Identification and taxonomical evaluation of the germplasm resources

METHODOLOGY

Based on the lists of mulberry varieties that has been provided by the Aracne partners within the first year of the project (D1.4 – Progress report on the collected mulberry samples, see Table 1 and Suppl. Tables I-IV), selected trees from CREA, SCS Vratsa, UM and IMIDA germplasm collections were included in the monitoring of morphological descriptors and silica dried leaf material was send to CREA for molecular analyses.
High yielding sericultural and local varieties from the following collections were included:

  • CREA Germplasm: The evaluation included 57 varieties from diverse taxonomical and geographical origins (Italy, China, France, India, Japan).
  • Vratsa Germplasm: A total of 70 mulberry varieties were evaluated. The selection focused on local Bulgarian varieties and those from Eastern Europe and Central Asia (Armenia, Azerbaijan, Georgia, Romania, Ukraine, and Uzbekistan) to trace the possible spread of mulberries (and sericultural activity) from the East to the West. These varieties, introduced and planted at the SCS-Vratsa germplasm between 1965 and 2004, offer a unique insight into regional diversity.
  • UM Germplasm: 166 distinct varieties were included in the analyses, representing 132 local historical white mulberry ( M. alba s.l.) trees inventoried in Slovenia (69) and in Hungary (63), along with 34 reference varieties used in sericulture and fruit processing. The varieties were planted mainly between years 2015-2018.
  • IMIDA germplasm: 35 varieties varieties originating from Italy, Spain and Japan were included in the analysis.

The main issue was that most varieties are labelled with old traditional taxonomic names, synonyms (syn.), that apply to a taxon that now goes by a different scientific name. Moreover, most modern sericultural varieties originate from simple or complex spontaneous and/or artificial crosses between M. alba, M. indica and M. rubra. Therefore, most of the varieties belonging to M. alba and M. indica have been concurrently defined as sensu lato (s.l.), which means “in the broad sense”, whereas M. rubra varieties have been concurrently asigned as confer (cf.). This qualifier indicates that most of the diagnostic characters correspond to M. rubra, but some characters are unclear. The identification is provisional but is likely to be definitive after comparing with reference material. Varieties that are noted as M. rubra cf. could originate from simple or complex spontaneous and/or artificial crosses involving M. alba. Their genetic relationship is going to be analysed under the scope of ARACNE mulberry research. The collections also include forms (f.) of M. alba (M. alba f. pendula, M. alba f. pyramidalis), which denote a taxonomic category bellow the species level and refer to specific morphological differences. Variety (var.) is a taxonomic category that ranks between the subspecies and forma level, its members differing from others in minor but permanent or heritable characteristics.

According to the currently accepted classification, the varieties identified, evaluated, and sampled in the germplasm were taxonomically organised as shown in Table 1, taking known synonyms into account.

Table 1 - Current taxonomical status of M. alba s.l., M. indica s.l. and M. rubra cf. along with synonyms.

Current taxonomical status

synonyms

M. alba s.l.

M. atropurpurea,

M. alba var. atropurpurea

M. alba f. pendula

M. pendula

M. alba f. pyramidalis 

M. pyramidalis

M. indica

M. australis

 

M. bombycis

M. kagayamae

M. latifolia

 

M. multicaulis,

M. indica var. multicaulis

M. planifolia,

M. multicaulis var. planifolia

M. rotundiloba

M. rubra cf.

 

Varieties that are noted as M. rubra cf. could originate from simple or complex spontaneous and/or artificial crosses involving M. alba. Their genetic relationship is going to be analysed under the scope of ARACNE mulberry research.

Methodology for maintenance of mulberry accessions

Conservation of mulberry accessions begins with their collection, multiplication, and subsequent establishment in ex-situ conservation sites. Different maintenance practices are essential to this process, including propagation methods, pruning techniques, and fertilisation strategies. The upcoming chapters will detail the maintenance methodologies employed by the germplasm collections of CREA, SCS Vratsa, and UM. These institutions serve as foundational models for other partners, providing a backbone for the effective conservation and maintainance of mulberry collections.

Methodology for mulberry germplasm characterisation and evaluation 

Morphological traits of mulberry accessions maintained in the ARACNE partners’ collections (CREA, Vratsa, IMIDA, UM) were analyzed using UPOV Mulberry descriptors, basically modified according to the Guidance model to collect mulberry samples. To standardise the database of morphological descriptors across all partners, a template for an Excel file pivot table was developed. This template has been utilized by CREA, SCS Vratsa, IMIDA, and UM to ensure consistency in data recording and analysis. The list of descriptors is summarized in Table 2.

Table 2 - The list of mulberry descriptors employed for the evaluation of the mulberry genetic resources maintained in the germplasm collections of ARACNE partners.

Category

Descriptor

Options

Taxonomical and basic information

Identification/ Accession n.

Data entry

Species

M. alba, M. indica, M. cathayana, M. celtidifolia, M. insignis, M. kordesiana, M. loboensis, M. macroura, M. mesozygia, M. microphylla, M. miyabeana, M. nigra, M. notabilis, M. rubra, M. serrata, M. trilobata, M. wittiorum, interspecific hybrid

Varietal name

Data entry

Date of sampling

Data entry  

Date of sampling

Data entry  

Number of individuals in the repository

Individual, Mulberry plantation, Mulberry row

Method of observation

Single measurement of a group of plants or part of plants, Measurement of a number of individual plants or parts of plants, Visual assessment by a single observation of group of plants or part of plants, Visual assessment by observation of individual plants

Mode of origin/biological status

Natural, Traditional cultivar/landrace, Advanced or improved cultivar, Hybrid

Ploidy level (if known)

Haploid, Diploid, Triploid, Tetraploid, Hexaploid, Docosaploid, Polyploid, Ploidy level not yet determined

Geographical origin lat.

Data entry  

Geographical origin long.

Data entry  

Availability

Public, Street, Square, Private Garden, Botanical Garden or Gene bank, Agricultural landscape

Areas of cultivation

Temperate, tropical, sub-tropical

Morphological characteristics

Trunk circumference (cm)

<180 cm, 180-249 cm, 250-299 cm, >300 cm

Exact measurement of trunk circumference (cm)

Data entry  

Tree growth habit

Upright, Semi-upright, Weeping

Tree vigour

Bad condition, Good condition

Pruning practices

Unpruned tree, Frequently pruned, Yearly pruned tree

Trunk

Trunk colour

Greyish brown, Light brownish-grey, Reddish brown

Trunk irregularities/damage

Curved, Hollow (pipe tree), Longitudinally cracked, External split

Shoots

Current years shoots: length (cm)

Data entry  

Current years shoot: number of lateral shoots

Data entry  

Current years shoot: length of internode

Data entry  

Colour of one-year old shoot

Greyish brown, Greenish brown, Yellowish brown, Medium brown, Reddish brown, Dark brown

Lenticel density

High, Medium, Sparse

Lenticel shape

Elliptical, Oval, Round

Buds

Bud shape

Broad triangular, Medium triangular, Narrow triangular, Ovate

Bud colour

Greyish brown, Yellowish brown, Reddish brown, Medium brown, Dark brown

Bud size

Small, medium, large

Leaves

Phyllotaxis

Alternate spiral, Alternate distichous, Opposite decussate

Leaf shape

Simple, Lobed

Peduncle length (cm)

Data entry  

Average leaf size: length (cm)

Data entry  

Average leaf size: width (cm)

Data entry  

Average leaf size: area (cm2)

Data entry  

Leaf blade

Low (<1.2 broad leaves), Medium (1.3-1.5), High (>1.6 long)

Petiole range

Absent or very short (< 10 mm), Short (11-20 mm), Medium (21-40mm), Long (41-70 mm), Very long (>71 mm)

Petiole size (cm)

Data entry  

Shape of leaf base

Cuneate, Truncate, Retuse, Cordate

Shape of leaf apex

Acute, Obtuse, Obcordate

Leaf blade tip

Absent, Caudate, Acuminate

Leaf blade margin

Repand, Crenate, Dentate, Serrulate, Biserrate, Serrate, Aristate

Hairiness

Glabrous, Midrib and veins, Evenly pubescent

Glossiness

Glossy, Matte

Phenological descriptors

Date of swollen bud (week)

Data entry  

Date of bud burst (week)

Data entry  

Flowering date (week)

Data entry  

Date of first leaf (week)

Data entry  

Date of first leaf (week)

Data entry  

Early yellowing due to disease (week)

Data entry  

Abundant autumn colouring (week)

Data entry  

Leaf fall (week)

Data entry  

Time of infructescence ripening

Early, Early to medium, Medium, Medium to late, Late

Uniformity of infructescence ripening 

Early, Early to medium, Medium, Medium to late, Late

Reproductive structures

Sexual dimorphism

Monoecious, Dioecious

Inflorescence type

Male, Female, Predominantly female with some male flowers at the base, Predominantly male with some female flowers at the apex, Predominantly male with some female flowers at the base, Predominantly female with some male flowers at the apex

Inflorescence size

Female inflorescence length (mm)

Data entry  

Female inflorescence diameter (mm)

Data entry  

Female inflorescence stalk length (mm)

Data entry  

Male inflorescence length (mm)

Data entry  

Male inflorescence length (mm)

Data entry  

Male inflorescence stalk length (mm)

Data entry  

Polygamous  inflor. length (mm)

Data entry  

Polygamous  inflor. diameter (mm)

Data entry  

Polygamous  inflor. diameter (mm)

Data entry  

Infructescence

Fruit weight (g)

Data entry  

Length (mm)

Data entry  

Width (mm)

Data entry  

Length of peduncle

Short, Medium, Long

Colour

Yellowish white, Light pink, Purple brown, Reddish black, Black

Taste

Acidic, Sweet, Balanced

Infructescence shape 

Ovoid/globose, Ellipsoid, Cylindrical, Irregular

Growth and yield attributes

Growth

Slow, Medium, Fast

Length of primary branch (cm)

Data entry  

Internodal distance (cm)

Data entry  

Leaf No./meter

Data entry  

Weight of 100 fresh leaves (g)

Data entry  

Chemical composition of leaves

Total protein (g/100 g)

Data entry  

Mineral (g/100 g)

Data entry  

Fibre (g/100 g)

Data entry  

Response to different physological conditions

Response to drought

Resistant, Tolerant, Susceptible

Response to salinity

Resistant, Tolerant, Susceptible

Response to chilling stress

Good, Medium, Poor

Response to repeated pruning

Good, Medium, Poor

Diseases and pests

Disease option

Fungal leaf spot, Bacterial leaf spot (Pseudomonas syringae pv. mori), Soft rot (Pectobacterium carotovorum), Ringspot virus

Pest option

Mulberry moth (Hyphantria cunea), Thrips (5 species), Mites (Tetranychus sp.), Scale insects (Hemiptera), Mealy bugs (Maconellicoccus hirsutus), Red scale (Aonidella aurantii), Hairy caterpillar (Spilarctia obliqua), Jassids (Empoasca Flavescens)

Any other disease or pest

Data entry  

Leaf necrotic spots

Low, medium, frequent

Bark lesions

Frequent, Few

Additional remarks

Optional

Data entry  

For the evaluation of morphological descriptors, varieties were meticulously selected from each germplasm collection to ensure a comprehensive and comparable analysis. The main morphological features were studied during the years 2023-2025. Supporting schematic and pictorial information of the studied descriptors are shown in Figure 1-5.

Figure 1: Morphological characterization of the one-year old shoots and buds. A) Colour of the one year old shoot, B) Bud colour, C) bud shape, D) Lenticel shape.

Figure 2: Morphological characterization of the leaves. Phyllotaxis, leaf shape, hairiness, glossiness, leaf blade, petiole.

Figure 3: Morphological characterization of the leaves. Shape of leaf base, shape of leaf apex, leaf blade tip, leaf blade margin.

Figure 4: Morphological characterization of the reproductive structures. Inflorescence types, stigma persistency length of infructescence peduncle, uniformity of infructescence ripness.

Figure 5: Morphological characterization of the reproductive structures. Colour of infructescence, infructescence shape.

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