horticulturae

Communication
Wild Malus niedzwetzkyana Dieck ex Koehne as a Genetic
Resource for Fire Blight Resistance
Mariya Kolchenko 1 , Aidana Nurtaza 2,3 , Alexandr Pozharskiy 1,4 , Damira Dyussembekova 2 ,
Anastasiya Kapytina 1 , Gulnaz Nizamdinova 1 , Marina Khusnitdinova 1 , Aisha Taskuzhina 1,4 ,
Almagul Kakimzhanova 2 and Dilyara Gritsenko 1, *

1 Laboratory of Molecular Biology, Institute of Plant Biology and Biotechnology, Almaty 050040, Kazakhstan
2 Laboratory of Plants Biotechnology and Breeding, National Center for Biotechnology,
Astana 010000, Kazakhstan
3 Department of General Biology and Genomics, L.N. Gumilyov Eurasian National University,
Astana 010000, Kazakhstan
4 Department of Molecular Biology and Genetics, Faculty of Biology and Biotechnology, Al Farabi Kazakh
National University, Almaty 050040, Kazakhstan
* Correspondence: [email protected]

Abstract: Wild apples and their hybrids are valued as a source of genetic resistance to biotic and
abiotic stress. Malus niedzwetzkyana is an endangered ornamental apple species endemic to Southeast
Kazakhstan, the center of Malus domestication. To test the fire blight resistance of M. niedzwetzkyana,
eight plant genotypes were inoculated with a local strain of Erwinia amylovora. The genotypes possess
different genetic backgrounds, which was confirmed via SSR profiling. Four out of eight displayed
moderate to severe symptoms of fire blight infection, while the three wild genotypes proved resistant.
To search for the source of the resistance, the samples were tested for the presence of FBF7 QTL using
SCAR markers, where seven genotypes tested positive for one of the markers (AE10-375) and one for
the other (GE80-19). No correlation between resistance phenotype and FBF7 QTL was confirmed,
Citation: Kolchenko, M.; Nurtaza, A.; indicating the source lies elsewhere. Developing detailed genetic and phenotypic profiles of wild
Pozharskiy, A.; Dyussembekova, D.;
apple species helps advance both the preservation efforts and marker-assisted selection in apple
Kapytina, A.; Nizamdinova, G.;
breeding.
Khusnitdinova, M.; Taskuzhina, A.;
Kakimzhanova, A.; Gritsenko, D.
Keywords: Malus niedzwetzkyana; resistance; fire blight
Wild Malus niedzwetzkyana Dieck ex
Koehne as a Genetic Resource for Fire
Blight Resistance. Horticulturae 2023,
9, 1066. https://doi.org/10.3390/
horticulturae9101066 1. Introduction
Southeast Kazakhstan lies in the center of origin and natural habitat of wild apples.
Academic Editors: Nicoletta Pucci,
The fruit forests of Kazakhstan contain plant material important for botany, geography, and
Stefania Loreti and Scala Valeria
genetics. Wild apples and their hybrids were historically utilized as a source of resistance
Received: 18 August 2023 to biotic and abiotic stress in domestic apple breeding [1,2].
Revised: 21 September 2023 Malus niedzwetzkyana Dieck ex Koehne is an endemic wild apple species featured in
Accepted: 21 September 2023 the Red Book of Kazakhstan and the International Red List of Endangered Species [3,4].
Published: 22 September 2023 It naturally grows in Kazakhstan (Karatau and Zailijskei Alatau), Kyrgyzstan (Jalal-Abad
region), and western China (Xinjiang region) [1,2,5,6].
Malus niedzwetzkyana is integral to the mixed fruit and nut forests in Central Asia.
This species is an important genetic resource for apple breeding and developing new
Copyright: © 2023 by the authors.
advantageous cultivars [7,8]. The particular value of M. niedzwetzkyana hinges on its genetic
Licensee MDPI, Basel, Switzerland.
This article is an open access article
predisposition to fire blight resistance [9]. It is also an exquisite ornamental plant due to the
distributed under the terms and
pink color of its flowers, fruits, leaves, and trunk, which is attributed to anthocyanins. These
conditions of the Creative Commons
compounds have antioxidative, anti-carcinogenic, and anti-inflammatory properties [10].
Attribution (CC BY) license (https:// Improving domestic apple productivity and conserving global biodiversity rely on
creativecommons.org/licenses/by/ resistant genotypes whose defense mechanisms have adapted to rapidly evolving local
4.0/). pathogens. Higher plants evolve much slower than their parasites, especially perennial

Horticulturae 2023, 9, 1066. https://doi.org/10.3390/horticulturae9101066 https://www.mdpi.com/journal/horticulturae

Horticulturae 2023, 9, 1066 2 of 12

tree plants. Therefore, new sources of resistance for marker-assisted selection (MAS) are
ceaselessly sought after. One of the most widespread apple diseases is fire blight, caused
by a Gram-negative bacterium Erwinia amylovora. The infection enters through flowers or
vegetative parts and leads to leaf and shoot necrosis, ooze droplets, wilting, and trichome
development [11,12]. Three major QTLs associated with resistance to the pathogen have
been identified: FBF7 on LG7 (linkage group 7) of cultivar “Fiesta”, FB_E on LG12 of
“Everest”, and FB_MR5 on LG3 of Malus × robusta 5, which are considered suitable for
MAS due to their stability [13–17]. Among them, QTL FBF7 accounted for up to 46%
of the observed phenotypic variation in fire blight resistance, and resistant variants are
characterized by two SCAR markers, AE10-375 and GE80-19 [14]. This QTL has previously
been identified among the local apple lineages, and the positive cultivars for both markers
exhibited moderate phenotypic resistance in the field [18].
This work evaluates the resistance of M. niedzwetzkyana genotypes growing in the
Republic of Kazakhstan to fire blight both phenotypically via an inoculation test and
molecularly using SCAR markers linked to one of the major resistance QTL (FBF7). Each
sample was genotyped and introduced into a culture medium to investigate its potential
for further selection and to preserve its biodiversity.

2. Materials and Methods

2.1. Plant Material
A total of eight M. niedzwetzkyana trees were identified according to the botanical
description and had leaf samples collected: five samples from four populations across
Astana and three specimens from a wild population inside the Tscherkesay Canyon (near
Tekeli, Kazakhstan).
A collection of endangered M. niedzwetzkyana was conducted following the decision
of the Council of the Eurasian Economic Commission of 26 January 2018 No. 15. “On the
approval of Rules of proper practice of cultivation, collection, processing and storage of
initial raw materials of plant origin”. The plant material was collected from the Djungar
Alatau mountain range (Tscherkesay) by the authors acting with permission from the
Forestry and Wildlife Committee and deposited at the Main Botanical Garden (AA) under
ID No. 3023/20-3028/20. The specimens collected in the Astana Botanical Garden (NUR)
were deposited under ID No. 536/20-552/20.

2.2. SSR Profiling

DNA was extracted from leaves using a modified CTAB protocol [19]. The quantity
and quality of extracted DNA were analyzed using a spectrophotometer (NanoDrop1000,
Thermo Scientific, Waltham, MA, USA).
Twelve SSR markers were used, namely GD12, GD147, CH01h10, CH01h01, CH04c07,
Hi02c07, CH01f03b, CH02d08, CH02c11, CH04e05, CH01f02, and CH02c09 [20–23]. These
markers are widely applied in apple genotyping and are suggested by the European
Cooperative Programme for Plant Genetic Resources (ECPGR) [24]. CH04e05, CH02c11,
CH02c09, CH02d08, CH04c07, CH01h01, Hi02c07, and CH01h10 are highlighted as priority
group 1 of the ECPGR marker set, whereas CH01f02, CH01f03b, GD12, and GD147 belong
to priority group 2. The primer sequences, fluorescent dye, and multiplex group are
described in Table A1 (Appendix A).
Amplification of each SSR marker was conducted in a 15 µL reaction mix containing a
1× DreamTaq buffer (Thermo Scientific, Waltham, MA, USA), 0.2 мM dNTPs, 0.2 мM of
each of the respective primers for each SSR marker, and 1 unit of DreamTaq polymerase
(Thermo Scientific, Waltham, MA, USA). The amplification program for each multiplex
group was as follows: 94 ◦ C for 3 min, followed by 10 cycles of denaturation at 94 ◦ C
for 30 s, annealing for 90 s at 60 ◦ C with a 1 ◦ C decrease in temperature each cycle, and
elongation at 72 ◦ C for 60 s. The second step of 30 cycles was denaturation at 94 ◦ C for
30 s, followed by annealing at 50 ◦ C for 90 s and further elongation at 72 ◦ C for 60 s. Final
elongation continued for 10 min at 72 ◦ C. PCR was performed using a Mastercycler Pro S
Horticulturae 2023, 9, 1066 3 of 12

thermocycler (Eppendorf, Hamburg, Germany). Fragment analysis was conducted using

Applied Biosystems 3500 (Thermo Scientific, Waltham, MA, USA). SSR genotyping data
were analyzed using GeneMapper™ Software 6 (Thermo Scientific, Waltham, MA, USA).
The resulting genetic profiles of eight genotypes were analyzed in GenAlEx 6.5 [25].

2.3. In Vitro Cultivation of M. niedzwetzkyana Genotypes

All collected M. niedzwetzkyana genotypes were introduced into a culture medium
according to the previously developed protocol for microclonal propagation in the Lab-
oratory of Plant Biotechnology and Selection at the National Center of Biotechnology,
Astana [26]. Axillary buds from one-year-old shoots were used as source material. For
each genotype, we used no less than 20 buds from different branches. The buds were
subjected to aseptic treatment for 4 min using a 12% hydrogen peroxide solution, result-
ing in up to 80% viable sterile explants for introduction into an in vitro culture. The M.
niedzwetzkyana explants were cultivated on the QL medium with the addition of 0.5 mg/L
6-BAP (6-benzylaminopurine) and 1.5 mg/L kinetin; the main shoots formed on day 50.
For the multiplication of additional shoots, the explants were cultivated on a QL medium
with 0.5 mg/L 6-BAP and 0.01 mg/L IBA (indole butyric acid) for 50 days. The shoots
were rooted in a QL medium (half concentration) with the addition of 10 g/L sucrose and
1.5 mg/L IBA. Overall, the number of regenerants varied from 20 to 40 depending on the
genotype.

2.4. Screening of a Local E. amylovora Strain

The E. amylovora isolate was obtained from a symptomatic “Golden Delicious” tree
from a commercial garden growing cv. Idared, Golden Delicious, and Gala in the Almaty
region, where trees of all the grown cultivars were affected by fire blight. The visible
symptoms included scorching leaf necrosis, ooze droplets, and blackening of shoots. The
culture was plated on a PDA (potato dextrose agar) medium. After 48 h, colonies exhibiting
growth patterns similar to E. amylovora were plated onto the selective NSA (nutrient sucrose
agar) medium [27,28]. Colonies that were whitish, circular, domed, smooth, and mucoid
after 48 h were tested using a peer-reviewed qPCR protocol [29]. Positive samples were
carried into the next steps.
To confirm the results of the morphology test and discover related strains, we attempted
a whole genome sequencing of the bacterium. A log-phase cell suspension was obtained via
2-day cultivation at 28 ◦ C in LB media and was collected via centrifugation at 5000× g for
2 min, followed by resuspension of the pellet with 2 mL sterilized distilled water [30]. DNA
was extracted using the innuPREP Bacteria DNA Kit (Analytik Jena, Jena, Germany).
Library preparation was performed according to the “Ligation sequencing gDNA—
whole genome amplification (SQK-LSK109)” legacy protocol from the Nanopore Com-
munity tab. DNA libraries were sequenced on a FLO-MIN106D flow cell using MinION
(Oxford Nanopore Technologies, Oxford, UK). The sequenced reads were subsequently run
through “Fastq WIMP” analysis hosted on the Epi2ME platform (Metrichor Ltd., Oxford,
UK), confirming the DNA belonging to E. amylovora. Additionally, reads were searched
against the NCBI nt database via Blast+ to determine the most closely related strains [31].

2.5. E. amylovora Inoculation Test

Syringes bearing cell suspension from the previous step were inserted into locally
grown pear fruits in up to 10 different surface patches. Five days later, the pathogenicity
of the strain was phenotypically evaluated. The ooze accumulated inside the lesions on
the infected pears from the above pathogenicity test was used as an inoculant for the
apple regenerants.
Inoculation was conducted through a series of stem incisions via a sterile scalpel
dipped into the ooze; the location of the incision was wrapped in paraffin film to create
an anoxygenic environment for bacteria growth. Eight 5-month-old apple tree saplings
were inoculated on 30 June 2021. Phenotypic fire blight responses of the regenerants were
Horticulturae 2023, 9, 1066 4 of 12

evaluated under glasshouse conditions and scored using a categorical scale of severity of
infection (0 to 5) [32,33]:
• 0, no reaction;
• 1, slight trichome development, necrosis of less than 5% of leaf matter;
• 2, moderate trichome development, necrosis of up to 10% of leaf matter localized
around the lesion;
• 3, moderate to severe trichome development, necrosis of up to 25% of leaf matter,
necrosis progression rootward, severe wilting in adjacent leaves;
• 4, the occurrence of ooze droplets, severe witling of leaves away from the lesion,
necrosis both rootward and upward, and up to 35% plant matter affected;
• 5, necrosis affecting up to 50% of leaves, several instances of ooze droplets.

2.6. FBF7 QTL Identification

We used two SCAR markers linked with FBF7 QTL, AE10-375, and GE80-19 [14] (Table 1).
For each DNA sample, 60 ng DNA was amplified in a 25 µL reaction mix containing a 1× Taq
buffer (750 мM Tris HCl, pH 8.8, 200 мM (NH4 )2 SO4 , 0.1% Tween 20), 2.5 мM MgCl2 , 0.2 мM
dNTPs, 0.2 мM of each of the respective primers, and 1 unit Taq of polymerase (Thermo
Scientific, Waltham, MA, USA). The PCR cycling conditions for every marker are described in
Table 1. The amplification results were analyzed on 1.5% agarose gel.

Table 1. Marker, primer (F, forward and R, reverse) sequences, and PCR cycling for the amplification
with SCAR markers.

Gen, Locus Marker Primer Sequence (50 –30 ) PCR Cycling

F7 QTL AE10-375 F-CTGAAGCGCACGTTCTCC 1× 95 ◦ C—3 min, 35× (95 ◦ C—40 s; 60 ◦ C—40 s;
R-CTGAAGCGCATCATTTCTGATAG 72 ◦ C—60 s), 1× 72 ◦ C—10 min.
F7 QTL GE80-19 F-TTGAGACCGATTTTCGTGTG 1× 95 ◦ C—3 min, 35× (95 ◦ C—40 s; 60 ◦ C—40 s;
R-TCTCTCCCAGAGCTTCATTGT 72 ◦ C—60 s), 1× 720 ◦ C—10 min

3. Results
3.1. SSR Profiling
To confirm the genetic diversity of M. niedzwetzkyana genotypes, each plant was
profiled using SSR markers (Figure 1). It revealed five different amplicons for markers
CH01f02 and GD12 and three amplicons for markers CH01h01, CH04c07, CH02d08, and
CH04e05. Four amplicons were identified for each of the remaining markers. Of particular
interest2023,
Horticulturae is 9,the
1066 3-W genotype, which differs significantly in its genetic profile: it amplified
5 of 12

unique alleles in eight SSR markers.

Figure 1. SSR profiling of in vitro propagated genotypes with resistance to fire blight. Fragment
Figure 1. SSR profilingsizes
of amplified
in vitroforpropagated
each marker aregenotypes
non-homogenous,with resistance
though to fire
pa erns emerge blight.
among Fragment
samples with
sizes amplified for eachsimilar
markerorigins.
areThe color of each fragment though
non-homogenous, corresponds to its length:
patterns the longer
emerge the fragment,
among samplesthe with
darker its shade.
similar origins. The color of each fragment corresponds to its length: the longer the fragment, the
darker its shade.
Horticulturae 2023, 9, 1066 5 of 12

Figure 1. SSR profiling of in vitro propagated genotypes with resistance to fire blight. Fragment
sizes amplified for each marker are non-homogenous, though pa erns emerge among samples with
M.origins.
similar niedzwetzkyana
The colorgenotypes were introduced
of each fragment correspondsinto a culture
to its medium,
length: the longer propagated,
the fragment,and
the
darker its to
adapted shade.
ex vitro conditions for further screening and preservation (Figure 2).

Figure 2. In vitro cultivation of M. niedzwetzkyana genotypes for conservation.

Figure 2. In vitro cultivation of M. niedzwe kyana genotypes for conservation.
3.2. Screening of a Local E. amylovora Strain
Horticulturae 2023, 9, 1066 3.2. Screening of a Local E. amylovora Strain 6 of 12
Resistance to fire blight in apples was reported to be strain-specific; moreover, arti-
ficialResistance
inoculationtooffire blight in
different apples was
accessions, reported
even those to be strain-specific;
belonging moreover,
to one species, can leadartifi-
to
cial inoculation of different accessions, even those belonging to one
symptoms of varying severity [16,34]. A local strain of E. amylovora was chosen for thespecies, can lead to
symptoms
To of
confirm varying
the severity
results of [16,34].
the first A local
positive strain of
screening E. amylovora
and was
investigate
experiment as posing the most consistent threat to apple trees within the region, against chosen
the for
bacterium’sthe
experiment
geneticwild
which as posing
makeup,
forests we
havethe most consistent
a evolved.
empted threat to whole-genome
Nanopore-based apple trees within the region,Over
sequencing. against
the
which
course wild forests
of sequencing,
E. amylovora have evolved.
33,285,592
was isolated frombases were generated.
a cultivated apple treeCoverage
exhibiting ofleaf
the necrosis
chromosome was
and ooze
E. amylovora
approximately
production was
8.09
and then andisolated
platedof on from
thePDA
plasmida cultivated
and bothapple
19.8; mediums.
NSA wereIttree exhibiting
calculated
demonstrated leaf necrosis
via Samtools
steady [35].and
growth Ac-
on
ooze
the production
cording
PDA to Blast+,and
medium the then
most plated
characterized by on
frequent PDAwhitish
matches
circular andforNSA mediums.
contigs were
colonies theItFN434113.1
demonstrated
coalescing togetherCFBP steady
(Figure1430
3).
growth
strain
The on the
[36],
colonies on PDA
FN666575.1
the NSA medium
ATCC
medium characterized
49,946
werestrain by circular
(chromosome),
less prone whitish
andand
to coalescence colonies
HF560649.1
appearedcoalescing
MR1 to-
strain
as whitish,
gether (Figure
(plasmid).
circular, domed,3).smooth,
The colonies on thebeads
and mucoid NSA following
medium were less prone
the EPPO to coalescence
Standard [27]. and
appeared as whitish, circular, domed, smooth, and mucoid beads following the EPPO
Standard [27].

Figure3.3.Colony
Figure Colony morphology
morphology on
on NSA
NSA medium
medium (left)
(left) and
andPDA
PDA medium
medium (right)
(right)after
afterincubation
incubation at
at
28◦°C
28 for 48
C for 48 h.
h. Off-white,
Off-white, bead-like
bead-like formations
formations on
on NSA
NSA medium
medium are
arecharacteristic
characteristicof
ofE.
E.amylovora.
amylovora.

3.3. E. amylovora Inoculation Test

Because ooze exuded from infected plant tissue is the primary method of dispersal
for E. amylovora in vivo [37], it was chosen as the inoculant. To obtain the ooze, five pear
fruits of a local susceptible cultivar, “Lesnaya krasavitsa”, were injected with the same E.
Horticulturae 2023, 9, 1066 6 of 12

To confirm the results of the first positive screening and investigate the bacterium’s
genetic makeup, we attempted Nanopore-based whole-genome sequencing. Over the
course of sequencing, 33,285,592 bases were generated. Coverage of the chromosome
was approximately 8.09 and of the plasmid 19.8; both were calculated via Samtools [35].
According to Blast+, the most frequent matches for contigs were the FN434113.1 CFBP 1430
strain [36], FN666575.1 ATCC 49,946 strain (chromosome), and HF560649.1 MR1 strain
Figure 3. Colony morphology on NSA medium (left) and PDA medium (right) after incubation at
(plasmid).
28 °C for 48 h. Off-white, bead-like formations on NSA medium are characteristic of E. amylovora.

3.3.
3.3. E.
E. amylovora InoculationTest
amylovora Inoculation Test
Because oozeexuded
Because ooze exudedfrom
frominfected
infected plant
plant tissue
tissue is the
is the primary
primary method
method of dispersal
of dispersal
for E. amylovora in vivo [37], it was chosen as the inoculant. To obtain the ooze,
for E. amylovora in vivo [37], it was chosen as the inoculant. To obtain the ooze, five pear five pear
fruits
fruits of a local susceptible cultivar, “Lesnaya krasavitsa”, were injected with the same E.same
of a local susceptible cultivar, “Lesnaya krasavitsa”, were injected with the
E. amylovora
amylovora cellcell suspension,
suspension, with
with one oneleft
pear pear lefta negative
out as out as acontrol
negative
(LBcontrol
medium) (LB medium)
(Figure
(Figure 4). Six
4). Six days afterdays after inoculation,
inoculation, pointsstarted
points of injection of injection started
excreting excreting
opaque opaque
ooze, which wasooze,
which wasfor
then used then
theused for the of
inoculation inoculation of apple plantlets.
apple plantlets.

Figure 4.
Figure 4. Local
Local pears
pears66days
daysafter
afterinoculation with
inoculation withananE. E.
amylovora cellcell
amylovora suspension. “-” indicates
suspension. “-” indicates
negative control (LB medium), “+”—replications of inoculation with the same strain. Whitish ooze
negative control (LB medium), “+”—replications of inoculation with the same strain. Whitish
droplets forming inside the lesions are characteristic of fire blight infection and were used for inoc-
ooze droplets
ulation formingkyana
of M. niedzwe inside the lesions are characteristic of fire blight infection and were used for
genotypes.
inoculation of M. niedzwetzkyana genotypes.
Results were recorded and photographed after 5 and 39 days after inoculation (dai),
Results
which can bewere
seenrecorded and
in Table 2. photographed
All five apple after 5 and 39
genotypes days after
sampled frominoculation
a cultured(dai),
which can be seen in Table 2. All five apple genotypes sampled from a cultured environment
exhibited mild to severe symptoms of fire blight infection, while all three wild genotypes
appeared to resist the pathogen. This phenomenon is exemplified by genotypes 12 and
2-W (Figure 5). The inoculated regenerant of genotype 2-W bears no signs of infection
aside from slight trichome development, and even young leaves present at the moment of
inoculation were able to mature over the course of observation. On the contrary, genotype
12 was characterized by a classic fire blight infection profile, complete with the occurrence
of ooze and wilting of five top leaves on the shoot.

Table 2. Symptoms of fire blight detected in genotypes bearing resistance markers.

Sample Fire Blight Marker Symptoms Infection Severity

2 AE10-375 Severe necrosis in five adjacent leaves 3
3 AE10-375 Small area of leaf necrosis, moderate trichome development 2
Necrosis of 30% of shoot, severe wilting, moderate trichome
9 AE10-375 4
development
Necrosis of 7% of adjacent leaf, wilting of the shoot
10 AE10-375 2
upwards from the lesion, limited effect rootward
Ooze production, severe wilting incl. downwards from the
12 AE10-375 4
lesion, trichome development
1-W AE10-375 Necrosis of less than 1% of leaves 1
2-W AE10-375 Slight trichome development 1
3-W GE80-19 Slight trichome development 1
 environment exhibited mild to severe symptoms of fire blight infection, while all three
wild genotypes appeared to resist the pathogen. This phenomenon is exemplified by gen-
otypes 12 and 2-W (Figure 5). The inoculated regenerant of genotype 2-W bears no signs
of infection aside from slight trichome development, and even young leaves present at the
moment of inoculation were able to mature over the course of observation. On the con-
Horticulturae 2023, 9, 1066 7 of 12
trary, genotype 12 was characterized by a classic fire blight infection profile, complete
with the occurrence of ooze and wilting of five top leaves on the shoot.

Figure5.5. Phenotypic
Figure Phenotypic response
response to
to fire
fire blight
blightatat0,0,5,5,and
and3939
days after
days inoculation
after inoculation(dai) with
(dai) E. amylo-
with
E.vora of all collected
amylovora M. niedzwe
of all collected kyana genotypes.
M. niedzwetzkyana The representative
genotypes. resistantresistant
The representative genotype 2-W displays
genotype
no visible
2-W displayssigns of infection
no visible at infection
signs of day 5. Slight trichome
at day 5. Slightdevelopment around the
trichome development lesionthe
around onlesion
day 39. The
on day 39. The representative susceptible genotype 12 has an open lesion-producing ooze by day 5.
Note the white trichome visible on the shoot around the lesion. By day 39, six of the top leaves had
dried out and wilted.
 2-W AE10-375 Slight trichome development 1
3-W GE80-19 Slight trichome development 1

3.4. FBF7 QTL Identification

Horticulturae 2023, 9, 1066 To search for the source of the resistance, the plants were tested for 8 ofthe
12 prese

FBF7 QTL using SCAR markers AE10-375 and GE80-19 [13,14,38]. This QTL has
ously been identified among the local apple lineages and the cultivars, a few of
3.4. FBF7 QTL Identification
originated from wild apple species [18].
To search for the source of the resistance, the plants were tested for the presence of
FBF7TheQTLresistant
using SCAR alleles
markersof AE10-375
markersand AE10-375 and GE80-19
GE80-19 [13,14,38]. arehas
This QTL characterized
previously by a
cons of 375 bp and 397 bp, respectively [13,14]. Seven genotypes tested positive
been identified among the local apple lineages and the cultivars, a few of which originated for
375 (Table
from 2), and
wild apple only[18].
species the 3-W genotype from the wild forest in the Tscherkesay C
The resistant alleles
amplified GE80-19 (Figure of markers AE10-375
6). The GE80-19 are characterized
andamplifying
plants both markers by amplicons
had been prev
of 375 bp and 397 bp, respectively [13,14]. Seven genotypes tested positive for AE10-375
characterized with higher resistance to the pathogen compared with the genotypes
(Table 2), and only the 3-W genotype from the wild forest in the Tscherkesay Canyon
ing only the
amplified resistance
GE80-19 (Figureallele forplants
6). The one of the twoboth
amplifying markers
markers[14].
had been previously
characterized with higher resistance to the pathogen compared with the genotypes bearing
only the resistance allele for one of the two markers [14].

Figure 6. Analysis of M. niedzwetzkyana genotypes using SCAR markers AE10-375 and GE80-19.
For fire6.blight
Figure resistance,
Analysis of M.theniedzwe
key alleles are 397
kyana bp (GE80-19)
genotypes andSCAR
using 375 bp (AE10-375) long. See also
markers AE10-375 and GE80-
Supplementary File S1 for the original full-size images.
fire blight resistance, the key alleles are 397 bp (GE80-19) and 375 bp (AE10-375) long. See als
plementary File S1 for the original full-size images.
4. Discussion
Wild plants are an important source of genetic material for marker-assisted selection
4. Discussion
(MAS), aiming to improve crops’ resistance to pathogens [39]. One of the ways to prevent in-
fection is the introgression of strong resistance factors originating from wild species [40]. For
Wild plants are an important source of genetic material for marker-assisted sel
example, involving wild apple trees in the breeding of Golden Delicious made it possible
(MAS), aiming
to intensify to improve
the world’s crops’ resistance
apple production more thantotwofold
pathogens
over 25[39].
yearsOne
[41].of the ways to p
Therefore,
infection is the
conservation andintrogression
rational use ofof strong
wild plantsresistance
are integralfactors originating
for germplasm from wild
biodiversity and specie
thriving agroindustry [42].
For example, involving wild apple trees in the breeding of Golden Delicious m
Wild apple, apricot, and walnut forests are indigenous to Central Asia [41]. The
species forming these communities are classified as endangered and require protection [3].
However, up to 70% of apple forests have suffered habitat degradation over the past
40 years. Wild apple forest recultivation is the main priority of the country’s wild flora
conservation program, which requires fundamental research of their genomics. To date,
no wide-scale studies of M. niedzwetzkyana genomics have been conducted, neither in
Kazakhstan nor elsewhere.
In the present study, four out of eight plant genotypes showed moderate to severe
susceptibility to fire blight infection, while the three wild genotypes 1-W, 2-W, and 3-W
proved to be resistant. Phenotypic expression was evaluated based on an inoculation test,
which revealed that the presence of any of the two markers of FBF7 does not correlate
with a tangible resistance profile. The fact that all the domesticated genotypes displayed
varied levels of susceptibility, while the wild population was characterized by a lack of
symptoms, suggests that the wild M. niedzwetzkyana population from the Tscherkesay
Canyon possesses resistance traits not covered by FBF7. Indeed, major QTLs associated
with resistance to fire blight originating from wild species have been described in linkage
groups LG3 [16,34], LG10 [43,44], and LG12 [15]. However, only FBF7 has been found
among the local Kazakhstani apple varieties, many of which are derived from various
wild apples growing in the vicinity, such as M. sieversii and M. niedzwetzkyana [18]. On the
Horticulturae 2023, 9, 1066 9 of 12

contrary, when the same cultivars were tested for FB_E and FB_MR5 via SNP-genotyping,
all markers appeared monomorphic [45].
In the case of 3-W, it tested positive for the GE80-19 marker and lacked AE10-375, which
others shared. We could not detect any measurable difference in its infection response from
the other wild regenerants. However, the origin of GE80-19-SCAR and AE10-375-SCAR as
markers for commercial apples [13] may explain the reduced specificity when applied to a
wild Malus species.
SSR analysis confirmed the genetic individuality of the specimens bearing fire blight
resistance markers. Previously, three genotypes of M. niedzwetzkyana from the Krutoye tract
(Kazakhstan) were fingerprinted using 16 microsatellite markers and clustered together
with M. sieversii [46]. Previously, investigations of fire blight resistance in M. sieversii
revealed accessions with a high degree of resistance, although the overall percentage of
resistant individuals was low [47,48]. In this study, for the first time, three M. niedzwetzkyana
specimens proved the resistance phenotypically in the inoculation test.
Tscherkesay Canyon genotypes represent a prospective genetic pool for the breeding
of new cultivars bearing loci associated with resistance to fire blight. The results of the work
would help to preserve the important genetic material of endangered M. niedzwetzkyana
species and would assist its further use in apple breeding, conservation, and revival of wild
apple populations.

5. Conclusions
In the present work, the fire blight resistance of the eight genotypes of M. niedzwet-
zkyana from different populations was investigated using genetic SCAR markers and an
inoculation test. While all of the specimens bore either AE10-375 or GE80-19, their phe-
notypic reaction to E. amylovora differed dramatically, suggesting a new avenue should
be pursued in search of the origin of its resistance, such as a cross with a known sus-
ceptible background, generating a population to investigate the genetic control of the
resistant accessions.

Supplementary Materials: The following supporting information can be downloaded at https:

//www.mdpi.com/article/10.3390/horticulturae9101066/s1. Supplementary File S1: The original
image of the electrophoretic gel featured in Figure 6.
Author Contributions: Conceptualization: D.G.; methodology—A.N. and D.D.; formal analysis—
A.K. (Anastasiya Kapytina); investigation: G.N. and A.T.; writing—original draft preparation: A.N.
and M.K. (Mariya Kolchenko); writing—review and editing: M.K. (Mariya Kolchenko) and D.G.;
resources: A.K. (Almagul Kakimzhanova); software: A.P.; data curation: M.K. (Marina Knusnitdi-
nova); funding acquisition: D.G. All authors have read and agreed to the published version of the
manuscript.
Funding: The work was funded by the Ministry of Science and Higher Education of the Republic of
Kazakhstan, AP14871004 “Investigation of phytopathological and botanical aspects of wild apple
populations growing in the Northern Tien Shan” 2022–2024 and 0.0809 “Creation of a biobank of
microorganisms, cell cultures, genomic, and genetically engineered materials to preserve biodiversity
and provide a resource base for biotechnology”.
Data Availability Statement: The dataset supporting the conclusions of this article is available in the
Open Science Foundation repository: https://osf.io/dz5gf (accessed on 15 September 2023).
Acknowledgments: We are grateful to the Astana Botanical Garden and the Forestry and Wildlife
Committee of the Ministry of Ecology, Geology, and Natural Resources of the Republic of Kazakhstan
for supporting the collection of plant materials for this study.
Conflicts of Interest: The authors declare no conflict of interest.
Horticulturae 2023, 9, 1066 10 of 12

Appendix A

Table A1. Sequences of primers (F—forward, R—reverse), fluorescent dye type, linkage group (LG),
and multiplex group numbers for SSR genotyping [24].

Multiplex
Marker Primer Sequence (50 –30 ) Fluorescent Dye Linkage Group
Group
F-TTGAGGTGTTTCTCCCATTGGA
GD12 TAMRA 3 III
R-CTAACGAAGCCGCCATTTCTTT
F-TCCCGCCATTTCTCTGC
GD147 ATTO565 13 III
R-GTTTAAACCGCTGCTGCTGAAC
F-TGCAAAGATAGGTAGATATATGCCA
CH01h10 HEX 8 II
R-AGGAGGGATTGTTTGTGCAC
F-GAAAGACTTGCAGTGGGAGC
CH01h01 TAMRA 17 II
R-GGAGTGGGTTTGAGAAGGTT
F-GGCCTTCCATGTCTCAGAAG
CH04c07 6-FAM 14 II
R-CCTCATGCCCTCCACTAACA
F-AGAGCTACGGGGATCCAAAT
Hi02c07 ATTO565 1 II
R-GTTTAAGCATCCCGATTGAAAGG
F-GAGAAGCAAATGCAAAAC CC
CH01f03b HEX 9 III
R-CTCCCCGGCTCCTATTCTAC
F-TCCAAAATGGCGTACCTCTC
CH02d08 HEX 11 I
R-GCAGACACTCACTCACTATCTCTC
F-TGAAGGCAATCACTCTGTGC
CH02c11 TAMRA 10 I
R-TTCCGAGAATCCTCTTCGAC
F-AGGCTAACAGAAATGTGGTTTG
CH04e05 6-FAM 7 I
R-ATGGCTCCTATTGCCATCAT
F-ACCACATTAGAGCAGTTGAGG
CH01f02 6-FAM 12 III
R-CTGGTTTGTTTTCCTCCAGC
F-TTATGTACCAACTTTGCTAACCTC
CH02c09 ATTO565 15 I
R-AGAAGCAGCAGAGGAGGATG

References
1. Janick, J. Wild Apple and Fruit Trees of Central Asia. In Horticultural Reviews; John Wiley & Sons: Hoboken, NJ, USA, 2003;
Volume 29, p. 416. ISBN 978-0-47065-086-8.
2. Wilson, B.; Mills, M.; Kulikov, M.; Clubbe, C. The Future of Walnut–Fruit Forests in Kyrgyzstan and the Status of the Iconic
Endangered Apple Malus niedzwetzkyana. Oryx 2019, 53, 415–423. [CrossRef]
3. Eastwood, A.; Lazkov, G.; Newton, A.C. The Red List of Trees of Central Asia; Fauna and Flora International: Cambridge, UK, 2009;
ISBN 978-1-90370-327-4.
4. The Red Data Book of the Republic of Kazakhstan, 4th ed.; DPS: Almaty, Kazakhstan, 2010; Volume 2, ISBN 9-96-532738-6.
5. Ji, X.-H.; Wang, Y.-T.; Zhang, R.; Wu, S.-J.; An, M.-M.; Li, M.; Wang, C.-Z.; Chen, X.-L.; Zhang, Y.-M.; Chen, X.-S. Effect of Auxin,
Cytokinin and Nitrogen on Anthocyanin Biosynthesis in Callus Cultures of Red-Fleshed Apple (Malus sieversii f. niedzwetzkyana).
Plant Cell Tiss. Organ. Cult. 2015, 120, 325–337. [CrossRef]
6. Yan, G.; Long, H.; Song, W.; Chen, R. Genetic Polymorphism of Malus sieversii Populations in Xinjiang, China. Genet. Resour. Crop
Evol. 2008, 55, 171–181. [CrossRef]
7. Harris, S.A.; Robinson, J.P.; Juniper, B.E. Genetic Clues to the Origin of the Apple. Trends Genet. 2002, 18, 426–430. [CrossRef]
8. Omasheva, M.E.; Chekalin, S.V.; Galiakparov, N.N. Evaluation of Molecular Genetic Diversity of Wild Apple Malus sieversii
Populations from Zailiysky Alatau by Microsatellite Markers. Russ. J. Genet. 2015, 51, 647–652. [CrossRef]
9. Yang, M.; Che, S.; Zhang, Y.; Song, W.; Yan, G.; Yu, W. Malus niedzwetzkyana (Dieck) Langenf Transcriptome Comparison and
Phylogenetic Analysis with Malus sieversii (Ledeb) Roem. Genet. Resour. Crop Evol. 2020, 67, 313–323. [CrossRef]
10. Wang, N.; Jiang, S.; Zhang, Z.; Fang, H.; Xu, H.; Wang, Y.; Chen, X. Malus sieversii: The Origin, Flavonoid Synthesis Mechanism,
and Breeding of Red-Skinned and Red-Fleshed Apples. Hortic. Res. 2018, 5, 70. [CrossRef]
11. Flachowsky, H.; Szankowski, I.; Fischer, T.C.; Richter, K.; Peil, A.; Höfer, M.; Dörschel, C.; Schmoock, S.; Gau, A.E.; Halbwirth, H.;
et al. Transgenic Apple Plants Overexpressing the Lc Gene of Maize Show an Altered Growth Habit and Increased Resistance to
Apple Scab and Fire Blight. Planta 2010, 231, 623–635. [CrossRef]
12. Oh, C.-S.; Beer, S.V. Molecular Genetics of Erwinia amylovora Involved in the Development of Fire Blight. FEMS Microbiol. Lett.
2005, 253, 185–192. [CrossRef]
Horticulturae 2023, 9, 1066 11 of 12

13. Calenge, F.; Drouet, D.; Denancé, C.; Van de Weg, W.E.; Brisset, M.-N.; Paulin, J.-P.; Durel, C.-E. Identification of a Major QTL
Together with Several Minor Additive or Epistatic QTLs for Resistance to Fire Blight in Apple in Two Related Progenies. Theor.
Appl. Genet. 2005, 111, 128–135. [CrossRef]
14. Khan, M.A.; Durel, C.-E.; Duffy, B.; Drouet, D.; Kellerhals, M.; Gessler, C.; Patocchi, A. Development of Molecular Markers Linked
to the ‘Fiesta’ Linkage Group 7 Major QTL for Fire Blight Resistance and Their Application for Marker-Assisted Selection. Genome
2007, 50, 568–577. [CrossRef] [PubMed]
15. Durel, C.-E.; Denancé, C.; Brisset, M.-N. Two Distinct Major QTL for Resistance to Fire Blight Co-Localize on Linkage Group 12 in
Apple Genotypes “Evereste” and Malus Floribunda Clone 821. Genome 2009, 52, 139–147. [CrossRef] [PubMed]
16. Peil, A.; Emeriewen, O.F.; Khan, A.; Kostick, S.; Malnoy, M. Status of Fire Blight Resistance Breeding in Malus. J. Plant Pathol.
2021, 103, 3–12. [CrossRef]
17. Papp, D.; Békefi, Z.; Balotai, B.; Tóth, M. Identification of Marker Alleles Linked to Fire Blight Resistance QTLs in Apple
Genotypes. Plant Breed. 2015, 134, 345–349. [CrossRef]
18. Omasheva, M.Y.; Pozharskiy, A.S.; Maulenbay, A.D.; Ryabushkina, N.A.; Galiakparov, N.N. SSR Genotyping of Kazakhstani
Apple Varieties: Identification of Alleles Associated with Resistance to Highly Destructive Pathogens. Eurasian J. Appl. Biotechnol.
2016, 14, 1–16. [CrossRef]
19. Amraee, L.; Rahmani, F. Modified CTAB Protocol for RNA Extraction from Lemon Balm (Melissa officinalis L.). Acta Agric. Slov.
2020, 115, 53–57. [CrossRef]
20. Hemmat, M.; Weeden, N.F.; Brown, S.K. Mapping and Evaluation of Malus × domestica Microsatellites in Apple and Pear. J. Am.
Soc. Hortic. Sci. 2003, 128, 515–520. [CrossRef]
21. Hokanson, S.C.; Szewc-McFadden, A.K.; Lamboy, W.F.; McFerson, J.R. Microsatellite (SSR) Markers Reveal Genetic Identities,
Genetic Diversity and Relationships in a Malus × domestica Borkh. Core Subset Collection. Theor. Appl. Genet. 1998, 97, 671–683.
[CrossRef]
22. Liebhard, R.; Gianfranceschi, L.; Koller, B.; Ryder, C.D.; Tarchini, R.; Van De Weg, E.; Gessler, C. Development and Characterisation
of 140 New Microsatellites in Apple (Malus × domestica Borkh.). Mol. Breed. 2002, 10, 217–241. [CrossRef]
23. Richards, C.M.; Volk, G.M.; Reilley, A.A.; Henk, A.D.; Lockwood, D.R.; Reeves, P.A.; Forsline, P.L. Genetic Diversity and
Population Structure in Malus sieversii, a Wild Progenitor Species of Domesticated Apple. Tree Genet. Genomes 2009, 5, 339–347.
[CrossRef]
24. Fernandez-Fernandez, F. Common Set of ECPGR SSR Markers for Malus Characterization; Bioversity International: Weggis, Switzer-
land, 2013.
25. Peakall, R.; Smouse, P.E. GenAlEx 6.5: Genetic Analysis in Excel. Population Genetic Software for Teaching and Research—An
Update. Bioinformatics 2012, 28, 2537–2539. [CrossRef] [PubMed]
26. Nurtaza, A.; Magzumova, G.; Yessimseitova, A.; Karimova, V.; Shevtsov, A.; Silayev, D.; Lutsay, V.; Ramankulov, Y.;
Kakimzhanova, A. Micropropagation of the Endangered Species Malus niedzwetzkyana for Conservation Biodiversity in
Kazakhstan. Vitr. Cell. Dev. Biol.-Plant 2021, 57, 965–976. [CrossRef]
27. PM 7/20 (3) Erwinia amylovora. EPPO Bull. 2022, 52, 198–224. [CrossRef]
28. Schaad, N.W.; Jones, J.B.; Chun, W. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd ed.; American Phytopatho-
logical Society (APS Press): St Paul, MI, USA, 2001; ISBN 0-89-054263-5.
29. Gottsberger, R.A. Development and Evaluation of a Real-time PCR Assay Targeting Chromosomal DNA of Erwinia amylovora.
Lett. Appl. Microbiol. 2010, 51, 285–292. [CrossRef] [PubMed]
30. De Lima, J.E.O.; Miranda, V.S.; Hartung, J.S.; Brlansky, R.H.; Coutinho, A.; Roberto, S.R.; Carlos, E.F. Coffee Leaf Scorch Bacterium:
Axenic Culture, Pathogenicity, and Comparison with Xylella fastidiosa of Citrus. Plant Dis. 1998, 82, 94–97. [CrossRef]
31. Cock, P.J.A.; Chilton, J.M.; Grüning, B.; Johnson, J.E.; Soranzo, N. NCBI BLAST+ Integrated into Galaxy. GigaScience 2015, 4,
s13742-015-0080-7. [CrossRef]
32. Cabrefiga, J.; Montesinos, E. Lysozyme Enhances the Bactericidal Effect of BP100 Peptide against Erwinia amylovora, the Causal
Agent of Fire Blight of Rosaceous Plants. BMC Microbiol. 2017, 17, 39. [CrossRef]
33. Mendes, R.J.; Sario, S.; Luz, J.P.; Tassi, N.; Teixeira, C.; Gomes, P.; Tavares, F.; Santos, C. Evaluation of Three Antimicrobial Peptides
Mixtures to Control the Phytopathogen Responsible for Fire Blight Disease. Plants 2021, 10, 2637. [CrossRef]
34. Peil, A.; Bus, V.G.M.; Geider, K.; Richter, K.; Flachowsky, H.; Hanke, M.-V. Improvement of Fire Blight Resistance in Apple and
Pear. Int. J. Plant Breed. 2009, 3, 1–27.
35. Danecek, P.; Bonfield, J.K.; Liddle, J.; Marshall, J.; Ohan, V.; Pollard, M.O.; Whitwham, A.; Keane, T.; McCarthy, S.A.; Davies, R.M.
Twelve Years of SAMtools and BCFtools. GigaScience 2021, 10, giab008. [CrossRef]
36. Smits, T.H.M.; Rezzonico, F.; Kamber, T.; Blom, J.; Goesmann, A.; Frey, J.E.; Duffy, B. Complete Genome Sequence of the Fire
Blight Pathogen Erwinia Amylovora CFBP 1430 and Comparison to Other Erwinia Spp. Mol. Plant-Microbe Interact. 2010, 23,
384–393. [CrossRef] [PubMed]
37. Slack, S.M.; Zeng, Q.; Outwater, C.A.; Sundin, G.W. Microbiological Examination of Erwinia amylovora Exopolysaccharide Ooze.
Phytopathology 2017, 107, 403–411. [CrossRef] [PubMed]
38. Khan, M.A.; Duffy, B.; Gessler, C.; Patocchi, A. QTL Mapping of Fire Blight Resistance in Apple. Mol. Breed. 2006, 17, 299–306.
[CrossRef]
Horticulturae 2023, 9, 1066 12 of 12

39. Rauzin, E. The Role of Wild Fruit Species in the Development of Modern Horticulture and the Experience of Preserving Their Gene Pool;
DPS: Almaty, Kazakhstan, 2007.
40. Bus, V.; Brewer, L.; Morgan, C. Observations on Scab Resistance in Interspecific Pear Seedling Families. Acta Hortic. 2013, 976,
493–498. [CrossRef]
41. Aleksanyan, S.; Ponomarenko, V.; Burmistrov, L.; Smekalova, T.; Sorokin, A. Modern Methods and International Experience of
Preserving the Gene Pool of Wild Plants (on the Example of Wild Fruits); United Nations Development Program in Kazakhstan: Almaty,
Kazakhstan, 2011; ISBN 978-6-01703-220-3.
42. Vitkovsky, V. Fruit Plants of the World; Lan’: Saint-Petersburg, Russia, 2003.
43. Emeriewen, O.; Richter, K.; Kilian, A.; Zini, E.; Hanke, M.-V.; Malnoy, M.; Peil, A. Identification of a Major Quantitative Trait
Locus for Resistance to Fire Blight in the Wild Apple Species Malusfusca. Mol. Breed. 2014, 34, 407–419. [CrossRef]
44. Emeriewen, O.F.; Richter, K.; Berner, T.; Keilwagen, J.; Schnable, P.S.; Malnoy, M.; Peil, A. Construction of a Dense Genetic Map of
the Malus Fusca Fire Blight Resistant Accession MAL0045 Using Tunable Genotyping-by-Sequencing SNPs and Microsatellites.
Sci. Rep. 2020, 10, 16358. [CrossRef]
45. Kairova, G.; Daulet, N.; Solomadin, M.; Sandybayev, N.; Orkara, S.; Beloussov, V.; Kerimbek, N.; Gritsenko, D.; Sapakhova, Z.
Identification of Apple Varieties Resistant to Fire Blight (Erwinia amylovora) Using Molecular Markers. Horticulturae 2023, 9, 1000.
[CrossRef]
46. Omasheva, M.Y.; Flachowsky, H.; Ryabushkina, N.A.; Pozharskiy, A.S.; Galiakparov, N.N.; Hanke, M.-V. To what extent do wild
apples in Kazakhstan retain their genetic integrity? Tree Genet. Genomes 2017, 13, 52. [CrossRef]
47. Baumgartner, I.O.; Patocchi, A.; Franck, L.; Kellerhals, M.; Broggini, G.A.L. Fire Blight Resistance from “Evereste” and Malus
sieversii Used in Breeding for New High Quality Apple Cultivars: Strategies and Results. Acta Hortic. 2011, 896, 391–397.
[CrossRef]
48. Harshman, J.M.; Evans, K.M.; Allen, H.; Potts, R.; Flamenco, J.; Aldwinckle, H.S.; Wisniewski, M.E.; Norelli, J.L. Fire Blight
Resistance in Wild Accessions of Malus sieversii. Plant Dis. 2017, 101, 1738–1745. [CrossRef]

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