Skip to main content
Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Definition of Eight Mulberry Species in the Genus Morus by Internal Transcribed Spacer-Based Phylogeny

  • Qiwei Zeng,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Hongyu Chen,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Chao Zhang,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Minjing Han,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Tian Li,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Xiwu Qi,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Zhonghuai Xiang,

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China

  • Ningjia He

    Affiliation State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, 216 Tiansheng Road, Beibei District, Chongqing, 400716, China


Mulberry, belonging to the order Rosales, family Moraceae, and genus Morus, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus Morus has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify Morus species, resulting in varied numbers of designated Morus spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus Morus. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. M. alba and M. notabilis showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the M. notabilis genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the Morus genus should be classified into eight species, including M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus Morus.


Mulberry (Morus spp.), belonging to the order Rosales, family Moraceae, and genus Morus [1], is distributed in a wide range of areas worldwide [2]. It is an important economic woody plant with a significant impact on human society due to its economic value in sericulture as well as for its nutritional benefits and medicinal values [3]. This plant also plays significant ecological roles in the prevention and control of sand erosion, and in stony desertification and saline-alkali land treatments [4]. However, the taxonomy of Morus has been disputed [5] because of its wide geographical distribution, morphological plasticity [6], hybridization among species [7], long history of domestication, and introduction and naturalization of species [8]. Different studies have recognized a variable number of Morus species, leading to an uncertain taxonomy. In 1753, Linnaeus established the first taxonomy of Morus [9]. The first comprehensive Morus taxonomy was created by Bureau [10] based on features of its leaves and pistillate catkins, and recognized five species, 19 varieties, and 13 sub-varieties. Since that time, the taxonomy has undergone many further modifications based on morphological and phenological characteristics [5]. In 1917, Schneider [11] described a new species, Morus notabilis C. K. Schneid in China. Subsequently, Koidzumi [12], Leroy [13], and Hotta [14] classified the genus Morus into 24 species and 1 subspecies, 19 species, and 35 species, respectively. More recently, Zhou and Gilbert [15] described 16 species in Morus, 12 of which were found in China. Because of this variable taxonomy, 260 validated published names in Morus have been deposited in the International Plant Names Index (, with many of them being synonymous. Therefore, although as many as 68 [16,17] or 150 [18,19] species have been reported in Morus, only 10–16 are generally cited and accepted [2024].

Molecular markers that are heritable, fast, and easy to measure and evaluate have been used to classify Morus species in order to improve the taxonomy of Morus based on phenotypic characteristics. Using amplified fragment length polymorphism (AFLP) markers, Sharma [25] and Kafkas [26] concluded that the results from both marker and conventional methods were consistent to a large extent and that M. nigra and M. rubra were molecularly distinct from M. alba. Rao [27] grouped domesticated species (M. alba and M. indica) into one cluster by building a phylogenetic tree of 80 wild mulberries from four species using four isozymes (polyphenol oxidase [PPO], peroxidase [PoX], esterase [EST], and diaphorase [DIA]). Zhao [2832] placed Chinese domesticated species, including M. alba, M. multicaulis, M. bombycis, M. australis, M. atropurpurea, and M. rotundiloba, into one cluster by creating phylogenetic trees using ITS sequences, microsatellite loci, and sequence related amplified polymorphism (SRAP), inter simple sequence repeat (ISSR), and SSR markers. Recently, the genus Morus was classified into 13 species using a combination of ITS and trnL-trnF intergenic spacer markers, these included eight Asian species (M. alba, M. australis, M. cathayana, M. macroura, M. mongolica, M. nigra, M. notabilis, and M. serrata), four New World species (M. celtidifolia, M. insignis, M. microphylla, and M. rubra), and one African species (M. mesozygia) [24]. However, the M. notabilis sample they collected was not a true M. notabilis sample because the ITS sequence (GenBank accession number HM747175) is actually identical to that of M. alba. The fact that these studies did not include M. notabilis, as reported by Schneider [11], limits our full understanding of Morus taxonomy.

In plants, the ITS locus has been widely used in phylogenetic inference [33] and has shown the highest discriminatory power for species [34]. Furthermore, ITS has been recently proposed for use as the primary DNA barcode marker in fungi [35] and its use has enabled the estimation of highly accurate phylogenies of closely related organisms in yeast [36]. In this study, which includes data from M. notabilis, we characterized the ITS locus in Morus spp. including length and SNP. Using this ITS data, we redefined the taxonomy of Morus into eight species (M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia).

Materials and Methods

Plant materials

All the Plant materials used in this study were listed in the Table 1. M. notabilis located at 29°45.278' north latitude, 102°53.878' east longitude, a wild mulberry species with seven pairs of chromosomes, was collected by the author (Ningjia He) from a pristine forest in Ya’an, Sichuan province, Southwest China. M. yunnanensis was kindly provided by associate-researcher Yining Chu from the Institute of Sericulture and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi, Yunnan. M. nigra located at 36°59.606' north latitude, 81°14.651' east longitude, was collected by the author (Qiwei Zeng) from Yutian County, Xinjiang Uygur Autonomous Region, China. The collection locations of M. notabilis and M. nigra in this study are not protected lands. Other samples were from the Mulberry Germplasm Nursery in Southwest University, China. No special permits were required for the collection of the additional Morus spp. for this study as these species of Morus, which is distributed worldwide, are not considered to be endangered and are not listed as protected species, and the samples were obtained from public lands without restrictive ordinance (M. alba, M. mongolica, M. macroura, M. cathayana, M. australis, M. wittiorum, M. bombycis, M. indica, M. multicaulis, and M. atropurpurea).

DNA isolation, amplification, and sequencing

Total DNA was extracted from fresh leaves using a DNeasy plant mini kit (Qiagen Corp., Valencia, California, USA). The target sequence regions (the partial 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2, and the partial 28S rRNA gene) were amplified with mulberry-specific forward (5′-GTA ACA AGG TTT CCG TAG GTG-3′) and reverse (5′-TAA ACT CAG CGG GTA GCC-3′) primers, which were designed from the highly conserved regions flanking the 3′ end of the 18S rRNA gene and the 5′ end of the 28S rRNA gene, respectively, based on available Morus rRNA gene sequences in GenBank (National Center for Biotechnology Information (NCBI), Polymerase chain reaction (PCR) amplification was conducted in a reaction mixture of 20 μL containing 20 ng genomic DNA, 2 mM PCR buffer, 0.2 mM each primer, 0.2 mM each dNTP, and 1 unit Taq polymerase with 1.25 mM or 2.5 mM MgCl2 (Takara Bio-technology (Dalian) Co. Ltd., Dalian, China). The PCR amplification protocol was as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, followed by a final 10 min extension at 72°C. The PCR products were separated on a 1% agarose gel. The target fragment was gel-purified using the Takara Agarose Gel DNA Purification Kit (Takara Bio-technology (Dalian) Co. Ltd., Dalian, China), cloned into the vector pUCm-T (Sangon, Shanghai, China) by AT cloning according to manufacturer’s instructions, and individual clones were sequenced by BGI-Shenzhen (Shenzhen, China).

Data analyses

We downloaded an additional 187 Morus ITS sequences from NCBI (, and determined the sequence boundaries of ITS1, 5.8S, and ITS2 within all Morus sequences by comparison to known Arabidopsis thaliana ITS sequences (X52320). All data containing the full ITS1, 5.8S rRNA and ITS2 sequences were used for analysis. The sequence length and GC contents of all samples were analyzed by PerlScript.

Detection of SNPs in M. notabilis

In order to detect M. notabilis ITS SNPs, the improved Short Oligonucleotide Alignment Program (SOAP2) [37] was used to map all of the M. notabilis genome reads from 12 libraries [38] onto the reference sequence, a 3709-bp fragment located at scaffold2247 (GenBank accession number KF784877). SNPs were detected using SoapSNP essentially as described [37], but with the following modifications: SoapSNP calls were discarded if the quality score of the consensus genotype was less than 20, if the sequencing depth of the site was less than 5, or if the distance of a neighbor candidate SNP was less than 5.

Phylogenetic analysis and estimation of divergence times

Phylogenetic analysis was performed using a Bayesian method with MrBayes V3.1.2 software [39]. The phylogenetic tree of the ITS locus (ITS1-5.8S-ITS2) was rooted by the corresponding homologous sequences in Broussonetia papyrifera and Ficus adhatodifolia, which are sister genera of Morus, and in Cannabis sativa, which is another member of the Rosales order in the Cannabaceae. The phylogenetic tree was reconstructed and the estimations of the divergence times were determined according to the method described by Sun et al [40]. The divergence time of M. notabilis and C. sativa [38] was used to calibrate the estimation.


Sequence characteristics of the ITS region

From our analysis, an additional 187 ITS sequences of Morus that included generally accepted Morus species were deposited in NCBI, including 56 sequences that contained the complete ITS region (ITS1-5.85S-ITS2), 60 that contained the partial 18S region (3′ end, “AGG ATC ATT G”), 156 covering the complete 5.8S and ITS2 region, and 57 sequences that contained the partial 28S region (5′ end, “G(T/C)G ACC CCA G”). After adding the newly sequenced ITS regions, we characterized the structures of the ITS nucleotide sequences (Table 2) along with their sequence lengths and GC contents (Table 3). The first 10 nucleotides of the ITS1 region were highly conserved in Morus, and a transversion (G/T) and a single nucleotide deletion (T) were found in the last 10 nucleotides (Table 2). The first transition of the ITS region (A/G, where G was only identified in M. rubra, M. serrata, and M. insignis) was detected in the 13th nucleotide. The first 10 nucleotides in the 5.8S region were also highly conserved, with the exception of a single-nucleotide substitution (A/T) at the first position of M. notabilis [38] (GenBank accession number KF784877) and M. yunnanensis (GenBank accession number KF850474) and a transition (T/C) at the fifth position of M. mesozygia (GenBank accession number HM747171). The last 10 nucleotides of 5.8S exhibited a transition (C/T) and a transversion (A/C) at 4th and 9th positions, respectively. In total, there were 8 variable positions in the 5.8S region in Morus. The 5.8S subunits of 121 Morus spp. including M. alba, M. mongolica, M. macroura, M. cathayana, M. australis, M. wittiorum, M. bombycis, M. indica, M. multicaulis (M. lhou), and M. atropurpurea were uniform in size (163 bp) with a GC content of 52.147%, except for M. australis (GenBank accession number AM042004), which had a GC content of 51.533%. In the genus Morus, M. alba and M. notabilis contained the shortest (215 bp) and longest (233 bp) respective ITS1 sequence lengths, and M. mesozygia and M. notabilis (or M. celtidifolia) contained the shortest (217 bp) and longest (235 bp) ITS2 sequence lengths, respectively (Table 3). In contrast to other Morus species, a 13 bp deletion of the ITS1 region in M. alba was located at the 55th nucleotide position. The comparison of the 5.8S region sequences of M. alba (121 sequences), M. rubra (9 sequences), M. notabilis (6 sequences), and M. celtidifolia (4 sequences) found that the intraspecific 5.8S regions of these species were completely identical. In addition, we found that the ITS1 sequences of interspecific hybrids between M. alba and M. rubra consisted of 215 bp (GenBank accession numbers HQ144171 and HQ144170) and 229 bp (GenBank accession numbers HQ144175 and HQ144187) sequences, respectively. However, the ITS2 lengths of these hybrids were unique (Table 3).

Table 2. Nucleotide sequences at the beginning and end of the Morus ITS region.

Sequence length is 10 bp. ITS, internal transcribed spacers.

Table 3. Sequence length (bp) and percentage GC content (100%) of the ITS region in the genus Morus.

Detection of SNPs in M. notabilis and ITS genetic heterogeneity in Morus

To detect SNP sites in the ITS region in the draft genome of M. notabilis, we employed the algorithm SOAP2 to map all of the genome reads, identifying six SNPs in the ITS region of M. notabilis (Table 4). In the M. alba lineage, only M. australis (GenBank accession number AM042004) showed variation in the 5.8S region. Comparison of all M. australis 5.8S region data deposited in GenBank indicated that ten sequences from different specimens were identical. In the full collection of ITS1 and ITS2 sequences of M. alba, we identified one SNP (A/G) existing at the 82nd site of ITS1 (4 out of 41 sequences) and one SNP (T/G) existing at the 219th site of ITS2 (21 out of 107 sequences). In M. rubra, 4 and 1 variable loci existed in the ITS1 and ITS2 regions of 9 sequences, respectively. Among these 5 variable loci, 4 occurred in two sequences from different samples, which suggested the possibility of 4 SNPs in the ITS region of M. rubra. In M. celtidifolia, we identified 3 variable loci in the ITS1 and ITS2 regions out of five ITS sequences.

Table 4. Predicted SNP sites in the ITS region of M. notabilis.

Phylogenetic analysis and estimation of divergence time

Bayesian inference of phylogeny based on ITS sequences separated Morus species into four large clades, as shown in Fig 1. The A clade was a polyphyletic group comprised of two Asian species (M. nigra and M. serrata) and two American species (M. rubra and M. celtidifolia). The A clade also included two clones (GenBank accession numbers HQ144175 and HQ144187) that were a red mulberry parent of a hybrid (M. rubra x M. alba). The calculated estimated divergence time between M. celtidifolia and M. nigra was 2.25 Mya. The B clade, which was the largest clade, was a monophyletic taxa comprised of only Chinese white mulberry (M. alba). The B clade also included two clones (GenBank accession numbers HQ144170 and HQ144171, amplified from the same hybrid mentioned above), which were a white mulberry parent of the hybrid. The divergence time between the A and B clades was estimated to be 5.34 Mya. The C clade consisted of M. notabilis and M. yunnanensis, with a 0.44 Mya estimated divergence time. Similar to M. notabilis, M. yunnanensis, which used to be recognized as variety of M. mongolica, was also found to contain 14 chromosomes. Finally, the D clade consisted of two species including M. insignis and M. mesozygia. The estimate of the divergence time between the Morus genus and the Ficus and Broussonetia genera was 39.91 Mya.

Fig 1. Phylogenetic tree and chronology of divergence times based on ITS as estimated using Bayesian methods implemented in BEAST.

Assignments into clades A (light red; A1, A2, A3 and A4 represent M. celtidifolia, M. nigra, M. rubra and M. serrata, respectively), B (light pink; B represents M. alba), C (light sky blue; C represents M. notabilis), D (light blue, D1 and D2 represent M. mesozygia and M. insignis, respectively) and an outgroup (light green) are shown. Values represent the divergence times with the 95% highest posterior density intervals shown in parentheses.


Mulberry is not only an important economic woody tree [24] but also plays significant roles in the recovery and restoration of damaged ecosystems [4], and was planted by famers as early as 5000 years ago [38]. Although the genus Morus is widely cultivated in the Eurasian, African, and American continents, its taxonomy is complex and has been disputed. Since Linnaeus [9], researchers have used morphological structures of the leaf, winter bud, bark, pistil, syncarp, and leaf idioblasts to infer evolutionary relationships among Morus species [1014].

DNA-based markers have been developed to better classify Morus species [2432,41]. Currently, ITS data have the highest discriminatory power for plant species determination [34] and this marker is widely used for taxonomic and phylogenetic analyses [42,43]. Although ITS data have previously been used to classify Morus species [24,28,4446], one recognized species, M. notabilis [11], was not included in these studies. In this study, we supplemented the 24 ITS sequences (GenBank accession numbers including KF784875-KF784897, KF850474) of Morus by including data for M. notabilis and M. yunnanensis (14 chromosomes), and M. nigra (308 chromosomes) collected using the method of cloning and sequencing.

Previously reported lengths of the Morus 5.8S gene were 152, 159, or 100 bp [28,45,47]. Here, we found the 5.8S length to be 163 bp, which was consistent with that of other species such as Malus, Triticum, and Canella [4854]. We also found that the samples with a 215 bp ITS1 sequence carried identical 5.8S sequences, these included M. alba, M. mongolica, M. cathayana, M. australis, M. macroura, M. wittiorum, M. bombycis, M. indica, M. trilobata, and M. multicaulis. To explore this phenomenon, we sorted the species names of these samples and found that all of them are synonyms or subspecies/varieties of M. alba [10,15,19,47]. Currently, there are 10–16 generally accepted Morus species, including M. alba, M. australis, M. cathayana, M. macroura, M. mongolica, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. microphylla, M. rubra, M. mesozygia, M. bombycis, M. wittiorum, and M. trilobata [15,24]. M. mongolica, M. cathayana, M. australis, M. wittiorum, and M. macroura used to be widely accepted as distinct Morus species, however, in this study, we found that they should be attributed to M. alba. Furthermore, we found that the 5.8S sequences of M. notabilis (6 clones, originating from different samples), M. rubra (9 clones from different samples), and M. celtidifolia (4 clones from different samples) were also identical. M. insignis (GenBank accession number HM747169), M. mesozygia (GenBank accession number HM747171), and M. serrata (GenBank accession number HM747176) had only one ITS sequence each deposited in NCBI, so it was necessary to obtain additional data to test the identification of their 5.8S sequences. Based on characterization of the 5.8S sequences from M. alba, M. rubra, M. notabilis, and M. celtidifolia, we found that the intraspecific 5.8S sequence was invariable in these species, whereas the interspecific 5.8S sequences were variable in Morus species exhibiting no evidence of hybridization. Therefore, the inclusion of the 5.8S region as part of the ITS sequence is critically important for taxonomic purposes [34].

In the present study, we found that M. serrata (GenBank accession number HM747176), referred to as M. serrata Roxb and also known as the Himalayan Mulberry [55], was distinct from M. serrata Wall., a synonym of M. alba. M. yunnanensis was previously regarded as a variant of M. mongolica, but M. yunnanensis Koidz. S. S. Chang was a synonym for M. notabilis, which was supported by our results. In addition, M. microphylla was used as a synonym for M. celtidifolia [56]. Therefore, with no regard for synonyms, subspecies, or varieties, the generally accepted Morus species identified in our study included four species native to Asia (M. alba, M. serrata, M. nigra, and M. notabilis), three New World species (M. celtidifolia, M. insignis, and M. rubra), and one species from Africa (M. mesozygia).

The use of ITS as a species barcode has been criticized due to its limitations, which include intraspecific variation [42] and heterogeneous copies [43,57]. However this heterogeneity did not affect the molecular identification of species [57]. The heterogeneity of ITS could be detected by both direct sequencing and sequencing after cloning [42]. In this study, we analyzed the heterogeneity of four Morus species. The results showed that there were SNPs in the ITS regions of M. notabilis, M. alba, M. rubra, and M. celtidifolia. Among these, the proportion of SNPs in the ITS region of M. notabilis was similar to that in the entire M. notabilis genome [38]. For Morus species without genome samples or repetitive ITS data in GenBank, ITS heterogeneity cannot be studied. Although intraspecific ITS SNPs were detected in Morus spp., the lengths of their intraspecific ITS regions and the positions of the Morus spp. with intraspecific ITS SNPs in the tree were invariable.

In this study, we demonstrated that interspecific hybrids of Morus species could be identified by ITS sequences. For example, two M. alba (GenBank accession numbers HQ144171 and HQ144170) and two M. rubra (GenBank accession numbers HQ144175 and HQ144187) clones were detected in a hybrid sample. Another example was an ITS sequence of M. rubra (FJ605516) whose sequence was identical to sequences of a hybrid (HQ144170) in the ITS1 and 5.8S regions, except for five separate locus differences in the ITS2 region. This suggested that the sample was possibly a hybrid (M. rubra x M. alba) because interspecific hybrids are quite commonly misidentified by morphological criteria [58].

We reconstructed the phylogenetic tree of the genus Morus based on ITS sequences. According to the phylogenetic analysis, the largest clade, B, was defined by a 215 bp ITS1 sequence and was a monophyletic group (M. alba), which agreed with the results obtained from sorting Morus species names. The C clade included M. notabilis from Ya’an county and M. yunnanensis from Dawei Mountain in China, each of which contained 14 chromosomes and an ITS1 sequence length of 233 bp. The distribution of parents of interspecific hybrids into separate clades (GenBank accession numbers HQ144175 and HQ144187, HQ144171 and HQ144170) indicated that ITS could be used to investigate Morus interspecific hybrids. The results of the phylogenetic analysis also indicated that there were only eight species in the genus Morus. Furthermore, the calibrated point of 63.5 (46.9–76.6) Mya for estimating the divergence time of the genus Morus was adopted from the mulberry genome [38]. This calibrated point is consistent with the divergence time of Moraceae (55 Mya) estimated by nuclear and chloroplast DNA analysis combined with data from multiple fossils [59], which confirmed that the estimated divergence time of the Morus genus was reliable.

Therefore, from the results obtained in this study after including new data for ITS sequences and reconstructing the phylogenetic tree of the genus Morus, we suggest that the genus Morus can be classified into eight species and that ITS might to be used to discover hybridization in this genus.


The authors thank Yining Chu from the Institute of Sericulture and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi, Yunnan, China and Tianlong Ding from the Sericultural Research Institute of Xinjiang, Hetian, China for providing the materials of M. yunnanensis and M. nigra, respectively. We are also grateful to editor and the anonymous reviewers who provided valuable comments that greatly improved the manuscript.

Author Contributions

Conceived and designed the experiments: QZ NH. Performed the experiments: QZ HC CZ. Analyzed the data: QZ MH TL. Contributed reagents/materials/analysis tools: ZX TL XQ. Wrote the paper: QZ NH.


  1. 1. Zhang SD, Soltis DE, Yang Y, Li DZ, Yi TS (2011) Multi-gene analysis provides a well-supported phylogeny of Rosales. Molecular Phylogenetics and Evolution 60: 21–28. pmid:21540119
  2. 2. Tutin GT (1996) Morus L. In: Tutin GT, Burges NA, Chater AO, Edmondson JR, Heywood VH et al., editors. Flora Europa. 2 ed. Psilotaceae to Platanaceae: Cambridge University Press, Australia.
  3. 3. Priya S (2012) Medicinal values of mulberry–an overview. Journal of Pharmacy Research 5: 3588–3596.
  4. 4. Qin J, He NJ, Huang XZ, Xiang ZH (2010) Development of mulberry ecological industry and sericulture. Science of Sericulture 36: 984–989. (In Chinese with English abstract)
  5. 5. Vijayan KS, Saratchandra B, T J. A.. (2011) Germplasm conservation in mulberry (Morus spp.). Scientia Horticulturae 128: 371–379.
  6. 6. Gray E, Gray RE (1987) Leaf lobation patterns in mulberry. Castanea 52: 216–224.
  7. 7. Burgess KS, Morgan M, Deverno L, Husband BC (2005) Asymmetrical introgression between two Morus species (M. alba, M. rubra) that differ in abundance. Molecular Ecology 14: 3471–3483. pmid:16156816
  8. 8. Toyo I (1985) Research of polyploidy and its application in Morus. Japan Agricultural Research Quarterly 18: 222–228.
  9. 9. Linnaeus C (1753) Morus. Species Plantarum. Stockholm Impensis Laurentii Salvii. pp. 968.
  10. 10. Bureau LÉ (1873) Moraceae. In: DeCandolle AP, editor. Prodromus systematis naturalis regni vegetabilis. Paris, France: Tuettel and Wurtz. pp. 211–288.
  11. 11. Schneider CK (1917) Moraceae. In: Sargent CS, editor. Plantae Wilsonianae: an enumeration of the woody plants collected in western China for the Arnold arboretum of Harvard university during the years 1907, 1908, and 1910. Cambridge: The University Press. pp. 293–294.
  12. 12. Koidzumi G (1917) Taxonomical discussion on Morus plants. Bulletin of Sericultural Experimental Station 3: 1–62.
  13. 13. Leroy JF (1949) Les Muriers sauvages et cultives. La sericiculture sous les tropiques Rev. Revue Internationale de Botanique aaplique'e et d' agriculture Tropicale 29: 481–496.
  14. 14. Hotta T (1954) Fundamentals of Morus plants classification. Kinugasa Sanpo 390: 13–21.
  15. 15. Zhou ZK, Gilbert MG (2003) Moraceae. In: Wu ZY, Raven PH, Hong DY, editors. Flora of China. Beijing, China & Saint Louis, Missouri: Science Press & Missouri Botanical Garden Press. pp. 22–26.
  16. 16. Sanjappa M (1989) Geographical distribution and exploration of the genus Morus L.(Moraceae). In: Sengupta KD, SB editor. Genetic resources of mulberry and utilization. Central Sericultural Research and Training Institute (CSRTI), Mysore, India. pp. 4–7.
  17. 17. Datta RK. Mulberry cultivation and utilization in india. In: Sánchez MD, editor. Mulberry for animal production; 2000; Rome, Italy.
  18. 18. Butt MS, AkmalSultan , TauseefSchroën M, Karin (2008) Morus alba L. nature's functional tonic. Trends in Food Science & Technology 19: 505–512.
  19. 19. Wikipedia, (2015) Morus (plant).
  20. 20. Kapche GD, Fozing CD, Donfack JH, Fotso GW, Amadou D, Tchana AN, et al. (2009) Prenylated arylbenzofuran derivatives from Morus mesozygia with antioxidant activity. Phytochemistry 70: 216–221. pmid:19147162
  21. 21. Pawlowska AM, Oleszek W, Braca A (2008) Quali-quantitative analyses of flavonoids of Morus nigra L. and Morus alba L.(Moraceae) fruits. Journal of Agricultural and Food Chemistry 56: 3377–3380. pmid:18412362
  22. 22. Berg CC (2001) Moreae, Artocarpeae, and Dorstenia (Moraceae): With Introductions to the Family and Ficus and with Additions and Corrections to Flora Neotropica Monograph 7. New York: New York Botanical Garden Press.
  23. 23. Berg CC. Moraceae diversity in a global perspective. In: Skrifter B, editor; 2005; Copenhagen, Denmark. pp. 423–440.
  24. 24. Nepal MP, Ferguson CJ. (2012) Phylogenetics of Morus (Moraceae) inferred from ITS and trnL-trnF sequence data. Systematic Botany 37: 442–450.
  25. 25. Sharma A,Sharma R, Machii H (2000) Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers. Theoretical and Applied Genetics 101: 1049–1055.
  26. 26. Kafkas S, Özgen M, Doğan Y, Özcan B, Ercişli S, Serçe S (2008) Molecular characterization of mulberry accessions in Turkey by AFLP markers. Journal of the American Society for Horticultural Science 133: 593–597.
  27. 27. Rao AA, Vijayan K, Krubakaran M, Borpujari MM, Kamble CK (2011) Genetic diversity in mulberry (Morus spp.) revealed by isozyme markers. Journal of Horticultural Science & Biotechnology 86: 291–297.
  28. 28. Zhao WG, Pan YL, Zhang ZF (2004) Phylogenetic relationship of genus Morus by ITS sequence data. Science of Sericulture 30: 11–14. (In Chinese with English abstract)
  29. 29. Zhao WG, Miao XX, Jia SH, Pan YL, Huang YP (2005) Isolation and characterization of microsatellite loci from the mulberry, Morus L. Plant Science 168: 519–525.
  30. 30. Zhao WG, Zhou ZH, Miao XX, Zhang Y, Wang SB, Huang JH, et al. (2007) A comparison of genetic variation among wild and cultivated Morus Species (Moraceae: Morus) as revealed by ISSR and SSR markers. Biodiversity and Conservation 16: 275–290.
  31. 31. Zhao WG, Fang RJ, Pan YL, Yang YH, Chung JW, Chung IM, et al. (2009) Analysis of genetic relationships of mulberry (Morus L.) germplasm using sequence-related amplified polymorphism (SRAP) markers. African Journal of Biotechnology 8: 2604–2610.
  32. 32. Zhao WG, Zhou ZH, Miao XX, Wang SB, Zhang L, Pan YL, et al. (2006) Genetic relatedness among cultivated and wild mulberry (Moraceae: Morus) as revealed by inter-simple sequence repeat analysis in China. Canadian Journal of Plant Science 86: 251–257.
  33. 33. Álvarez I, Wendel JF (2003) Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. pmid:14615184
  34. 34. Group CPB, Li DZ, Gao LM, Li HT, Wang H, Ge XJ, et al. (2011) Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proceedings of the National Academy of Sciences of the USA 108: 19641–19646. pmid:22100737
  35. 35. Schoch CL, Seifertb KA, Huhndorfc S, Robert V, Spouge JL, Levesque CA, et al. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences 109: 6241–6246.
  36. 36. West C, James SA, Davey RP, Dicks J, Roberts IN (2014) Ribosomal DNA sequence heterogeneity reflects intra-species phylogenies and predicts genome structure in two contrasting yeast species. Systematic Biology 63(4): 543–554. pmid:24682414
  37. 37. Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, et al. (2009) SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 25: 1966–1967. pmid:19497933
  38. 38. He N, Zhang C, Qi X, Zhao S, Tao Y, Yang G, et al. (2013) Draft genome sequence of the mulberry tree Morus notabilis. Nature Communications 4.
  39. 39. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. pmid:12912839
  40. 40. Sun W, Yu H, Shen Y, Banno Y, Xiang Z, Zhang Z (2012) Phylogeny and evolutionary history of the silkworm. Science China Life Sciences 55: 483–496. pmid:22744178
  41. 41. Vijayan K, Srivastava PP, Awasthi AK (2004) Analysis of phylogenetic relationship among five mulberry (Morus) species using molecular markers. Genome 47: 439–448. pmid:15190361
  42. 42. Kiss L (2012) Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for Fungi. Proceedings of the National Academy of Sciences of the USA 109: E1811. pmid:22715287
  43. 43. Simon UK, Weiss M (2008) Intragenomic variation of fungal ribosomal genes is higher than previously thought. Molecular Biology and Evolution 25: 2251–2254. pmid:18728073
  44. 44. Chen RF, Chen XP, Fan JY, Chen XM, Liu ZT, Yu ZW (2012) An analysis on phylogenetic relationship and divergence time of yunnan guangye sang and qinzhou changguo sang in Morus genus based on its sequence. Science of Sericulture 38: 002–007. (In Chinese with English abstract)
  45. 45. Chen RF, Yu MD, Liu XQ, Chen LQ (2010) Analysis on the internal transcribed spacers (ITS) sequences and phylogenetics of mulberry (Morus). Scientia Agricultura Sinica 43: 1771–1781. (In Chinese with English abstract)
  46. 46. Zhao WG, Pan YL, Zhang ZF, Miao XX, Huang YP (2005) Phylogeny of the genus Morus (Urticales: Moraceae) inferred from ITS and trnL-F sequences. African Journal of Biotechnology 4: 563–569.
  47. 47. Nepal MP (2008) Systematics and reproductive biology of the genus Morus L.(Moraceae). Manhattan, Kansas: Kansas State University.
  48. 48. Yang ZY, Chao Z, Huo KK, Xie H, Tian ZP, Pan SL (2007) ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China. Phytomedicine 14: 416–423. pmid:17509842
  49. 49. Sukrong S, Zhu S, Ruangrungsi N, Phadungcharoen T, Palanuvej C, Komatsu K (2007) Molecular analysis of the genus Mitragyna existing in Thailand based on rDNA ITS sequences and its application to identify a narcotic species: Mitragyna speciosa. Biological and Pharmaceutical Bulletin 30: 1284–1288. pmid:17603168
  50. 50. Suh Y, Thien LB, Zimmer EA (1992) Nucleotide sequences of the internal transcribed spacers and 5.8 S rRNA gene in Canella winterana (Magnoliales; Canellaceae). Nucleic Acids Research 20: 6101–6102. pmid:1461743
  51. 51. Vollmer SV, Palumbi SR (2004) Testing the utility of internally transcribed spacer sequences in coral phylogenetics. Molecular Ecology 13: 2763–2772 pmid:15315687
  52. 52. Chemeris AV, Vakhitov VA (1989) Primary structure of the 5.8 S gene of rRNA and internal transcribed spacers of rDNA in diploid wheat Triticum urartu thum. ex. gandil. Molekuliarnaia Biologiia 23: 320–326. pmid:2739648
  53. 53. Vakhitov VA, Gimalov FR, Shumiatskiĭ GP (1989) Nucleotide sequences of 5S rRNA genes of polyploid species of wheat and Aegilops species. Molekuliarnaia Biologiia 23: 431–440. pmid:2770725
  54. 54. Robinson JP, Harris SA, Juniper BE (2001) Taxonomy of the genus Malus Mill.(Rosaceae) with emphasis on the cultivated apple, Malus domestica Borkh. Plant Systematics and Evolution 226: 35–58.
  55. 55. Basavaiah SBD, Rajan MV (1989) Microsporogenesis in hexaploid Morus serrata Roxb. Cytologia 54: 747–751.
  56. 56. Buckley SB. Morus microphylla Buckley is a synonym of Morus celtidifolia Kunth 1863; Philadelphia. pp. 8.
  57. 57. Li W, Sun H, Deng Y, Zhang A, Chen H (2014) The heterogeneity of the rDNA-ITS sequence and its phylogeny in Rhizoctonia cerealis, the cause of sharp eyespot in wheat. Current Genetics 60: 1–9. pmid:23839120
  58. 58. Saar D. Red and white mulberries (Morus rubra and M. alba: Moraceae) and their interspecific hybrids: the "real" red mulberry is not as previously thought.; 2011; Austin Peasy State University, clarksville, Tennessee.
  59. 59. Zerega NJC, Clement WL, Datwyler SL, Weiblen GD (2005) Biogeography and divergence times in the mulberry family (Moraceae). Molecular Phylogenetics and Evolution 37: 402–416. pmid:16112884