.D. Tanksley and T.J. Orton (eds.), Isozymes in Plant Genetics and Breeding. Part A. Elsevier, New York. Bajpai, A., Srivastava, N., Chandra, R. and Rajan, S. 2008. Genetic diversity and discrimination of Mangifera indica L. accessions using ISSR and RAPD markers. Ind. J. Horticul. 65:377-382. Bompard, J.M. 1993. The genus Mangifera rediscovered: the potential contribution of wild species to mango cultivation. Acta Hort. 341:69-77. Brettell, R.I.S., Johnson, P.R., Kulkarni, V.J., Muller, W. and Bally, I.S.E. 2004. Inheritance of fruit characters in hybrid mangoes produced through controlled pollination. Acta Hort. 645:319-326. Burns, W. and Prayag, S.H. 1921. The Book of Mango Bombay. Dept. Agric. Bull. Degani, C., El-bastri, R. and Gazit, S. 1990. Enzyme polymorphism in mango. J. American Soc. Hort Sci. 115:844-847. Desai, A.R. and Dhandar, D.G. 2000. Variation in physico-chemical and morphogenetic characters of some mango cultivars of Goa. Acta Hort. 509:243-252. Dinesh, M.R. 2003. Genetical studies in mango (Mangifera indica L.). J. Appl. Hort. 5:27-28. Dinesh, M.R. and Vasugi, C. 2002. Catalogue of Mango Germplasm. Published by IIHR. Duval, M.F., Bunel, J., Sitbon, C. and Risterucci, A.M. 2005. Development of microsatellite markers for mango (Mangifera indica L.). Mol. Ecol. Notes 5(4):824826. Eiadthong, W., Yonemori, K., Kanzaki, S. and Sugiura, A. 2000. Amplified fragment length polymorphism analysis for studying genetic relationship among Mangifera species. J. Amer. Soc. Hort. Sci. 125:160-164. Eiadthong, W., Yonemori, K., Sugiura, A., Utsunomiya, N. and 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 Hort. 82:57-66. Feret, P.P. and Bergmann, F. 1976. Gel electrophoresis of proteins and enzymes. p.49-77. In: J.P. Miksche (ed.), Modern Methods in Forest Genetics. Springer-Verlag, New York. Gan, Y.Y., Zaini, S. and Idris, A. 1981. Genetic variation in the grafted vegetatively propagated mango (Mangifera indica). Pertanika 4:53-62. Gangolly, S.R., Ranjit Singh, Katyal, S.L. and Singh, D. 1957. The Mango. Published by ICAR, New Delhi. Gazit, S. and Kadman, A. 1980. 13-1 mango rootstock selection. J. HortScience 15(5):669. Gupta, J.H. 1976. Reaction of mango cultivars to powdery mildew (Oidium mangiferae Berthet) in Uttar Pradesh. Progressive Hort. 8:63-64. Hemanthkumar, N.V., Ravishankar, K.V., Shivashankar, K.S., Lalitha, A., 43 Narayanaswamy, P., Prasad, T.G. and Mukunda, G.K. 2001. Poster presented at the Plant and Animal Genomes XII Conference, San Diego, CA. Honsho, C., Hishiyama, K., Eiadthong, S. and Yonemori, K. 2005. Isolation and characterization of new microsatellite markers in mango (Mangifera indica). Molecular Ecology Notes 5:152-154. IPGRI. 2006. Descriptors for Mango (Mangifera indica L). International Plant Genetic Resources, Rome, Italy. Iyer, C.P.A. 1991. Recent advances in varietal improvement in mango. Acta Hort. 291:109-112. Iyer, C.P.A. 2001. Genetic variation and breeding patterns in some tropical fruits. Regional Training Course “Characterization, Evaluation and Conservation of Tropical Fruits Genetic Resources”, organized by IPGRI, ICAR and IIHR. Iyer, C.P.A. and Subramanyam, M.D. 1987. Improvement of mango by selection and hybridization. Annual Report of the Indian Institute of Horticultural Research, Bangalore. p.11. Kaemer, D., Weising, K., Beyemann, B., Bomer, T., Epplen, J.T. and Kahl, G. 1995. Oligonucleotide fingerprinting of tomato Dann. Plant Breed. 114:12-17. Karihaloo, J.L., Dwivedi, Y.K., Archak, S. and Gaikwad, A.B. 2003. Analysis of genetic diversity of Indian mango cultivars using RAPD markers. Hort. Sci. and Biotech. 78:285-289. Kashkush, K., Jinggui, F., Tomer, E., Hillel, J. and Lavi, U. 2001. Cultivar identification and genetic map of mango (Mangifera indica L.). Euphytica 122:129-136. Lavi, U., Kashkush, K., Saada, D., Shats, H., Ravid, U. and Tomer, E. 2004. Mango breeding and the potential of modern biology. Acta Hort. 645:51-59. Lavi, U., Tomer, E. and Gazit, S. 1989. Inheritance of agriculturally important traits in mango. Euphytica 44:5-10. Lavi, U., Tomer, E., Gazit, S. and Hillel, J. 1998. Components of the genetic variance and genetic correlations between traits in mango. Scientia Hort. 75:11-25. Lopez-Valenzuela, J.A., Martinez, O. and Paredes-Lopez, O. 1997. Geographic differentiation and embryo type identification in Mangifera indica L. cultivars using RAPD markers. HortScience 32(6)1105-1108. Mukherjee, S.K. 1948. The cultivars of mango (M. indica L.) and their classification. Bull. Bot. Soc., Bengal 2:101-33. Mukherjee, S.K. 1963. Cytology and breeding of mango. Punjab Hort. J. 3:107-115. Naik, K.C. and Gangolly, S.R. 1950. Monograph on Classification and Nomenclature of South Indian Mangos. Madras, Supt. of Government Press. Om Prakash and Srivastava, K.C. 1987. Mango Diseases and Their Management – a World Review. Today and Tomorrows Printers, New Delhi. Palaniappan. 2001. Germplasm screening for salinity stress in tropical fruit species. Regional Training Course “Characterization, Evaluation and Conservation of Tropical Fruits Genetic Resources”, organized by IPGRI, ICAR and IIHR. Pandit, S.S., Mitra, S.S., Giri, A.P. and Gupta, V.S. 2007. A quick method for isolating RNA from raw and ripe fleshy fruits as well as for co-isolating DNA and RNA from polysaccharide- and polyphenol-rich leaf tissues. J. Plant Biol. 50:60-64. Rajan, S., Kumar, Ram and Negi, S.S. 2001. Variation in canopy characteristics of mango (Mangifera indica L.) cultivars from diverse eco-geographical regions. J. Appl. Hort. 3:95-97. Ram Nath, Kamalwanshi, R.S. and Sachin, I.P. 1987. Studies on mango malformation. Ind. J. Mycol. Plant Pathol. 17:29-33. Rami Reddy, P.V. and Dinesh, M.R. 2005. Evaluation of mango exotic collections for resistance to hopper, Idiosopus niveosparsus Leth. I.J. Plant Genetic Resources 18:6970. Ravishankar, K.V., Anand, L. and Dinesh, M.R. 2000. Assessment of genetic relatedness among mango cultivars of India using RAPD markers. J. Hort. Sci. Biotechnol. 75:198-201. 44 Ravishankar, K.V., Chandrashekara, P., Sreedhara, S.A., Dinesh, M.R., Lalitha, A. and Sai Prasad, G.V.S. 2004. Diverse genetic bases of Indian polyembryonic and monoembryonic mango (Mangifera indica L.) cultivars. Curr. Sci. 7:870-01. Ravishankar, K.V., Mani, B.H.R., Lalitha, A. and Dinesh, M.R. 2011. Development of new microsatellite markers from mango (Mangifera indica) and cross-species amplification. American Journal of Botany, p.96-99. Rick, C.M and Holle, M. 1990. Andean Lycopersicum esculentum var. cerasiformie. Genetic variation and its evolutionary significance. Ecol. Bot. 44:69-78. Schnell, R.J. and Knight, R.J. 1993. Genetic relationship among Mangifera spp. based on RAPD markers. Acta Hort. 341:86-92. Schnell, R.J. and Knight, R.J., Jr. 1992. Frequency of zygotic seedlings from five polyembryonic mango rootstocks. HortScience 27:174-176. Schnell, R.J., Brown, J.S., Olano, C.T., Meerow, A.W., Campbell, R.J. and. Kuhn, D.N. 2006. Mango genetic diversity analysis and pedigree inferences for Florida cultivars using microsatellite markers. J. Amer. Soc. Hort. Sci. 131:214-224. Schnell, R.J., Olano, C.T., Quintanilla, W.E. and Meerow, A.W. 2005. Isolation and Characterization of 15 microsatellite loci from mango (Mangifera indica L.) and cross-species amplification in closely related taxa. Mol. Ecol. Notes 5:625-627. Schnell, R.J., Ronning, C.M. and Knight, R.J. Jr. 1995. Identification of cultivars and validation of genetic relationships in Mangifera indica L., using RAPD markers. Theor. Appl. Genet. 90(2):269-274. Sharma, D.K. 1987. Mango breeding. Acta Hort. 196:61-67. Sharma, D.K. and Majumder, P.K. 1988a. Further studies on inheritance in mango. Acta Hort. 231:106-111. Singh, L.B. and Singh, R.N. 1956. A Monograph on the Mangoes of Uttar Pradesh, Vol. I & II, Superintendent of Printing, Lucknow. Torres, A.M. and Bergh, B.O. 1980. Fruit and leaf isozymes as genetic markers in avocado. J. Amer. Soc. Hort. Sci. 105:614-619. Truscott, M., Visser, G.J. and Anderson, S.H. 1994. Isoenzyme description of mango genotypes. J. S. Afr. Soc. Hort. Sci. 4:16-18. Vasugi, C., Dinesh, M.R. and Sekar, K. 2009. Studies on inter and intra specific crossability of Mangifera using cryopreserved pollen. The Icfai University Journal of Genetics and Evolution, p.33-39. Viruel, M.A., Escribano, P., Barbieri, M., Ferri, M. and Hormaza, J.I. 2005. Fingerprinting, embryo type and geographic differentiation in mango (Mangifera indica L., Anacardiaceae) with microsatellites. Mol. Breeding 15:383-393. Weber, W.E. and Wricke, G. 1994. Genetic markers in plant breeding. p.16. In: Advances in Breeding. J. Plant. Breed, Suppl. Welsh, J. and McClelland, M. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:7213-7218. Williams, J.G.K., Kubelik, A.R., Livak, J.J., Rafalski, J.A. and Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535. Yeshitela, T. and Nessel, T. 2003. Characterization and classification of mango ecotypes grown in eastern Hararghe (Ethiopia). Sarhad Journal of Agriculture 19:179-183. 45 Tables Table 1. Segregation pattern for color in various combinations. Sl. no Combination Segregation for skin color 1 Alphonso × Kalapadi Gradation of greenish yellow to yellow 2 Alphonso × Banganapalli Gradation of yellow, one progeny had red blush 3 Alphonso × Janardhan Pasand One progeny had red blush 4 Raspuri × Neelum Greenish yellow in all 5 Alphonso × Neelum One progeny had red blush 6 Alphonso × Kerala Dwarf One progeny had red blush 7 Amrapali × Arka Anmol All had greenish yellow 46