Mulberry (Morus spp.) is a cross-pollinating and highly hybridized plant belonging to the genus Morus, which is widely distributed in nature, having been evolved by natural crossing, artificial hybridization and mutation.
Mulberry is a sole food plant, of which productivity is varied in different varieties with the intraspecific distinction value of 169.8% in the early years of the last century (1928–1930), whereas 253.5% in recent years (2013–2019). Therefore, the importance of elite mulberry varieties for high silk cocoon and mulberry leaf production is much higher than any cultivation conditions. Hence, it is substantial to reorder collectively and classify more scientifically many culticvated mulberry varieties in order to produce a large number of new varieties by using good resources efficiently and subsequently to contribute to the silk cocoon production.
The genus Morus, which is widely distributed in tropical, sub-tropical and tempereate regions, is cultivated extensively in East, Central and South Asia such as DPR Korea, China, Vietnam and Thai for silk production. The genus Morus has 68 recognised species including M. mongolica Schneid., M. bombycis Koidz., M. alba Linn., M. multicaulis Perr., M. nigra L., M. rubra L., M. tartarica L., M. indica L., M. papyrifera and M. tinctoria (Sanjappa, 1989).
In DPR Korea M. mongolica Schneid. is cultivated in mountainous areas of South and North Phyongan provinces, north Hamgyong province, Kangwon Province, Jagang Province and it exhibits high cold tolerance, having remarkablely long style (1 ~ 2mm) in female flowers.
M. bombycis Koidz. is grew naturally in the mountains and fields of DPR Korea, China and Japan, being dioecious or androgynous and having similarly long style (1 ~ 2mm) like M. mongolica Schneid.
M. alba Linn. is most widely distributed in the world such as DPR Korea, China (Chang, 1984), Vietnam (Chang, 1984; Kansumata, 1973), India (Dhar and Ahsan, 1989; Ravindran et al., 1997) and Japan (Maichii, 1999), and is a only cultivar in European countries. This species is dioecious or androgynous and the style in female flowers is absent or indistinct.
M. multicaulis Perr. is originated from China and has been cultivated during many years in DPR Korea. It is dioecious and shown only female plants in DPR Korea with a very short style (0.2mm).
So far, using morphological and biochemical characters (Mala et al., 1998; Fotedar and Dandin, 1998; Vijayan et al., 1999; Tikader and Roy, 2001) there were many reports to classify mulberry species and cultivars, but these methods were time consuming and even might be made a mistake because of environmental variation.
Generally, the classification of mulberry is performed by comparing some morphological traits such as floral characters. For example, the length of catkins (4.1 ~ 7.2cm in some varieties while 1.7 ~ 2.4cm in other varieties) (Das et al., 1970; Tikader et al., 1995) and the style length in female flowers and the nature of stigma in male flowers (1 ~ 2mm in M. mongolica Schneid. and M. bombycis Koidz. wheras absent or 0.2 ~ 0.5mm in M. alba Linn. and M. multicaulis Perr.) (Koidzumi, 1917). But such floral characters are greatly changed depending on environmental conditions including temperature, day length and daylight, and cultivating conditions such as hormonal application or pruning of branches of the plant (Das and Mukherjee, 1992).
As above mentioned, in mulberry floral characters could not be utilized to identify species and cultivars as an only diagnostic marker. Especially, it is difficult to identify species and cultivars in highly hybridized plants like mulberry. So it is critical to use not only morphological and biochemical traits but also molecular markers to solve disruptions in species, cultivation classification and identification.
Additionally, using molecular markers the accurate classification is important in the breeding of a cross pollinating mulberry plant. Because the cross hybridization between mulberry species represent a high seed fertility (Das and Krishnaswami, 1965; Dandin et al., 1987), the interspecific crossing is favor to mulberry breeding to increase leaf yield potential.
Earlier studies have been focused on elucidation of genetic distinction among mulberry species and varieties by molecular markers (Bhattacharya and Ranade, 2001; Vijayan, 2003; Vijayan et al., 2004a, b, c; Vijayan et al., 2005; Vijayan et al., 2006; Kar et al., 2008).
There were reports on phylogenetic relationship between a little number of local indian genotypes using ISSR markers(Vijayan and Chatterjee, 2003), eleven species of M. alba L. by ISSR and RAPD technique (Prem et al., 2004), and Chinese wild and cultivated mulberry cultivars based on ISSR and SSR markers (Zhao et al., 2006, 2007ª, b). By employing RAPD markers, the work has been also performed to reveal genetic relationship among some mulberry genotypes (Bhattacharya and Ranade, 2001; Chatterjee et al., 2003; Muzaffer et al., 2012; Nataraj and Chikkaswamy 2017). And recently, there were attempts to solve the problems remaining in the classificational identification of mulberry species based on RAPD and ISSR markers (Chikkaswamy and Rabin Chandra, 2014; Girish Naik et al., 2015; Rita et al., 2016).
Besides, ITS sequence has been used as a potential nuclear DNA marker to discriminate each other on the level of species in most plants (Walker and David, 2012). Recent study revealed that the nuclear ribosomal ITS sequence was more efficient than the chloroplastic psbA-trnH sequence in differentiating medicinal plant Boerhavia diffusa from other Boerhavia species (Dhivya et al., 2012). So far, there have been some attempts to use ITS sequences for phylogenetic classification and evolutionary research (Zhao et al., 2005; Zeng et al, 2015; Yahui et al., 2019).
This study aimed to elucidate genetic distinction among four mulberry species such as M. mongolica Schneid., M. bombycis Koidz., M. alba Linn. and M. multicaulis Perr., including fourteen varieties by using ITS sequence and ISSR markers.
Variable sites of ITS seuennces differing in species level among 4 mulbery species used in our study were not found and it shows that ITS sequences can’t be used to classify these mulberry species.
ITS sequences didn’t show a considerable distinction among four studied mulberry species, indicating that it could not be applied to classify mulberry species.
In M. multicaulis Perr. the highest ISSR polymorphism (31.62%) and observed number of alleles (1.34), the highest effective number of alleles (1.27), Shanon’s index (0.21) and Nei’s gene diversity (0.15) were founded. Nei’s genetic distance is the most genetically close between M. mongolica Schneid. and M. bombycis Koidz. (0.1377), while the most distant between M. bombycis Koidz. and M. multicaulis Perr. (0.3244). ISSR results showed that inter-specific genetic variation (55.34%) is somewhat higher than intra-specific genetic variation(44.66%), with relatively low average number of migrants per generation (Nm) among populations (0.3886).
This result suggests that ISSR markers might be superior to ITS sequences to distinct diffrent species as well as mulberry varieties used in this study.
Depending on the genetic distinction among fourteen mulberry varieties cultivated in DPR Korea obtained in this study, in the future the selection of more suitable crossing pairs will be conducted to expect a disirable hybrid vigor in the breeding of a highly heterozygous mulberry plant.