Genetic Resources and Crop Evolution (2006) 53: 873–882 DOI 10.1007/s10722-004-6148-3
# Springer 2006
Assessment of genetic relationships between wild and cultivated mulberry (Morus) species using PCR based markers K. Vijayan1,*, A. Tikader2, P.K. Kar2, P.P. Srivastava1, A.K. Awasthi1, K. Thangavelu2 and B. Saratchandra1 1
Seri-Biotech Research Laboratory, Central Silk Board, Kodathi, Carmelram Post, Bangalore 560 035, Karnataka, India; 2Central Sericultural Germplasm Resources Centre, Hosur, Tamil Nadu 635 109, India; *Author for correspondence (e-mail:
[email protected]; phone: 808 4406 51; fax: 808 4300 60) Received 24 September 2003; accepted in revised form 13 February 2004
Key words: Genetic relationship, ISSR markers, Morus, Sericulture, Wild mulberry
Abstract In order to formulate appropriate strategies for the conservation and utilization of the wild mulberry genetic resources available in India, a study was undertaken with 20 mulberry genotypes from the four different species. Seventeen intersimple sequence repeat primers were used to generate a total of 114 markers, of which 98 (85.96%) were polymorphic. Seven unique bands for Morus serrata Roxb. and one for both M. serrata Roxb. and Morus macroura Miq. were identified, of which one fragment has been sequenced and deposited in the EMBL-GeneBank (AJ-585512). The genetic dissimilarity coefficients varied from 0.078 to 0.530 among these genotypes and from 0.168 to 0.465 among the species. The dendrograms realized from these markers clustered the genotypes into three groups. The outermost group was M. serrata Roxb., which was followed by the group of M. macroura Miq. and the innermost group contained genotypes of Morus indica L. and Morus alba L. This intermixing of genotypes of M. indica and M. alba supports the view that M. indica is merely a synonym of M. alba. Distribution of the genotypes on a two-dimensional figure upon multidimensional scaling with ALSCAL program, further, confirmed the genetic divergence between the cultivated and wild mulberry groups. On the basis of the results a few potential wild mulberry genotypes were identified for its conservation and utilization in breeding programs to confer the stress tolerance to the cultivated varieties of mulberry. Introduction Intensive agriculture has resulted in the loss of much of the genetic diversity that local and traditional varieties of cultivated crops possessed. The new varieties developed through modern techniques are becoming genetically more homogenous than ever before and are, thus, more vulnerable to pathogens and adverse environmental conditions (Asins and Carbonell 1989). This has prompted scientists to look for new sources of variations to widen the genetic base of the high yielding varieties
to make them more adaptable to the local environment. Wild populations found within the same agro climatic conditions of the cultivated varieties offer the advantage of being utilized more easily for the breeding purposes than those found in far away places. Thus, the precious genetic resources of the wild population present indigenously are to be conserved for its proper utilization. In order to achieve this, it is imperative to assess the genetic variability present among wild populations along with their genetic inter-relationships with the cultivated relatives (De Bustos et al. 1998).
874 Mulberry (Morus L. Family Moraceae), is a tree crop used in sericulture industry to feed the silkworm Bombyx mori L. (Lepidoptera). Mulberry plants in India were earlier designated as Morus alba, Morus indica Linn., Morus atropurpurea Roxb., Morus nigra, Morus macroura Miq. (synonym: Morus laevigata Wall. ex Brandis) and Morus serrata Roxb. However, later the first three viz M. alba, M. indica Linn., M. atropurpurea Roxb. were considered as mere synonyms of M. alba and the other three species namely M. nigra, M. macroura Miq. and Morus serrata Roxb. were kept as separate species (Anonymous 1962; Sastry 1984). Plants of M. alba and M. indica are generally grown for silkworm rearing while the same from M. macroura Miq. and M. serrata Roxb. are mostly grown as ornamental plants due to the rough, hairy and thick leaves, which are not suitable for silkworm feeding (Tikader and Dandin 2001). M. macroura Miq. has a wide distribution throughout India where as M. serrata Roxb., is mostly confined to the forest areas in the northwestern parts of the Himalayas (Anonymous 1962; Ravindran et al. 1997). Hence, plants of M. serrata Roxb. are commonly known as ‘‘Himalayan mulberry’’. Plants of these two species harbour a number of agronomically desirable traits like resistance to biotic and abiotic stresses (Ravindran et al. 1997; Tikader and Dandin 2001), which are of much use for improving the mulberry varieties cultivated for silkworm rearing. Since, the hybridization studies in mulberry (Das and Krishnaswami 1965; Dwivedi et al. 1989; Tikader and Rao 2003) showed no reproductive barrier among these species, widening of the narrowing genetic base of the cultivated varieties through interspecific hybridization could be attempted. However, knowledge on the genetic divergence and inter-relationships of the parental materials is an essential factor, which decides success of the breeding programs. Hence, the present study was undertaken with an objective to unravel the genetic relationships among the genotypes of these four Indian mulberry species so as to utilize them in different breeding programs to enhance the quality leaf production per unit area in tropical sericultural belts. Molecular DNA techniques like polymerase chain reaction (PCR) has been used extensively
to amplify genomic DNA to study the phylogeny and evolution of species and to characterize germplasm to select desirable genotypes for breeding and other utilization purposes, due to its superiority over the conventional methods (Tosti and Negri 2002). Random amplified polymorphic DNA (RAPD) (Williams et al. 1990) technique is extensively used to assess levels of genetic diversity and phylogenetic relationships in wide range of plants (Nair et al. 1999; Rodriguez et al. 1999). However, RAPD has been widely criticized because the origin of polymorphism is not understood, band homology cannot be assured to phylogenetic construction (Rieseberg 1996), and banding patterns are not reliable and reproducible in many cases (Perez et al. 1998). Thus, of late, intersimple sequence repeat (ISSR) marker system is extensively used for investigating phylogeny, evolutionary relationships and genetic variability among plants (Zietkiewicz et al. 1994; Tsumura et al. 1996; Jones et al. 1997; Bornet et al. 2002). Because of the greater length of ISSR primers they show greater repeatability and stability. Further, in mulberry ISSR has been successfully used to study the genetic divergence among selected cultivars (Vijayan and Chatterjee 2003; Vijayan et al. 2005). Hence, in this study ISSR primers were selected to unravel the genetic relationships of the wild mulberry species (M. serrata Roxb. and M. macroura Miq.) with its cultivated relatives (M. indica L. and M. alba L.).
Materials and methods Mulberry genotypes, morphology and anatomy Twenty mulberry genotypes (Table 1) comprising six M. serrata Roxb., five M. macroura Miq., six M. indica L. and three M. alba L., collected from different geographic regions of India (Figure 1), were used for the study. These accessions are presently maintained at Central Sericultural Germplasm Resources Center, Hosur, Tamil Nadu, India (77.5 E, 12.45 N; 942 m above mean sea level (AMSL)). Morpho-biochemical characters of the plants were recorded from five plants from each genotypes maintained under similar cultural practices. Data on branching, plant height and leaf yield were recorded on 90th day since
875 Table 1. Geographic distribution of 20 mulberry genotypes along with their species status. Sl. no.
Genotype
Species
Place of collection
States
Latitude ( N)
Longitude ( E)
Altitude (m ASL)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Mussorie Dunda Gangnani Rampur Joharji Nainitikar Sung valley Bilaspur-2 Resham majri-1 Resham majri-8 Saravathi S36 RFS-175 Kanva-2 Kajli Bombay piasbari Surat Almora local Sujanpur-5 Punjab local
M. serrata Roxb. M. serrata Roxb. M. serrata Roxb. M. serrata Roxb. M. serrata Roxb. M. serrata Roxb. M. macroura Miq. M. macroura Miq. M. macroura Miq. M. macroura Miq. M. macroura Miq. M. indica L. M. indica L. M. indica L. M. indica L. M. indica L.
Dehradun Uttarkashi Uttarkashi Rudraprayag Solan Sirmour JH district Bilaspur Dehradun Dehradun Jalpaiguri Mysore Mysore Mysore Murshidabad Malda
Uttaranchal Uttaranchal Uttaranchal Uttaranchal H’chal Pradesh H’chal Pradesh Meghalaya Chhattisgarh Uttaranchal Uttaranchal West Bengal Karnataka Karnataka Karnataka West Bengal West Bengal
30.19 30.45 30.45 30.16 30.54 30.30 25.26 22.03 30.19 30.19 26.30 12.18 12.18 12.18 24.61 25.02
78.03 78.19 78.19 78.59 77.06 77.40 92.14 82.12 78.03 78.03 88.50 76.42 76.42 76.42 88.15 88.12
1900 900 2100 1600 1380 1500 1000 400 680 680 300 767 767 767 19 16
M. indica L. M. alba L. M. alba L. M. alba L.
Surat Almora Gurudaspur Gurudaspur
Gujarat Uttaranchal Punjab Punjab
21.10 29.36 32.19 32.19
72.54 79.40 75.38 75.38
17 18 19 20
pruning, for three consecutive years. The leaf anatomy for stomatal size and frequency was from fully expanded leaves (5th–7th position in descending order from the top). Small rectangular pieces were taken out from the middle portion of the leaf blade avoiding veins and veinlets and preserved in FAA (formalin 5 mL, glacial acetic acid 5 mL and 70% ethanol, 90 mL) solution. Stomatal size and frequency were studied by taking a thin layer of Wimbley’s quickfix impression on the abaxial (lower) surface of the leaf and observed under a Leica Leitz DMRB microscope. The stomatal size was measured with the help of ocular micrometre attached to the microscope. The number of stomata per unit area was calculated and expressed as stomatal frequency per mm2. DNA extraction Seventh leaves from the top of 90-days-old five ramets were collected from each genotype and stored immediately at 80 C for DNA extraction. Total genomic DNA was isolated from the leaf samples using modified cetyl trimethyl ammonium bromide protocols (Vijayan 2003). Quantification of the DNA was done by electrophoresis of 0.8% agarose gel (1 TBE) stained with ethidium
150 19.00 200 200
bromide (0.5 g/mL) and using uncut DNA (10 ng/L) as standard. PCR amplification of the DNA with ISSR primers Seventeen ISSR primers comprising of di-, triand tetra-nucleotide repeats, obtained from the University of British Columbia, Vancouver, Canada (Set # 9), were selected for the study based on the results of earlier studies (Vijayan and Chatterjee 2003; Vijayan et al. 2005). The PCR amplification was carried out on an MJ Research Thermal-Cycler, PTC-200, (MJ Research Inc. Watertown, Massachusetts, USA) using 20 L of reaction mixture containing 2.0 L of 10 PCR buffer (750 mM Tris–HCl pH 8.8; 200 mM (NH4)2SO4; 0.1% Tween-20), 0.2 mM dNTP, 2 mM MgCl2; 200 nM Primer; 50 ng genomic DNA and 1 U Taq DNA polymerase (MBI Fermentas Inc, Hanover, MD-21076, USA). The PCR schedule included an initial cycle at 94 C for 2 min followed by 35 cycles of 94 C for 30 s, 50 C for 30 s, 72 C for 2 min and a final extension of 10 min at 72 C. The PCR product was separated on 2.0% agarose gel in 1 tris–boric acid buffer containing
876
Figure 1. A map of India showing the place of collection of mulberry genotypes.
0.5 g/mL ethidium bromide as a stain. Scoring of bands was on the basis of presence (1) or absence (0) of a particular band. Statistical analysis Pair-wise genetic similarity coefficients (GS) for each pair of the accessions were calculated follow-
ing the methods of Nei and Li (1979), which is briefly stated as 2Nij/Ni + Nj, where Nij is the number of common bands in ith and jth accessions and Ni and Nj are the number of bands for accessions i and j, respectively and 1-GS is used as a dissimilarity index (genetic distance). Cluster analysis was carried out with dissimilarity matrix using unweighted pair group method using
877 Table 2. Morpho-anatomical characteristics of the 20 mulberry genotypes. Sl. no.
Genotype
No. of branches
Height of the plant (cm)
Leaf yield (kg/yr/plant)
Stomatal frequency/mm2
Stomatal size (m2)
Leaf thickness (m)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Mussorie Dunda Gangnani Rampur Joharji Nainitikar Sung valley Bilaspur-2 Resham majri-1 Rasham majri-8 Saravathi S36 RFS-175 Kanva-2 Kajli Bombay piasbari Surat Almora local Sujanpur-5 Punjab local
6.0 ± 0.58 7.0 ± 0.58 7.0 ± 0.58 6.3 ± 0.67 10.7 ± 0.67 12.3 ± 1.45 15.3 ± 0.88 20.0 ± 1.15 27.3 ± 1.45 21.3 ± 2.03 17.7 ± 1.45 35.0 ± 2.89 21.0 ± 1.15 27.3 ± 1.45 37.3 ± 1.45 27.7 ± 1.45
116 ± 8.8 93 ± 8.8 123 ± 3.3 100 ± 5.8 80 ± 5.8 100 ± 5.8 152 ± 4.4 128 ± 1.5 200 ± 5.8 163 ± 1.7 111 ± 1.7 125 ± 2.9 135 ± 2.9 145 ± 2.9 131 ± 2.3 202 ± 4.4
1.18 ± 0.09 0.95 ± 0.03 0.83 ± 0.03 0.70 ± 0.06 0.50 ± 0.06 0.57 ± 0.03 1.60 ± 0.06 1.58 ± 0.06 2.17 ± 0.09 1.90 ± 0.06 1.40 ± 0.06 1.82 ± 0.04 1.6 ± 0.06 1.67 ± 0.12 0.67 ± 0.04 1.20 ± 0.06
392 ± 30 467 ± 22 475 ± 14 450 ± 29 400 ± 29 423 ± 33 658 ± 30 550 ± 29 508 ± 46 475 ± 14 450 ± 29 758 ± 30 700 ± 28 667 ± 36 917 ± 30 558 ± 30
581 ± 20 633 ± 30 586 ± 29 600 ± 29 650 ± 28 650 ± 29 365 ± 9 356 ± 38 410 ± 0 220 ± 10 341 ± 4 306 ± 3 380 ± 6 205 ± 3 255 ± 5 240 ± 10
235 ± 3 241 ± 4 245 ± 9 243 ± 12 236 ± 9 243 ± 7 186 ± 19 211 ± 24 180 ± 3 175 ± 26 191 ± 44 137 ± 3 175 ± 3 155 ± 3 140 ± 3 163 ± 8
30.0 ± 1.73 20.0 ± 1.15 28.3 ± 0.88 37.3 ± 1.45
105 ± 2.9 155 ± 2.9 145 ± 2.9 181 ± 4.4
0.43 ± 0.04 1.0 ± 0.06 2.00 ± 0.06 1.83 ± 0.09
975 ± 43 775 ± 28 892 ± 30 858 ± 30
148 ± 3 240 ± 6 247 ± 9 160 ± 6
112 ± 2 132 ± 9 147 ± 16 142 ± 4
17 18 19 20
Table 3. Mean morpho-anatomical characteristics of four mulberry species. Parameters
M. serrata Roxb.
M. macroura Miq.
M. indica L.
M. alba L.
Branch (no) Height (cm) Leaf yield (kg/plant/yr) Stomatal frequency (No/ mm2) Stomatal size (m2) Leaf thickness (m) DNA polymorphism(%)
8.22 ± 0.16 102.22 ± 0.92 0.79 ± 0.01 410.7 ± 2.46
20.33 ± 0.22 150.93 ± 1.52 1.73 ± 0.01 528.3 ± 4.22
29.76 ± 0.29 140.34 ± 1.48 1.23 ± 0.03 762.5 ± 7.41
28.56 ± 0.37 160.56 ± 0.82 1.61 ± 0.02 841.6 ± 3.26
616.94 ± 7.40 240.83 ± 1.72 66.66
338.67 ± 3.38 189.00 ± 1.98 68.42
255.83 ± 3.62 146.94 ± 1.05 70.15
215.56 ± 2.05 140.00 ± 0.84 61.40
arithmetic average (UPGMA) and a clustering program, PHYLIP 3.5c software program (Felsenstein 1993), which compresses the patterns of variation into two-dimensional branch diagrams (phenograms) (Sneath and Sokal 1973). The robustness of the dendrogram was verified with cophenetic correlation values for the dendrogram and comparing them with the original genetic dissimilarity matrix using Mantel’s matrix correspondence test (Mantel 1967). The bootstrap values were calculated using the free software WINBOOT (Yap and Nelson 1996). In order to test the genetic variability further, multidimensional scaling of the data was done using the ALSCAL software program, available
in SPSS (SPSS Inc. Chicago, USA) In this method a dissimilarity matrix was created using Euclidean distance and the same was used for stimulus configuration of the data using the classical Young-Householder multidimensional scaling procedure (Young et al. 1984; Young and Harris 1990).
Results Morpho-anatomical characters The morphological data showed considerable variations for all characters (Tables 2 and 3).
878
Figure 2. PCR fingerprint of the twenty mulberry genotypes with the ISSR primer UBC-864; Names of the genotypes 1–20 as given in Table 1.
Table 4. Intra- (in bold) and interspecific genetic distance among four mulberry species. Species
M. serrata
M. serrata M. macroura Miq. M. indica M. alba
0.180 0.401
0.167
0.465 0.457
0.307 0.329
M. macroura Miq.
M. indica
M. alba
0.177 0.168
0.147
Multivariate analysis, using SPSS, showed high significance ( p < 0.000) for all characters. Number of branches sprouted from each plant varied from 6.0 ± 0.88 in Mussorie to 37.3 ± 1.45 in Kajli and Punjab local. The plant height also varied from 93 ± 8.8 cm in Dunda to 202 ± 4.4 cm in Bombay piasbari. Leaf yield was in the range of 0.43 ± 0.04 kg/yr/plant in Surat to 2.17 ± 0.09 kg/ yr/plant in Resham majri-1. Highest stomatal frequency was observed in Kajli (917 ± 30) and the lowest was in Mussorie (392 ± 30). The stomatal size also showed considerable variation among the genotypes. When we consider the species as a whole the number of branches was highest in M. indica L. (29.72 ± 0.29) and lowest in M. serrata Roxb. (8.22 ± 0.16). The plant height was highest in M. alba L. (160.56 ± 0.82 cm) and lowest in M. serrata Roxb. (102.22 ± 0.95 cm). Stomatal frequency was highest in M. alba L. (841 ± 3/mm2) and lowest in M. serrata Roxb. (410 ± 2/mm2). Stomatal size was maximum in M. serrata Roxb. (616.94 ± 2.39 m2) and minimum in M.alba L. (215.56 ± 2.05 m2). Leaf thickness showed wide variability as in M. serrata Roxb. the leaf thickness was 240.35 ± 0.57 m while in M. alba L. it was 140.00 ± 1.98 m. Thus, clear variations were observable among the species for all the morpho-anatomical characters studied in this experiment.
DNA polymorphism A total of 114 bands were generated by the 16 ISSR primers, of which 98 were polymorphic, thus generating 85.96% polymorphism among the genotypes. The PCR amplification products of the primer 864 (Figure 2) shows clear polymorphism among the genotypes. The polymorphism within a species was highest in M. indica L. (70.17%) followed by M. macroura Miq. (68.42%), M. serrata Roxb. (66.67%) and the least polymorphism was in M. alba L. (61.40%). A few unique bands were also observed among the species. Seven bands i.e., 856900, 856800, 8571350, 838900, 836550, 835650 and 830450 were unique for M. serrata Roxb., while 8382300 was present only in wild mulberry species (M. serrata Roxb. and M. macroura Miq.), 841300 was absent in M. macroura Miq. but present in all other species. Out of these bands, 835650 has been sequenced and deposited in the EMBL-GeneBank (AJ-585512). Genetic divergence among the species The genetic distances estimated among the genotypes varied from 0.078 in Bombay piasbari vs. Surat to 0.530 in Joharji vs. Kajli. The average genetic distance among the genotypes was 0.324. However, when the genetic distance was calculated between species, it could be seen that the interspecific distance was highest between M. serrata Roxb. and M. indica L. (0.465), which is followed by M. serrata Roxb. and M. alba L. (0.457), M. serrata L. and M. macroura Miq. (0.401). The genetic distance between M. macroura Miq. and M. indica L. was 0.307, which was higher than that between M. macroura Miq. and M. alba L. 0.339. The interspecific distance between M. indica L. and M. alba L. was 0.168. The intraspecific
879 1.0
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0.5
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20 15 14 19 16 13 18 17 12
4
5 1
0.0
3
6
–0.5
11
–1.0
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10 8 7
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Figure 4. Two dimensional figure showing the distribution of the 20 mulberry genotypes based on multidimensional scaling using the ALSCAL algorithm. Numbers showing the serial number of the genotypes as given in Table 1. & M. serrata Roxb., M. macroura Miq., * M. indica L., ^ M. alba L.
Figure 3. Dendrogram of the 20 mulberry genotypes realized from the dissimilarity matrix derived from the ISSR markers using UPGMA analysis. Numbers at the nodes indicate bootstrap values (%).
variability as calculated by averaging the genetic distance among the genotypes of a single species was highest in M. serrata Roxb. (0.180) and lowest in M. alba L. (0.147) (Table 4). Cluster analysis The dendrogram using UPGMA analysis has clustered the twenty mulberry genotypes into three broad groups (Figure 3). Group A comprised of all the six genotypes of M. serrata Roxb. and the Group B consisted of all the five genotypes of M. macroura Miq. However, in the Group C genotypes of M. indica L. and M. alba L., were grouped together without any distinct separation. Thus, the clustering of the genotypes was almost in accordance with their species status. The subclustering of the genotypes showed little correlation with their
geographic origin, though in one of the subgroup all the cultivated genotypes from Mysore grouped together as a small subgroup. The mantel test between the dissimilarity matrix and the cophenetic matrix derived from the dendrogram was highly significant (r ¼ 0.64; P < 0.000). The higher bootstrap values at the nodes further emphasis the genetic divergence of M. serrata Roxb. and M. macroura Miq. from the other two species.
Two-dimensional distribution of genotypes The multidimensional scaling of the ISSR data using ALSCAL program available in SPSS has clearly separated the mulberry genotypes into three distinct groups (Figure 4). The genotypes belong to M. serrata Roxb. were grouped as a distant clump at 1.5–2.0 in dimension-1 and 0.02–1.0 at dimension-2. Similarly, genotypes of M. macroura Miq. were grouped together at an orientation of 0.75–0.25 at dimension-1 and 0.5 to 1.5 at dimension-2. However, the genotypes of the cultivated species M. indica L. and M. alba L. did not show any separation, instead they grouped together at 1.5 to 0.9 in dimension-1 and 1.0–0.25 in dimension-2. Thus, the ALSCAL scaling of the data showed almost similar kind of genetic relationships that was expressed by clustering analysis.
880 Discussion Assessment of genetic relationships among the species of a crop has important consequences in plant breeding and conservation of genetic resources. It is particularly useful in selecting parents for breeding purposes as genetically divergent but sexually compatible parents produce greater heterosis. In mulberry, the sexual incompatibility among various species was reported to be very little as most of the interspecific hybridization produced good seed fertility (Das and Krishnaswami 1965; Dandin et al. 1987; Dwivedi et al. 1989; Tikader and Rao 2003). This, clearly indicates that interspecific hybridization in mulberry is quite possible. The results of the morpho-anatomical investigations revealed certain interesting features as the less stomatal frequency and higher leaf thickness in the wild mulberry species points to the fact that these wild mulberry species are more adaptable to stress conditions, as earlier reported by Ravindran et al. (1997). Susheelamma et al. (1990) conducted an extensive screening of mulberry genotypes for drought resistance and reported that low stomatal frequency and higher leaf thickness are intimately associated with drought resistance in mulberry. The geographic distribution of these mulberry species in India further reveals that natural populations of M. macroura Miq. were present in the forest regions of north-western part of India (Ommachan 1976; Dhar and Ahsan 1989; Dandin et al. 1993), Andaman Nicobar Islands (Parkinson 1923), West Bengal (Ravindran et al. 1997) and some parts of the South India (Yadav and Pavankumar 1996). The distribution of M. serrata Roxb. on the other hand confined mostly to the high altitude regions (1100–2200 AMSL) of the north-western part of India (Ravindran et al. 1997), Genotypes of M. alba L. was found distributed in Punjab, north-western part of the Himalayas, western Tibet. (Ravindran et al. 1997). Watt (1891) and Parker (1956) were of the opinion that this species is indigenous to China and extensively cultivated in the plains of India and in Himalayas. However, natural and cultivated forms of M. indica L. are commonly seen in the sericultural areas extending from temperate to subtropical Himalayas, from Kashmir to Sikkim ascending to 2100 m ASL. Presently most of the cultivated forms of this species are distributed in Uttar
Pradesh, West Bengal, Sikkim, Assam, Meghalaya, Arunachal Pradesh, Karnataka, Andhra Pradesh, Tamil Nadu and Kerala (Ravindran et al. 1997). Thus, it could be seen that though M. macroura Miq. and M. indica L. have a wider distribution than the other two species, M. macroura Miq. is generally present in wild condition mainly due to the unsuitability of its leaves for feeding the silkworms. The genotypes of M. indica L. on the other hand are chiefly grown by farmers for its leaf to feed the silkworm B. mori. Though, a number of higher yielding mulberry varieties have been developed from this species, most of these varieties are highly sensitive to both biotic and abiotic stresses (Sastry 1984; Sarkar et al. 2000). Thus, gene pools of the cultivated mulberry species have to be enriched by introgression of genes of better adaptation from the wild mulberry species. Genes for salt (M. macroura Miq. from Andaman), drought (genes from M. serrata Roxb. and M. macroura Miq.), frost tolerance (from M. serrata Roxb.) and disease resistance (from both M. serrata Roxb. and M. macroura Miq.) are to be transferred to the cultivated genotypes to expand sericulture to the marginal areas. The higher genetic diversity found between the wild and cultivated forms of mulberry further points to the possibility of harnessing the heterosis resulting from breeding between selective genotypes from these groups. The clustering of the genotypes according to their species status is an information, which needs special mention as two previous studies with ISSR primers (Vijayan and Chatterjee 2003; Vijayan et al. 2003) and the one with RAPD primers (Bhattacharya and Ranade 2001) could not differentiate the mulberry genotypes, collected from various parts of India, according to their species. However, a recent study with ISSR markers could discriminate Indian mulberry genotypes, mostly belonged to M. indica L. and M. alba L., from those of Japan origin, mostly belonged to Morus latifolia Poir. and Morus bombycis Koidz, (Vijayan 2003). In the present study also it could be seen that genotypes belonged to M. serrata and M. laevigata could be separated out while the genotypes of M. indica and M. alba L. could not be separated in distinct groups. This could be due to the mixing of the gene pools of these two species due to lack of reproductive isolation as reported by Das and
881 Krishnaswami (1965), Dandin et al. (1987), Dwivedi et al. (1989), Tikader and Rao (2003). Results from isozyme profiles of these species also revealed very less differences among genotypes of these species (Hirano 1977, 1982). Thus, from this study, it is once again confirmed that genotypes of M. indica L. and M. alba L. are genetically very close and M. indica should be treated as synonymous of M. alba as suggested by Gururajan (1960), Anonymous (1962) and Hirano (1977). The ALSCAL-multidimensional scaling (Young et al. 1984; Young and Harris 1990), further substantiate these findings by spatial distribution of the genotypes upon the Euclidean distances they hold one another. Further, it could be seen that out of the six genotypes of M. serrata Roxb., Joharji, Rampur and Mussorie showed close genetic similarity, hence any one of these genotypes along with the other three could be conserved. Regarding the genotypes of M. macroura Miq., both Resham majri-1 and -8 seemed to be duplicates as they had very short genetic distance. Similarly, the unique bands identified from M. serrata Roxb. could be of much use in breeding as well as conservation purposes, once its sequencing and association with any desired traits is established. The one marker (835650) sequenced was found to be a new DNA sequence (EMBL-GeneBank # AJ-585512). Thus, from the present study it can be concluded that genotypes like Mussorie, Gangnani, Sung valley and Rasham majri-1 can be used for breeding with cultivated varieties which are capable of high yielding but are sensitive to the biotic and abiotic stress, to confer stress resistance to these varieties. Further, these highly promising genetic resources of M. serrata Roxb. and M. macroura Miq. should be preserved properly for the genetic advancement of the presently cultivated varieties of mulberry.
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