horticulturae

Article
Genetic Diversity and Population Structure Analysis of
Excellent Sugar Beet (Beta vulgaris L.) Germplasm Resources
Fei Peng 1,2 , Zhi Pi 1,2 , Shengnan Li 1,2, * and Zedong Wu 1,2, *

1 Academy of Modern Agriculture and Ecological Environment, Heilongjiang University, Harbin 150080, China;
[email protected] (F.P.); [email protected] (Z.P.)
2 Key Laboratory of Sugar Beet Genetic Breeding, Heilongjiang University, Harbin 150080, China
* Correspondence: [email protected] (S.L.); [email protected] (Z.W.)

Abstract: This study analyzed the genetic diversity, population structure, and cluster analysis of
129 sugar beet germplasm resources to screen superior germplasms for breeding using the 27 simple
sequence repeat (SSR) and 33 pairs of insertion–deletion (InDel) molecular markers. After integrating
the phenotypic variation of 16 descriptive and 4 qualitative phenotypic variables, the genetic varia-
tion levels of the 129 sugar beet germplasms’ phenotypic traits were analyzed using the principal
component analysis (PCA), correlation analysis, and analysis of variance methods. The genetic
diversity examination of molecular markers showed a polymorphism information content (PIC)
of 0.419–0.773 (mean = 0.610). Moreover, the mean number of effective alleles detected via the
SSR and InDel markers was 3.054 and 2.298, respectively. Meanwhile, the PIC ranged from 0.130
to 0.602 (mean = 0.462). The population structure analysis revealed the most appropriate K-value,
indicating three populations (K = 3). The genetic distances of the 129 germplasm resources ranged
from 0.099 to 0.466 (mean = 0.283). The cluster analysis results demonstrated that the germplasms
were grouped into three primary classes. Based on the analysis of variance, the two qualitative
features with the highest coefficients of variation were petiole width (16.64%) and length (17.11%).
The descriptive trait root length index (1.395) exhibited the greatest genetic diversity. The PCA
reduced the 20 phenotypic traits into five principal components, contributing 51.151%. The results of
this study provide a theoretical foundation for the future selection and breeding of superior sugar
beet germplasm resources.
Citation: Peng, F.; Pi, Z.; Li, S.; Wu, Z.
Genetic Diversity and Population
Keywords: sugar beet; genetic diversity; population structure; phenotypic traits; molecular markers;
Structure Analysis of Excellent Sugar
breeding
Beet (Beta vulgaris L.) Germplasm
Resources. Horticulturae 2024, 10, 120.
https://doi.org/10.3390/
horticulturae10020120
1. Introduction
Academic Editor: Radu E. Sestras
The sugar beet (Beta vulgaris L.) is a biennial plant belonging to the Amaranthaceae
Received: 13 December 2023 family. The sea beet (Beta vulgaris ssp. maritima) is a wild ancestor of the sugar beet that
Revised: 23 January 2024 originates from the Mediterranean region [1]. Sugar beet is one of the largest sugar crops
Accepted: 23 January 2024 in the world, amounting to 20% of the total worldwide sucrose production, second only
Published: 25 January 2024 to sugar cane. It is a significant source of sucrose in the temperate parts of the northern
hemisphere [2,3]. Its byproducts can also serve as raw materials for producing other
commodities, such as bioethanol and animal feed [4]. Due to their self-incompatibility,
most commercial sugar beet varieties are hybrids descended from a single cytoplasmic
Copyright: © 2024 by the authors.
male sterility (CMS) lineage, resulting in a more restricted genetic base [5–7]. However,
Licensee MDPI, Basel, Switzerland.
introducing new characters into CMS seed parents is challenging [8]. Understanding the
This article is an open access article
genetic diversity of sugar beet germplasm resources and population structure for sugar
distributed under the terms and
beet breeding is critical. The crops may lose some genetic diversity found in their wild
conditions of the Creative Commons
ancestors due to the widespread adoption of genetically consistent variations [9–11]. The
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
acquisition, evaluation, conservation, and use of crop germplasm resources are integral
4.0/).
components of agricultural research and development since they serve as the basis for

Horticulturae 2024, 10, 120. https://doi.org/10.3390/horticulturae10020120 https://www.mdpi.com/journal/horticulturae

Horticulturae 2024, 10, 120 2 of 16

theoretical biological investigations and molecular genetics as well as for the cultivation
and choice of crop varieties.
The presence of diversity among various organisms, also known as biodiversity, can
be observed at three distinct levels: genetic, species, and ecological. Genetic diversity is the
term used to describe the overall genetic variation among individuals of different species
within or between populations. This phenomenon is an inherent characteristic of living or-
ganisms that emerges from evolutionary processes [12]. Plant species require a wide range
of genetic pools with high genetic variability to survive and adapt to the environment [13].
Sufficient genetic diversity is necessary to produce new species with desirable traits [14].
The probability of selecting high-quality germplasm from the germplasm resources avail-
able is enhanced as genetic diversity increases. To identify high-quality germplasms for
breeding, studying genetic diversity can provide valuable insights into the evolution of
genes. Crop genetic diversity can be established using lineage analysis, phenotypic eval-
uation, biochemical analysis, or molecular markers [15,16]. Phenotypic identification is
the easiest and most fundamental research method for analyzing genetic diversity. Its
application is intuitive and facilitates the quick understanding of crop genetic variation
by analyzing phenotypic traits. However, certain drawbacks are associated with pheno-
typic identification, such as instability and vulnerability to environmental factors [17,18].
Molecular markers are extensively employed in population genetics due to their utility
in evaluating genetic variation. They provide a direct measure of the extent of genetic
variation between genotypes at the level of the DNA and establish a connection between
phenotypic and genotypic variation [19–21]. Simple sequence repeats (SSRs) are repeating
patterns ranging from 1 to 6 bp generally present in eukaryotic organisms’ genomes [22]
and are frequently utilized in plant breeding as marker types. SSR markers are commonly
used to assess crop genetic diversity due to their consistency with Mendelian co-dominant
inheritance, good stability, reproducibility, convenience, and widespread availability within
the genome [10,23–26]. Various studies revealed that these markers have also been ex-
tensively utilized in the gene flow and genetic analysis of sugar beet populations [27,28].
Based on PCR amplification, insertion–deletion (InDel) markers are a type of base sequence
length polymorphism markers. These are useful for determining the relationship between
samples and allow the filtering out of single-base nonsense mutations. InDel molecular
markers have proven effective in plant genetic diversity, population structure analysis,
and assisted breeding. This is because they are highly reproducible, have co-occurring
inheritance, and provide extensive genome coverage. These markers are also less expensive
to develop and easier to design and detect [29–31].
Integrating phenotypic identification and molecular markers can enhance the accuracy
of genetic diversity studies. This is because molecular markers solely identify variations at
the DNA level, which may not necessarily manifest in observable traits (phenotypes) [32–34].
To select excellent hybrid combinations and obtain high-quality innovative varieties, it
is necessary to identify and analyze sugar beet germplasm resources. This will further
elucidate their germplasm’s genetic background and intrinsic genetic structure. Using
27 and 33 pairs of SSR and InDel primers, respectively, in addition to 20 phenotypic traits
such as leaf shape and color, the genetic diversity and population structure of 115 sugar
beet polyembryonic pollinated lines and seven pairs of sugar beet monoembryonic sterile
and maintained lines were examined in this study. This research, therefore, provides
resources and theoretical direction for the future selection and breeding of sugar beet
germplasm resources.

2. Materials and Methods

2.1. Plant Materials and Experimental Design
A total of 129 sugar beet lines, comprising seven pairs of sugar beet CMS and main-
tained lines as well as 115 polyembryonic pollinated lines, were utilized (Table S1). All the
materials were backbone parents that can be used in breeding and were supplied by the
Key Laboratory of Sugar Beet Genetic Breeding (Heilongjiang University, Harbin, China).
Horticulturae 2024, 10, 120 3 of 16

The test lines were grown at the experimental base of Heilongjiang University’s Hulan
Campus, where the soil was flat and black. This location is known for its mesothermal
continental monsoon climate. Observation and data collection of agronomic qualities were
conducted on ten plants from each line. The experiment was set up in randomized blocks,
with two rows planted in each experimental region, a row length of 10 m, a spacing of
0.67 m, and a sowing depth of 3 cm. The aboveground section of the beet leaf cluster during
its period of rapid growth was selected, specifically the largest true leaf, excluding the old
and young leaves. The data of the traits in the belowground portion of the sugar beets were
recorded throughout the sugar beets’ sucrose accumulation stage, commonly referred to as
the harvesting period. The test lines matched the criteria of the trait survey and exhibited
satisfactory progress throughout the growth cycle.

2.2. Phenotypic Trait Measurements

The phenotypic traits of sugar beet germplasm were qualified using the “Descriptors
and date standard for beet(Beta vulgaris L.)” reference [35]. The following parameters were
examined in the 129 sugar beet lines: sixteen qualitative traits (leaf shape, color, margin
shape, and surface, mesophyll thickness, leaf hairiness, petiole color and thickness, fascicled
leaf type, root length, width, length-to-width ratio, shape, and groove depth, crown size,
and skin roughness) and four quantitative traits (petiole width and length, plant height,
and width of leaf coverage). Ten randomly selected plants were examined from each line
for the above traits.

2.3. DNA Extraction and Genotyping

Total DNA was extracted from young sugar beet leaves at the 2–3 pairs of true leaf
stage using the Cetyl Tri-methyl Ammonium Bromide (CTAB) method [36]. DNA purity
and concentration were detected using a NanoDrop 2000/2000c Ultra-Micro UV-Vis Spec-
trophotometer (Thermo Fisher, Madison, WI, USA). The samples were diluted to 10 ng/µL
to prepare the working solution and stored at −20 ◦ C until further use.
A total of 129 sugar beet germplasms were examined as the test materials, from which
6 distinct germplasms were selected randomly as templates. These templates were then
used to screen 42 SSRs and 41 pairs of InDel primers. After evaluating these primers for
distinct bands, stable amplification, and significant polymorphisms, 27 pairs of SSR and
33 pairs of InDel primers were chosen for subsequent investigations (Table S2). Some of the
SSR markers came from earlier research [25,37], while the remainder were designed and
supplied by the Molecular Genetics Laboratory of Heilongjiang University using the sugar
beet’s whole genome sequence. All the InDel markers were designed and provided by
the Molecular Genetics Laboratory (Heilongjiang University) using the sugar beet’s whole
genome sequence. Shanghai Bioengineering Co., Ltd. (Shanghai, China) developed the
above-mentioned primers.
The 5 µL PCR amplification system contained 2.5 µL of 2 × Taq PCR Master Mix
(BioTeke Corporation, Wuxi, China), 0.4 µL of primers, 1.1 µL of double distilled water
(ddH2 O), and 1 µL of sugar beet genomic DNA. A Veriti 96-Well Thermal Cycler (Ther-
moFisher Scientific™, Shanghai, China) was used to perform PCR. The length, primer
concentration, and base composition all affect the annealing temperature. To increase the
PCR reaction’s specificity and decrease non-specific binding, distinct PCR programs were
employed for each primer, given that primers can only show amplified bands at their ideal
annealing temperature. The steps in the PCR reaction were as follows: pre-denaturation at
94 ◦ C for 3 min, followed by 35 cycles of 15 s at 94 ◦ C, 15 s at 58 ◦ C, 30 s at 72 ◦ C, and a
final extension at 72 ◦ C for 5 min. The touchdown program was used for some primers:
pre-denaturation at 94 ◦ C for 3 min, followed by 15 s at 94 ◦ C, annealing at 65 ◦ C for 15 s,
followed by two cycles of 65–56 ◦ C for every one degree down to 56 ◦ C, and extension
at 72 ◦ C for 30 s. After that, 20 cycles of 15 s at 94 ◦ C, 15 s at 55 ◦ C, 30 s at 72 ◦ C, and
a final extension at 72 ◦ C for 5 min. The PCR products were then separated using 8%
non-denaturing polyacrylamide gel electrophoresis, which was run for 1.5 h at a constant
Horticulturae 2024, 10, 120 4 of 16

180 V. The gel was stained with the non-toxic G-Red nucleic acid dye (BioTeke Corporation,
Wuxi, China) and was photographed using a gel imager.

2.4. Data Analysis

A binary data matrix was created using the 0/1 assignment method. The amplified
bands were manually read, and their types were recorded. A “1” was assigned to a band
at a specific point, while a “0” was given to no band. The following genetic diversity in-
dices were calculated using PopGene32 version 1.32 [38]: observed number of alleles (Na),
effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity
(He), Shannon’s information index (I), Nei’s expected heterozygosity, and gene flows (Nm).
The gene diversity and polymorphism information content (PIC) [39] among different
populations were calculated using PowerMarker 3.25 [40]. The STRUCTURE software
was used to conduct the population structure analysis of the sugar beet germplasm re-
sources [41], calculating the optimal number of subpopulations. This model-based software
uses a Bayesian clustering method [42]. The Markov chain Monte Carlo (MCMC) technique
computed the posterior probabilities. The MCMC chains were run using a model that
allowed for admixture and correlated allele frequencies, with a 100,000 burn-in period
followed by 100,000 iterations. Ten runs for each K-value were performed with K ranging
from 1 to 10 to obtain an accurate estimation of the number of populations. The optimal
number of subpopulations of the population was later determined by the rate of change in
the a posteriori probability values (∆K) [42] using the web-based program STRUCTURE
HARVESTER [43]. By combining the SSR and InDel data a clustered dendrogram of the
129 sugar beet genotypes was generated based on Nei’s genetic distance [44], using the
unweighted pair–group method with arithmetic averaging (UPGMA) as implemented
using the MEGA7 [45].
All phenotypic trait data were assembled in Microsoft Excel. Basic statistical parame-
ters were computed for each trait, including the minimum and maximum value, the mean,
the standard deviation (SD), and the coefficient of variation (CV) [46]. The Shannon–Wiener
diversity index (H0 ) [47,48] was calculated and correlated via the IBM Statistical Package
for the Social Sciences (SPSS) Statistics, version 20.0. Origin 2020 was used for the principal
component analysis (PCA) [49].

3. Results
This section presents the results of the genetic diversity analysis, population structure, ge-
netic distance, cluster structure, and phenotypic trait variation in 129 sugar beet germplasms.

3.1. Genetic Diversity Using SSR Primers

With the use of 27 pairs of carefully selected SSR primers, a total of 129 sugar beet
germplasm resources were thoroughly analyzed to identify any potential polymorphisms.
A total of 130 alleles were found, ranging from 3 to 9 alleles per primer, with an average
of 4.815 (Table 1). The mean Ne value was 3.054, with primer 26319 having the highest
effective number of alleles at 4.870. The I ranged from 0.787 to 1.810, with a mean value
of 1.231. The Ho of the primers was 1, and the minimum value was 0.781. The maximum
value of He was 0.798, while the minimum was 0.524. Nei’s expected heterozygosity ranged
from 0.52 to 0.795. The average value of Nm was 0.599. The variation in the gene diversity
index ranged from 0.529 to 0.798, with an average of 0.668. The PIC ranged from 0.419 to
0.773, with an average of 0.610. Primer 26319 demonstrated the maximum PIC value, while
the minimum value was observed in primer 2170.
Horticulturae 2024, 10, 120 5 of 16

Table 1. Genetic diversity was amplified by 27 pairs of SSR primers in the 129 sugar beet germplasms.

Nei’s Expected Genetic

Locus Na Ne I Ho He Nm PIC
Heterozygosity Diversity
14118 4 3.116 1.224 1.000 0.682 0.679 0.660 0.684 0.624
17623 5 3.124 1.349 0.961 0.683 0.680 0.603 0.680 0.640
2305 5 2.941 1.237 0.946 0.663 0.660 0.632 0.660 0.602
11965 5 4.104 1.455 0.985 0.759 0.756 0.466 0.756 0.713
27374 4 2.560 1.080 0.891 0.612 0.609 0.637 0.615 0.544
BQ588629 5 3.325 1.339 0.992 0.702 0.699 0.611 0.699 0.651
L37 4 2.765 1.114 1.000 0.641 0.638 0.904 0.638 0.566
L7 6 3.655 1.498 0.953 0.729 0.726 0.441 0.735 0.699
7492 4 2.595 1.097 0.929 0.617 0.615 0.634 0.632 0.574
L47 3 2.702 1.042 0.992 0.632 0.630 0.927 0.630 0.555
L57 6 2.492 1.107 0.977 0.601 0.599 1.107 0.599 0.523
L35 5 3.196 1.264 0.935 0.690 0.687 0.413 0.713 0.663
L48 4 2.878 1.182 0.953 0.655 0.653 0.639 0.658 0.600
L64 4 3.247 1.240 0.898 0.695 0.692 0.444 0.697 0.638
W21 5 2.372 1.078 0.781 0.581 0.578 0.380 0.615 0.561
2170 3 2.090 0.787 0.906 0.524 0.522 1.418 0.529 0.419
15915 5 2.773 1.182 0.960 0.642 0.639 0.561 0.665 0.610
17923 8 3.445 1.468 0.977 0.713 0.710 0.552 0.710 0.664
24552 5 2.825 1.128 0.961 0.649 0.646 0.682 0.652 0.582
26319 9 4.870 1.810 0.953 0.798 0.795 0.364 0.798 0.773
57236 4 3.315 1.249 0.954 0.701 0.698 0.538 0.698 0.637
SSD6 6 3.302 1.450 0.992 0.700 0.697 0.617 0.697 0.661
TC94 3 2.492 0.982 1.000 0.601 0.599 1.267 0.599 0.516
BVV23 3 2.950 1.090 0.961 0.664 0.661 0.666 0.661 0.587
W15 4 2.655 1.074 0.921 0.626 0.623 0.625 0.635 0.562
BVV21 4 2.587 1.080 0.952 0.616 0.613 0.656 0.636 0.571
TC122 7 4.079 1.627 0.985 0.758 0.755 0.469 0.755 0.722
Average 4.815 3.054 1.231 0.952 0.664 0.662 0.599 0.668 0.610
Na: Observed number of alleles; Ne: effective number of alleles; I: Shannon’s information index; Ho: observed
heterozygosity; He: expected heterozygosity; Nm: gene flow estimated from Fst = 0.25 (1 − Fst)/Fst; and PIC:
polymorphism information content.

3.2. Genetic Diversity Using InDel Primers

Polymorphism was assessed on 129 sugar beet germplasm resources using 33 pairs
of InDel primers. A total of 107 alleles were identified, with the number of detectable
alleles varying from 2 to 5 per primer and an average of 3.242 (Table 2). The mean Ne
value was 2.298, with ND129 having the highest value at 3.008. The I ranged from 0.268
to 1.175 (mean = 0.886). The maximum value of Ho of the primers was 0.985, while the
minimum was 0.057. The maximum He value was 0.670, while the minimum was 0.140.
Nei’s expected heterozygosity ranged from 0.140 to 0.668. The mean value of gene flow
was 0.660. The variation in the gene diversity index ranged from 0.140 to 0.668, with an
average of 0.543. The PIC ranged from 0.130 to 0.602, with an average of 0.462. Primers
ND129 and ND18 demonstrated the maximum and minimum PIC values.

Table 2. Genetic diversity was amplified by 33 pairs of InDel primers in the 129 sugar beet germplasms.

Nei’s Expected Genetic

Locus Na Ne I Ho He Nm PIC
Heterozygosity Diversity
ND31 3 2.486 1.002 0.734 0.600 0.598 0.380 0.598 0.531
ND75 3 1.876 0.729 0.636 0.469 0.467 0.533 0.467 0.378
ND109 3 2.269 0.898 0.892 0.561 0.559 0.982 0.559 0.465
Horticulturae 2024, 10, 120 6 of 16

Table 2. Cont.

Nei’s Expected Genetic

Locus Na Ne I Ho He Nm PIC
Heterozygosity Diversity
ND113 3 1.825 0.663 0.682 0.454 0.452 0.768 0.452 0.353
ND220 3 2.768 1.053 0.813 0.641 0.639 0.341 0.639 0.562
ND223 3 1.750 0.702 0.558 0.430 0.429 0.467 0.429 0.360
ND253 5 2.424 1.042 0.859 0.590 0.588 0.636 0.588 0.508
ND258 3 2.856 1.074 0.954 0.652 0.650 0.688 0.650 0.576
ND275 4 2.204 0.925 0.852 0.548 0.546 0.808 0.546 0.459
ND277 3 1.804 0.791 0.566 0.447 0.446 0.435 0.446 0.404
ND10 4 1.985 0.741 0.758 0.498 0.496 0.735 0.496 0.384
ND11 3 1.808 0.658 0.659 0.449 0.447 0.701 0.447 0.351
ND18 2 1.162 0.268 0.057 0.140 0.140 0.015 0.140 0.130
ND108 4 2.844 1.115 0.914 0.651 0.648 0.566 0.648 0.576
ND120 3 2.015 0.716 0.969 0.506 0.504 6.319 0.504 0.381
ND121 3 2.579 1.004 0.876 0.615 0.612 0.629 0.612 0.530
ND129 4 3.008 1.175 0.845 0.670 0.668 0.431 0.668 0.602
ND142 3 2.428 0.956 0.954 0.590 0.588 1.070 0.588 0.499
ND143 4 1.581 0.696 0.426 0.369 0.368 0.345 0.368 0.336
ND162 4 2.577 1.052 0.984 0.614 0.612 0.942 0.612 0.533
ND229 3 2.062 0.762 0.985 0.517 0.515 5.382 0.515 0.398
ND231 4 2.931 1.104 0.930 0.661 0.659 0.600 0.659 0.586
ND233 3 2.711 1.046 0.915 0.634 0.631 0.658 0.631 0.558
ND243 5 2.701 1.104 0.915 0.632 0.630 0.663 0.630 0.564
ND244 3 2.913 1.084 0.954 0.659 0.657 0.662 0.657 0.583
ND246 2 1.928 0.674 0.667 0.483 0.481 0.564 0.481 0.365
ND267 3 2.606 1.018 0.977 0.619 0.616 0.879 0.616 0.540
ND270 4 2.325 0.959 0.868 0.572 0.570 0.799 0.570 0.479
ND279 3 2.226 0.873 0.922 0.553 0.551 1.140 0.551 0.451
ND280 2 1.555 0.542 0.434 0.358 0.357 0.388 0.357 0.293
ND281 3 2.806 1.065 0.930 0.646 0.644 0.614 0.644 0.571
ND283 2 1.961 0.683 0.813 0.492 0.490 1.062 0.490 0.370
ND286 3 2.865 1.074 0.961 0.654 0.651 0.705 0.651 0.576
Average 3.242 2.298 0.886 0.796 0.545 0.543 0.660 0.543 0.462
Na: Observed number of alleles; Ne: effective number of alleles; I: Shannon’s information index; Ho: observed
heterozygosity; He: expected heterozygosity; Nm: gene flow estimated from Fst = 0.25 (1 − Fst)/Fst; and PIC:
polymorphism information content.

3.3. Population Structure Analysis

A total of 129 sugar beet germplasms were examined for their population genetic
structure using 27 and 33 pairs of SSR and InDel primers, respectively. The findings
indicated that the maximum value of ∆K occurred at K = 3 (Figure 1), indicating the
possibility of identifying three distinct populations from the 129 sugar beet germplasm
resources (Figure 2). Fifty-one germplasms were grouped in Pop1, and all of them were
polyembryonic pollinated lines. Fifty-seven germplasms were grouped in Pop2, and all
of them were polyembryonic pollinated lines. Pop3 consisted of a total of twenty-one
germplasms, which included both CMS lines and maintained lines.

3.4. Genetic Distance and Cluster Structure

The genetic distances (Nei’s genetic distance) of the 129 germplasms, measured by
60 pairs of SSR and InDel molecular markers, varied from 0.099 to 0.466 (mean = 0.283)
(Table S3). The findings indicated that 66 and 67 exhibited the lowest genetic distances,
suggesting their closest proximity, while 29 and 129 had the highest genetic distances,
marking the furthest separation. The 129 sugar beet germplasms could be distinguished
from one another using the SSR and InDel markers, according to the findings of the UPGMA
cluster analysis (Figure 3). These resources were grouped into three major taxa, which
could be related to the findings of the population structure analysis.
Horticulturae 2024,10,
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x FOR PEER REVIEW 77 of 7 of 16
Horticulturae 2024, 10, x FOR PEER REVIEW of 17
17

Figure
Figure 1.∆K
Figure 1.
1. ∆Kvalue
∆K valuechange
value change
change graph.
graph.
graph. The
TheThe corresponding
corresponding
corresponding ∆K ∆K statistics
∆K statistics
statistics identify
identify
identify the the optimal
the optimal
optimal STRUCTURE
STRUCTURE
STRUCTURE
assignment
assignment of
assignment of the
ofthe 129
the129 sugar
129sugar beet
sugarbeet germplasms
beetgermplasms
germplasms(K =
(K (K 3).
= 3).
= 3).

Figure 2. Population genetic structure of 129 sugar beet germplasms at K = 3. The highest Delta K is K is
Populationgenetic
geneticstructure
structure
of of
129129 sugar beet germplasms
at Kat K The
= 3. highest
The
Figure 2.
Horticulturae 2024, 10, x FOR PEER REVIEW 2. Population sugar beet germplasms = 3. 8 ofhighest
17Delta Delta
K is
obtained
obtained at
obtained atK
at KK===3,
3,3,suggesting
suggesting
suggestingthat
that the population
thethe
that population could
could
population be
be divided
could divided into three
into into
be divided subpopulations:
threethree
subpopulations: Pop1,
Pop1, Pop1,
subpopulations:
Pop2,
Pop2, and
and Pop3.
Pop3.
Pop2, and Pop3.
3.4.
3.4. Genetic
Genetic Distance
Distance and
and Cluster
Cluster Structure
Structure
The
The genetic distances (Nei’s genetic
genetic distances (Nei’s genetic distance)
distance) of
of the
the 129
129 germplasms,
germplasms, measured
measured byby
60
60 pairs of SSR and InDel molecular markers, varied from 0.099 to 0.466 (mean == 0.283)
pairs of SSR and InDel molecular markers, varied from 0.099 to 0.466 (mean 0.283)
(Table
(Table S3).
S3). The
The findings
findings indicated
indicated that
that 66
66 and
and 67
67 exhibited
exhibited the
the lowest
lowest genetic
genetic distances,
distances,
suggesting their closest proximity, while 29 and 129 had the highest genetic distances,
suggesting their closest proximity, while 29 and 129 had the highest genetic distances,
marking
marking the
the furthest
furthest separation.
separation. The
The 129
129 sugar
sugar beet
beet germplasms
germplasms could
could be
be distinguished
distinguished
from
from one
one another
another using
using the
the SSR
SSR andand InDel
InDel markers,
markers, according
according to to the
the findings
findings of
of the
the
UPGMA cluster analysis (Figure 3). These resources were grouped into three major taxa,
UPGMA cluster analysis (Figure 3). These resources were grouped into three major taxa,
which
which could
could be
be related
related to
to the
the findings
findings ofof the
the population
population structure
structure analysis.
analysis.

Figure 3. The 129 sugar beet germplasms clustered using UPGMA, based on SSR and InDel markers.
Figure 3. The 129 sugar beet germplasms clustered using UPGMA, based on SSR and InDel markers.
The materials are categorized into three taxa (indicated by blue, green, and red colors). The color of
The materials arecorresponds
the number labels categorized into
to the three
taxon taxa (indicated
findings by blue,
of the population green,
structure and red colors). The color of
analysis.
the number labels corresponds to the taxon findings of the population structure analysis.
3.5. Variation Analysis of the Phenotypic Traits
For each of the four qualitative traits, a statistical analysis of the 129 germplasms’
phenotypic characteristics was carried out (Table 3). The results demonstrated that petiole
length (17.11%) and width (16.64%) exhibited the highest coefficients of variation, while
plant height (9.73%) was the lowest. Out of the four characteristics evaluated, only the plant
height coefficient of variation had a value below 10%. The findings indicated a relatively
Horticulturae 2024, 10, 120 8 of 16

3.5. Variation Analysis of the Phenotypic Traits

For each of the four qualitative traits, a statistical analysis of the 129 germplasms’
phenotypic characteristics was carried out (Table 3). The results demonstrated that petiole
length (17.11%) and width (16.64%) exhibited the highest coefficients of variation, while
plant height (9.73%) was the lowest. Out of the four characteristics evaluated, only the plant
height coefficient of variation had a value below 10%. The findings indicated a relatively
low level of variability in plant height among the test lines. Conversely, the remaining three
qualitative characteristics showed significant variations across the genetic components.

Table 3. Statistical analysis of variation in measured traits of sugar beet germplasm resources.

Trait Max (cm) * Min (cm) ** Average (cm) SD CV (%)

Petiole width 2.760 1.075 1.596 0.266 16.64
Petiole length 38.940 18.030 26.663 4.562 17.11
Plant height 69.450 41.625 53.566 5.211 9.73
Width of leaf coverage 103.260 54.620 79.442 11.990 15.09
* Maximum values; ** minimum values; SD: standard deviation; and CV: coefficient of variation.

The lines exhibited variable degrees of different descriptive traits, as determined by

the frequency analysis of 16 descriptive traits in the 129 germplasms (Table 4). Petiole
color had the highest number of variation types among the six primary variation forms.
The petiole color percentages were highest for the “white-green” variety at 60.47% and
the “light green” variety at 27.13%. Root length was the second most common attribute,
with five distinct types of variation. Medium (37.21%) and long root (31.01%) lengths
exhibited the maximum percentages. The lines had many of the same leaf properties, such
as being primarily green in color, having large waves at the edges of the leaf margins, and
having slightly wrinkled leaf surfaces. Less hairy (96.90%) leaves were more common than
hairier (3.10%) ones, whereas a thin (58.91%) mesophyll was more prevalent than a medium
(33.33%) or thick (31.01%) mesophyll. The petiole thickness was most commonly medium,
and 61.24% of the fascicled leaves were erect. Most of the roots were medium in length,
broad in width, and high in their length-to-width ratio. A total of 51.94% of the roots were
cuneiform; the crown size was primarily small; the root groove depth was generally not
apparent; and the epidermis was very smooth.

Table 4. Frequency distribution and diversity index of descriptive characteristics of sugar beet
germplasm resources.

Characteristic Description (Proportion of Distribution, %)

Traits H0
1 2 3 4 5 6
Halberd Share Tongue
Leaf shape 1.074
23.26 38.76 37.98
Light green Green Dark green
Leaf color 1.057
20.93 43.41 35.66
Full margin Small wave Medium wave Big wave
Leaf margin shape 1.237
5.43 29.46 26.36 38.76
Smooth Wavy Slight crease More creases
Leaf surface 1.146
2.33 23.26 44.96 29.46
Thin Medium Thick
Mesophyll thickness 1.041
58.91 33.33 31.01
Little Much
Leaf hairiness 0.138
96.90 3.10
White White-green Light green Green Pink Purplish red
Petiole color 1.034
3.88 60.47 27.13 6.98 0.00 1.55
Slight Medium Thick
Petiole thickness 1.065
28.68 45.74 25.58
Horticulturae 2024, 10, 120 9 of 16

Table 4. Cont.

Characteristic Description (Proportion of Distribution, %)

Traits H0
1 2 3 4 5 6
Erect Semi-crawl Crawl
Fascicled leaf type 0.810
61.24 34.11 4.65
Very short Short Medium Long Very long
Root length 1.395
3.10 13.18 37.21 31.01 15.50
Narrow Medium Broad
Root width 1.082
26.36 32.56 41.09
Root length-to-width Small Medium Large
1.051
ratio 30.23 22.48 47.29
Cuneiform Conical Spindle
Root shape 1.000
51.94 31.78 16.28
Small Medium Large
Crown size 1.068
43.41 33.33 23.26
None Not obvious Shallow Deep
Root groove depth 1.240
27.91 37.98 28.68 5.43
Very smooth Smoother Very rough
Skin roughness 0.965
49.61 38.76 11.63
H0 : Shannon–Wiener diversity index.

Among the 16 descriptive traits, the genetic diversity indices were ranked as follows:
root length (1.395) > root groove depth (1.240) > leaf margin shape (1.237) > leaf surface
(1.146) > root width (1.082) > leaf shape (1.074) > crown size (1.068) > petiole thickness
(1.065) > leaf color (1.057) > root length-to-width ratio (1.051) > mesophyll thickness (1.041)
> petiole color (1.034) > root shape (1.000) > skin roughness (0.965) > fascicled leaf type
(0.810) > leaf hairiness (0.138).

3.6. Correlation Analysis

The correlation study of 129 sugar beet germplasm resources revealed seven pairs of
highly significant correlations between traits. Five of these pairs were positively associated,
and two were negatively correlated. The analysis was based on four qualitative and sixteen
descriptive traits (Table 5, Table S4, and Figure 4).
The results of the correlation analysis of the four quantitative traits showed that petiole
width was highly significant and positively correlated with petiole length (0.361 **), plant
height (0.392 **), and leaf cover width (0.441 **). The petiole length was highly significant
and positively correlated with plant height (0.788 **) and leaf cover width (0.642 **), and
plant height was highly significant and positively correlated with leaf cover width (0.444 **).
The leaf margin shape showed a significant negative association with the root length-to-
width ratio and a highly significant negative relationship with the petiole thickness among
the 16 qualitative features. A significant positive correlation was detected between leaf
blade thinness, petiole thinness, and root epidermal texture. Moreover, a highly significant
positive correlation was also detected with the root furrow depth, and a significant negative
correlation was identified with the leaf clump shape. The petiole thickness and the root
shape showed a significant negative association; however, the root epidermal texture
showed a significant positive correlation. A significant negative correlation was observed
between the petiole thickness and the leaf margin shape. A significant positive correlation
was identified between the root length-to-width ratio and the root length (correlation
coefficient = 0.661), and a highly significant negative correlation was observed with the
root width. The root shape and width demonstrated a significant positive correlation
(correlation coefficient = 0.267). Root groove depth was significantly negatively correlated
with mesophyll thickness, crown size, and skin roughness, with correlation coefficients of
0.322, 0.273, and 0.386, respectively.
 petiole width was highly significant and positively correlated with petiole length (0.361
**), plant height (0.392 **), and leaf cover width (0.441 **). The petiole length was highly
significant and positively correlated with plant height (0.788 **) and leaf cover width
(0.642 **), and plant height was highly significant and positively correlated with leaf cover
Horticulturae 2024, 10, 120 width (0.444 **). 10 of 16

Table 5. Correlation analysis of four quantitative characteristics of sugar beet germplasm resources.
Table 5. Correlation analysis of four quantitative characteristics of sugar beet germplasm resources.
Trait Petiole Width Petiole Length Plant Height
Petiole length Petiole Width 0.361 Petiole
Trait ** Length Plant Height
PetiolePlant
lengthheight 0.361 ** 0.392 ** 0.788 **
Plant height 0.392 ** 0.788 **
Width of leaf coverage 0.441 ** 0.642 ** 0.444 **
Width of leaf
** p < 0.01.coverage 0.441 ** 0.642 ** 0.444 **
** p < 0.01.

Figure 4. Heatmap of quality trait correlations.

Figure 4. Heatmap of quality trait correlations.
3.7. PCA Evaluation
ThePCA
leafwas performed
margin shape onshowed
20 traits ofa 129 sugar beet
significant germplasms
negative (Table 6, Figures
association 5 and
with the 6).length-
root
The first five major components of sugar beet, with a cumulative contribution rate of
to-width ratio and a highly significant negative relationship with the petiole thickness
51.151%, encompass the primary variable information of the correlated indices. The initial
among the 16ofqualitative
eigenvalue features. A
principal component significant
1 (PC1) positive
was 3.615, with acorrelation
contributionwasratedetected between
of 18.075%.
leafThe
blade
main thinness,
agronomic petiole thinness,
traits in PC1 were the and rootlength,
petiole epidermal texture.
the width of leaf Moreover,
coverage, anda highly
significant positive
the plant height, correlation
with was
characteristic also ofdetected
vectors with
0.434, 0.422, andthe root
0.373, furrow depth,
respectively. These and a
were classified
significant as the
negative overall aboveground
correlation was identifiedplant proportion.
with the leafTheclump
initial eigenvalue
shape. The of petiole
principal component 2 (PC2) was 1.903, with a contribution rate of 9.516%.
thickness and the root shape showed a significant negative association; however, the root The primary
agronomictexture
epidermal traits in showed
PC2 wereathesignificant
roots’ length (0.431), the
positive crown’s size
correlation. A (0.367), and the
significant negative
petiole’s thickness (0.330). The initial eigenvalue of principal component 3 (PC3) was
correlation was observed between the petiole thickness and the leaf
1.853, with a contribution rate of 9.263%. PC3’s principal agronomic characteristics had
margin shape. A
significant positive
characteristic correlation
vectors was identified
of 0.354, 0.333, and 0.329 for between
the rootthe rootdepth,
groove length-to-width
the mesophyllratio and
thickness, and the leaf shape, respectively. The initial eigenvalue of principal component 4
(PC4) was 1.480, with a contribution rate of 7.399%. The root length, the root shape, and
the fascicled leaf type were the primary agronomic features in PC4, with corresponding
characteristic vectors of 0.391, 0.389, and 0.277, respectively. The initial eigenvalue of
principal component 5 (PC5) was 1.380, with a contribution rate of 6.899%. The fascicled
leaf type (0.511), the skin roughness (0.429), and the plant height (0.258) were the primary
agronomic traits.
 Horticulturae 2024, 10, 120 11 of 16

Table 6. Eigenvectors and cumulative contribution rates of each principal component.

Principal Contribution Rate Cumulative

Eigenvalue
Component Number (%) Contribution Rate (%)
1 3.615 18.075 18.075
2 1.903 9.516 27.590
3 1.853 9.263 36.854
4 1.480 7.399 44.252
5 1.380 6.899 51.151
6 1.173 5.866 57.017
7 1.072 5.359 62.376
8 1.068 5.340 67.716
9 0.968 4.838 72.555
10 0.850 4.252 76.807
11 0.804 4.020 80.827
12 0.691 3.454 84.281
13 0.678 3.389 87.669
14 0.620 3.102 90.771
15 0.466 2.328 93.099
16 0.434 2.172 95.271
ticulturae 2024, 10, x FOR PEER REVIEW 12 of 17
17 0.395 1.977 97.249
18 0.295 1.476 98.725
19 0.142 0.710 99.435
20 0.113 0.565 100.000

Figure 5. Principal component analysis (PCA) of 129 sugar beet germplasms based on 20 phenotypic
Figure 5.traits. The coloration
Principal of each
component germplasm
analysis is determined
(PCA) by the
of 129 sugar population
beet structure
germplasms results
based on (Pop1 = red,
20 phenotypic
Pop2 = green, and Pop3 = blue). Populations one and two show significant overlap,
traits. The coloration of each germplasm is determined by the population structure results (Pop1 indicating a =
relatively high genetic similarity.
red, Pop2 = green, and Pop3 = blue). Populations one and two show significant overlap, indicating
a relatively high genetic similarity.
 Figure 5. Principal component analysis (PCA) of 129 sugar beet germplasms based on 20 phenotypic
traits. The coloration of each germplasm is determined by the population structure results (Pop1 =
red, Pop2 = green, and Pop3 = blue). Populations one and two show significant overlap, indicating
Horticulturae 2024, 10, 120 12 of 16
a relatively high genetic similarity.

Figure 6. PCA heatmap of sugar beet germplasms.

Figure 6. PCA heatmap of sugar beet germplasms.
4. Discussion
The development and utility of superior sugar beet germplasm resources are critical
4. Discussion
for effective breeding. The germplasms of sugar beets are essential for scientific study and
The development
breeding and utility
and for developing of superior
novel, high-yield,sugar beet germplasm
high-quality, resources
and disease-resistant are critical
cultivars.
for effective
Geneticbreeding. The germplasms
variety typically of sugar However,
ensures biodiversity. beets are essential
only a highfordiversity
scientific
of study
genes and
can guarantee biodiversity’s continuous survival. The collection, breeding,
breeding and for developing novel, high-yield, high-quality, and disease-resistant conservation,
genetic
cultivars. diversity
Genetic analysis,
variety and study
typically of sugar
ensures beet germplasm
biodiversity. resourcesonly
However, are of
a great
high value
diversity
in all developed nations. Crop genetic diversity is best investigated via joint usage of
of genes can guarantee biodiversity’s continuous survival. The collection, breeding,
morphological and molecular markers. Kleine et al. previously used morphological and
conservation, genetic diversity analysis, and study of sugar beet germplasm resources are
molecular methods to examine the potential of 30 different nematode-resistant lines as a
of great value
new sourcein of
allresistance
developed nations.
[50]. Crop
In another genetic diversity
investigation, is best
eight pairs investigated
of amplified via joint
fragment
length polymorphism (AFLP) markers and four root traits were used to examine the
molecular and morphophysiological properties of wild and cultivated sugar beet lines
from the Italian Adriatic coast [51]. Taguchi et al. investigated the genetic diversity of
63 of the best Japanese sugar beet inbred lines using 33 cleaved amplified polymorphic
sequences (CAPS). In their study, the contribution of genetic and molecular characteristics
to the phenotypic variance of agronomically significant traits was evaluated using 38 SSR
markers [52]. The genetic diversity of sugar beet in Kazakhstan was determined using
randomly amplified polymorphic DNA (RAPD) markers, geomorphological traits, root
yield, and sugar content [53].
The molecular markers we selected were not specific enough to identify specific
traits and only reflected variation at the DNA level that was not necessarily expressed in
the phenotype, so the relationship between traits and markers was not further analyzed.
The use of a combination of phenotypic identification and molecular markers has been
widely employed to analyze genetic diversity. Morphological markers were the first genetic
markers to be used for genetic diversity analyses and have the advantage of being simple
and intuitive, but are susceptible to environmental influences and less stable. In contrast,
the use of molecular markers better reflects the genetic differences of germplasm resources
and makes up for the shortcomings of morphological identification, and the combination
of the two methods can improve the resolution of genetic diversity analysis. This whole
study is assisting breeding work as, before configuring hybrid combinations, it is necessary
to understand the phenotypic trait characteristics of existing sugar beet polyembryonic
germplasms to screen sugar beet germplasm with excellent target traits and provide a basis
Horticulturae 2024, 10, 120 13 of 16

for the innovation of sugar beet germplasm resources and varietal selection and breeding,
avoiding the blindness of traditional formulation methods of hybrid combinations.
SSR markers have a more comprehensive range of applications and are more common
and polymorphic. The amplification product band patterns of InDel markers are clearer and
more accurate than those of SSR markers, preventing errors brought on by specificity and
complexity. Given the advantages of these two markers, we decided to combine them to
improve the efficacy of this study and yield superior and reliable findings when examining
the genetic diversity of sugar beet germplasm. As PIC may precisely reflect the marker
polymorphisms in the population under investigation, it is an essential tool in genetic
research. The investigation results showed that PIC values higher than 0.50 were present
in 68.33% of the 60 highly polymorphic molecular marker pairs. Collectively, the tested
primers were polymorphic and extremely valuable. At the molecular level, the 129 sugar
beet germplasm samples showed a significant degree of genetic variation. However, the PIC
values of the SSR markers were greater than those of the InDel markers, indicating that the
microsatellites were genotypically diverse and highly informative. STRUCTURE was used
to divide the population into subgroups with distinct structures. The results demonstrated
that the 129 germplasms were categorized into three groups. The germplasms were divided
into three groups using cluster analysis, and all CMS and maintained lines were grouped
into a single category. This was in line with the findings of the population structure
research. The average genetic distance between the sterile lines and the maintained and
polyembryonic sugar beet germplasm was 0.315 when the genetic distance results were
combined. These findings suggest that the genetic correlation between the seven pairs
of sterile lines and the maintained and polyembryonic sugar beet germplasm resources
was relatively distant. Therefore, in future breeding efforts, superior germplasms can be
screened in each population based on the findings of the sugar beet germplasm population
structure, and, by combining the findings of genetic distance analysis, hybrid combinations
can be configured to predict hybrid advantage.
The genetic variation indices of 16 qualitative qualities varied from 0.138 to 1.395
regarding phenotype. Almost every trait in the thirteen traits was covered by more than
one genetic diversity index. Leaf hairiness was the only trait with a lower genetic diversity
index. A variation coefficient of more than 10% was shown by three of the four quantitative
parameters, indicating that the sugar beet germplasm resources had a high breeding
potential and were genetically rich. The four qualitative traits demonstrated a highly
significant positive correlation, and the descriptive traits also showed varying degrees of
correlation. These results indicated mutual promotion and constraints among the different
traits of sugar beet lines. The indirect selection of target traits could potentially be based on
a correlation analysis. The correlation between traits should be fully considered during
sugar beet cultivation to promote the coordinated development of each trait [17]. The PCA
results demonstrated that the first five principal components reflected the most significant
information in the 20 phenotypic traits with a cumulative contribution of 51.151%. The
section of the plant aboveground was designated as the first principal component. The
size and shape of the roots and leaves were represented in the second and third principal
components. The overall shape of the plant’s aboveground and belowground portions was
primarily expressed in the fourth and fifth principal components.
This study provided a theoretical foundation for germplasm innovation and vari-
ety selection. Further selection of genetically distant germplasms for hybridization can
improve sugar beet genetic diversity by introducing excellent sugar beet germplasm re-
sources worldwide. This strategy will enhance germplasm resources with increased genetic
information for the better development and utilization of sugar beet lines.

5. Conclusions
This study examined 60 pairs of molecular markers and 20 phenotypic traits to deter-
mine the genetic diversity of sugar beet germplasms in China. The findings demonstrated
that sugar beet germplasms displayed high genetic variation at the molecular level. More-
Horticulturae 2024, 10, 120 14 of 16

over, due to the superior resolution of the SSR markers over the InDel markers, they were
deemed more appropriate for genetic diversity research. Rich levels of genetic variation
in phenotypic traits were also seen in the sugar beet germplasm, suggesting a rich genetic
background in Chinese sugar beet germplasm.
The characteristics of sugar beet germplasm can be considered through breeding
identification and genetic diversity analyses of sugar beet germplasm resources. This will
help filter out the best sugar beet germplasm, lay the groundwork for selecting superior
hybrid combinations and obtaining high-quality innovative varieties, and provide theoreti-
cal references for future breeding work. This is crucial in accelerating the production of
high-yield high-quality sugar beet in China and achieving sustainable development in the
sugar beet industry.

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

mdpi.com/article/10.3390/horticulturae10020120/s1: Table S1: Sugar beet (Beta vulgaris L.) germplasm
resources used in the study; Table S2: The DNA molecular markers used in the study; Table S3:
Genetic Distance of 129 sugar beet germplasms; Table S4: Correlation analysis of 16 descriptive
characteristics of sugar beet germplasms.
Author Contributions: Conceptualization, F.P. and Z.W.; methodology, Z.W.; software, F.P.; valida-
tion, F.P., S.L., Z.P. and Z.W.; formal analysis, F.P.; investigation, F.P.; resources, S.L., Z.P. and Z.W.; data
curation, F.P. and Z.W.; writing—original draft preparation, F.P.; writing—review and editing, S.L.,
Z.P. and Z.W.; visualization, F.P.; supervision, Z.W.; project administration, Z.W.; funding acquisition,
S.L. and Z.W. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Special Fund for the Improvement of High-quality Sugar
Beet Varieties of the National Sugar Modern Agricultural Industrial Technology System grant number
CARS-170111 and the Basic Scientific Research Projects of provincial colleges and universities in the
Heilongjiang Province grant number 2022-KYYWF-1039.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.
Conflicts of Interest: The authors declare no conflicts of interest.

References
1. Wascher, F.L.; Stralis-Pavese, N.; McGrath, J.M.; Schulz, B.; Himmelbauer, H.; Dohm, J.C. Genomic distances reveal relationships
of wild and cultivated beets. Nat. Commun. 2022, 13, 2021. [CrossRef] [PubMed]
2. Wolfgang, A.; Temme, N.; Tilcher, R.; Berg, G. Understanding the sugar beet holobiont for sustainable agriculture. Front. Microbiol.
2023, 14, 1151052. [CrossRef] [PubMed]
3. Stevanato, P.; Chiodi, C.; Broccanello, C.; Concheri, G.; Biancardi, E.; Pavli, O.; Skaracis, G.N. Sustainability of the Sugar Beet
Crop. Sugar Tech 2019, 21, 703–716. [CrossRef]
4. Duraisam, R.; Salelgn, K.; Berekete, A.K. Production of beet sugar and bio-ethanol from sugar beet and it bagasse: A review. Int. J.
Eng. Trends Technol. 2017, 43, 222–233. [CrossRef]
5. McGrath, J.M. Assisted Breeding in Sugar Beets. Sugar Tech 2010, 12, 187–193. [CrossRef]
6. Li, J.; Lühmann, A.K.; Weißleder, K.; Stich, B. Genome-wide distribution of genetic diversity and linkage disequilibrium in elite
sugar beet germplasm. BMC Genom. 2011, 12, 484. [CrossRef] [PubMed]
7. Ćurčić, Ž.; Taški-Ajduković, K.; Nagl, N. Relationship between hybrid performance and genetic variation in self-fertile and
self-sterile sugar beet pollinators as estimated by SSR markers. Euphytica 2017, 213, 108. [CrossRef]
8. McGrath, J.M.; Panella, L.W. Sugar Beet Breeding. Plant Breed. Rev. 2018, 42, 167–218. [CrossRef]
9. Monteiro, F.; Frese, L.; Castro, S.; Duarte, M.C.; Paulo, O.S.; Loureiro, J.; Romeiras, M.M. Genetic and Genomic Tools to Asssist
Sugar Beet Improvement: The Value of the Crop Wild Relatives. Front. Plant Sci. 2018, 9, 74. [CrossRef]
10. Veloso, M.M.; Simões-Costa, M.C.; Guimarães, J.B.; Ribeiro, C.M.; Evaristo, I.; Espírito-Santo, D.; Pinto-Ricardo, C.; Paulo, O.S.;
Duarte, M.C. Genetic Diversity and Population Structure of Wild Beets (Beta spp.) from the Western Iberian Peninsula and the
Azores and Madeira Islands. Diversity 2021, 13, 593. [CrossRef]
11. Fugate, K.K.; Campbell, L.G.; Covarrubias-Pazaran, G.; Rodríguez-Bonilla, L.; Zalapa, J. Genetic diversity is enhanced in Wild ×
Cultivated hybrids of sugarbeet (Beta vulgaris L.) despite multiple selection cycles for cultivated traits. Genet. Resour. Crop Evol.
2021, 68, 2549–2563. [CrossRef]
12. Ramanatha Rao, V.; Hodgkin, T. Genetic diversity and conservation and utilization of plant genetic resources. Plant Cell Tissue
Organ Cult. 2002, 68, 1–19. [CrossRef]
Horticulturae 2024, 10, 120 15 of 16

13. Guo, S.; Ji, P.; Wang, J.; He, Y.; Zhang, Y.; Zhang, F.; Yun, Y.; Zhang, G. Estimation of Genetic Diversity between and within
Biparental Clones and Full-Sib Families of the Chinese Pine Using SSR Markers. Horticulturae 2023, 9, 1205. [CrossRef]
14. Lee, H.Y.; Ro, N.Y.; Jeong, H.J.; Kwon, J.K.; Jo, J.; Ha, Y.; Jung, A.; Han, J.W.; Venkatesh, J.; Kang, B.C. Genetic diversity and
population structure analysis to construct a core collection from a large Capsicum germplasm. BMC Genet. 2016, 17, 142.
[CrossRef]
15. Mai, T.T.P.; Hardner, C.M.; Alam, M.M.; Henry, R.J.; Topp, B.L. Phenotypic characterisation for growth and nut characteristics
revealed the extent of genetic diversity in wild macadamia germplasm. Agriculture 2021, 11, 680. [CrossRef]
16. Placide, R.; Shimelis, H.; Laing, M.; Gahakwa, D. Application of principal component analysis to yield and yield related traits
to identify sweet potato breeding parents. J. Trop. Agric. 2015, 92, 1–15. Available online: https://www.researchgate.net/
publication/274958783 (accessed on 10 September 2023).
17. Liu, D.; Wang, X.; Li, W.; Li, J.; Tan, W.; Xing, W. Genetic Diversity Analysis of the Phenotypic Traits of 215 Sugar Beet Germplasm
Resources. Sugar Tech 2022, 24, 1790–1800. [CrossRef]
18. Liu, S.; Zhong, H.; Zhang, F.; Wang, X.; Wu, X.; Wang, J.; Shi, W. Genetic Diversity and Core Germplasm Research of 144 Munake
Grape Resources Using 22 Pairs of SSR Markers. Horticulturae 2023, 9, 917. [CrossRef]
19. Varshney, R.K.; Graner, A.; Sorrells, M.E. Genic microsatellite markers in plants: Features and applications. Trends Biotechnol.
2005, 23, 48–55. [CrossRef]
20. Abu-Ellail, F.F.B.; Salem, K.F.M.; Saleh, M.M.; Alnaddaf, L.M.; Al-Khayri, J.M. Molecular Breeding Strategies of Beetroot (Beta
vulgaris ssp. vulgaris var. conditiva Alefeld). Adv. Plant Breed. Strateg. Veg. Crops 2021, 8, 157–212. [CrossRef]
21. Stevanato, P.; Broccanello, C.; Biscarini, F.; Del Corvo, M.; Sablok, G.; Panella, L.; Stella, A.; Concheri, G. High-throughput
RAD-SNP genotyping for characterization of sugar beet genotypes. Plant Mol. Biol. Report. 2014, 32, 691–696. [CrossRef]
22. Simko, I.; Eujayl, I.; van Hintum, T.J.L. Empirical evaluation of DArT, SNP, and SSR marker-systems for genotyping, clustering,
and assigning sugar beet hybrid varieties into populations. Plant Sci. Int. J. Exp. Plant Biol. 2012, 184, 54–62. [CrossRef] [PubMed]
23. Kalia, R.K.; Rai, M.K.; Kalia, S.; Singh, R.; Dhawan, A.K. Microsatellite markers: An overview of the recent progress in plants.
Euphytica 2011, 177, 309–334. [CrossRef]
24. Taški-Ajduković, K.; Nagl, N.; Ćurčić, Ž.; Zorić, M. Estimation of genetic diversity and relationship in sugar beet pollinators
based on SSR markers. Electron. J. Biotechnol. 2017, 27, 1–7. [CrossRef]
25. Smulders, M.J.M.; Esselink, G.D.; Everaert, I.G.; de Riek, J.; Vosman, B. Characterisation of sugar beet (Beta vulgaris L. ssp. vulgaris)
varieties using microsatellite markers. BMC Genet. 2010, 11, 41. [CrossRef] [PubMed]
26. Srivastava, S.; Pathak, A.; Kumar, R.; Joshi, B. Genetic diversity of sugar beet genotypes evaluated by microsatellite DNA markers.
J. Environ. Biol. 2017, 384, 777–783. [CrossRef]
27. Li, J.; Schulz, B.; Stich, B. Population structure and genetic diversity in elite sugar beet germplasm investigated with SSR markers.
Euphytica 2010, 175, 35–42. [CrossRef]
28. Andrello, M.; Henry, K.; Devaux, P.; Verdelet, D.; Desprez, B.; Manel, S. Insights into the genetic relationships among plants of
Beta section Beta using SNP markers. Theor. Appl. Genet. 2017, 130, 1857–1866. [CrossRef]
29. Xiang, X.; Li, C.; Li, L.; Bian, Y.; Kwan, H.S.; Nong, W.; Cheung, M.K.; Xiao, Y. Genetic diversity and population structure of
Chinese Lentinula edodes revealed by InDel and SSR markers. Mycol. Prog. 2016, 15, 37. [CrossRef]
30. Wu, K.; Yang, M.; Liu, H.; Tao, Y.; Mei, J.; Zhao, Y. Genetic analysis and molecular characterization of Chinese sesame (Sesamum
indicum L.) cultivars using Insertion-Deletion (InDel) and Simple Sequence Repeat (SSR) markers. BMC Genet. 2014, 15, 35.
[CrossRef]
31. Lü, Y.; Cui, X.; Li, R.; Huang, P.; Zong, J.; Yao, D.; Li, G.; Zhang, D.; Yuan, Z. Development of genome-wide insertion/deletion
markers in rice based on graphic pipeline platform. J. Integr. Plant Biol. 2015, 57, 980–991. [CrossRef]
32. Mahmoodi, R.; Dadpour, M.R.; Hassani, D.; Zeinalabedini, M.; Vendramin, E.; Leslie, C.A. Composite core set construction and
diversity analysis of Iranian walnut germplasm using molecular markers and phenotypic traits. PLoS ONE 2021, 16, e0248623.
[CrossRef]
33. Abbasi, Z.; Arzani, A.; Majidi, M.M. Evaluation of Genetic Diversity of Sugar Beet (Beta vulgaris L.) Crossing Parents Using
Agro-morphological Traits and Molecular Markers. J. Agric. Ence Technol. 2014, 16, 1397–1411. Available online: https:
//jast.moares.ac.ir/article-23-6906-en.pdf (accessed on 10 September 2023).
34. Chalbi, A.; Chikh-Rouhou, H.; Mezghani, N.; Slim, A.; Fayos, O.; Bel-Kadhi, M.S.; Garcés-Claver, A. Genetic Diversity Analysis of
Onion (Allium cepa L.) from the Arid Region of Tunisia Using Phenotypic Traits and SSR Markers. Horticulturae 2023, 9, 1098.
[CrossRef]
35. Cui, P. Descriptors and Date Standard for Beet (Beta vulgaris L.); China Agriculture Press: Beijing, China, 2006; pp. 8–42.
36. Doyle, J. DNA Protocols for Plants. In Molecular Techniques in Taxonomy; Springer: Berlin/Heidelberg, Germany, 1991; pp. 283–293.
[CrossRef]
37. Fugate, K.K.; Fajardo, D.; Schlautman, B.; Ferrareze, J.P.; Bolton, M.D.; Campbell, L.G.; Wiesman, E.; Zalapa, J. Generation and
Characterization of a Sugarbeet Transcriptome and Transcript-Based SSR Markers. Plant Genome 2014, 7, plantgenome2013-11.
[CrossRef]
38. Yeh, F. Population genetic analysis of co-dominant and dominant markers and quantitative traits. Belg. J. Bot 1997, 129, 157.
39. Ross-Ibarra, J. Genetic Data Analysis II. Methods for Discrete Population Genentic Data. Econ. Bot. 2002, 56, 216. [CrossRef]
Horticulturae 2024, 10, 120 16 of 16

40. Liu, K.; Muse, S.V. PowerMarker: An integrated analysis environment for genetic marker analysis. Bioinformatics 2005, 21,
2128–2129. [CrossRef] [PubMed]
41. Pritchard, J.K.; Stephens, M.; Donnelly, P. Inference of population structure using multilocus genotype data. Genetics 2000, 155,
945–959. [CrossRef]
42. Evanno, G.; Regnaut, S.; Goudet, J. Detecting the number of clusters of individuals using the software structure: A simulation
study. Mol. Ecol. 2005, 14, 2611–2620. [CrossRef]
43. Earl, D.; Vonholdt, B.M. STRUCTURE HARVESTER: A website and program for visualizing STRUCTURE output and implement-
ing the Evanno method. Conserv. Genet. Resour. 2012, 4, 359–361. [CrossRef]
44. Nei, M. Genetic distance between populations. Am. Nat. 1972, 106, 283–292. [CrossRef]
45. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol.
Evol. 2016, 33, 1870–1874. [CrossRef] [PubMed]
46. Bedeian, A.G.; Mossholder, K.W. On the use of the coefficient of variation as a measure of diversity. Organ. Res. Methods 2000, 3,
285–297. [CrossRef]
47. Shannon, C.E.; Weaver, W. The Mathematical Theory of Communication; University of Illinois Press: Urbana, IL, USA, 1949; p. 117.
48. Ortiz-Burgos, S. Shannon-Weaver Diversity Index. In Encyclopedia of Estuaries; Kennish, M.J., Ed.; Springer: Dordrecht, The
Netherlands, 2016; pp. 572–573.
49. Patterson, N.J.; Price, A.L.; Reich, D. Population Structure and Eigenanalysis. PLoS Genet. 2006, 2, e190. [CrossRef] [PubMed]
50. Kleine, M.; Voss, H.H.; Cai, D.; Jung, C. Evaluation of nematode-resistant sugar beet (Beta vulgaris L.) lines by molecular analysis.
Theor. Appl. Genet. 1998, 97, 896–904. [CrossRef]
51. Saccomani, M.; Stevanato, P.; Trebbi, D.; McGrath, J.M.; Biancardi, E. Molecular and morpho-physiological characterization of sea,
ruderal and cultivated beets. Euphytica 2009, 169, 19–29. [CrossRef]
52. Taguchi, K.; Kuroda, Y.; Okazaki, K.; Yamasaki, M. Genetic and phenotypic assessment of sugar beet (Beta vulgaris L. subsp.
vulgaris) elite inbred lines selected in Japan during the past 50 years. Breed Sci 2019, 69, 255–265. [CrossRef]
53. Abekova, A.M.; Yerzhebayeva, R.S.; Bastaubayeva, S.O.; Konusbekov, K.; Bazylova, T.A.; Babissekova, D.I.; Amangeldiyeva, A.A.
Assessment of sugar beet genetic diversity in the Republic of Kazakhstan by using RAPD markers and agromorphological traits.
Sabrao J. Breed. Genet. 2022, 54, 67–78. [CrossRef]

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