Assessment of genetic diversity of Prunus salicina 'Shazikongxinli' by morphological traits and molecular markers

Prunus salicina 'Shazikongxinli' is one of China’s most economically valuable and reputable Prunus salicina cultivars. Understanding the genetic diversity and population structure of 'Shazikongxinli' is imperative for excellent germplasm breeding and the preservation of genetic resources. This work used morphological markers, inter-retrotransposon amplified polymorphism (IRAP), and inter simple sequence repeat (ISSR) to assess the genetic variation status of 50 'Shazikongxinli', with 18 plum cultivars selected as the control outgroup. The results showed that the average Shannon–Weaver diversity index (H') of 32 qualitative traits in 50 tests 'Shazikongxinli' was 0.557, the average coefficient of variation of 11 quantitative traits was 15.57%, and a total of 232 and 105 polymorphic loci were obtained from 22 IRAP and 15 ISSR primers, respectively. All three marker systems showed relatively rich polymorphism, especially the IRAP markers. This might be related to the nature of the retrotransposons in the IRAP markers, which might be more suitable for intraspecific variability detection than ISSR. In addition, all three markers clustered the 68 tested germplasms into two groups. All of 'Shazikongxinli' clustered into one group, and most outgroup plum cultivars clustered into another group. This suggested a relatively narrow genetic base within the 'Shazikongxinli' population. These results would be useful in understanding the genetic diversity of the germplasm resources of 'Shazikongxinli' and provide a basic theoretical reference for breeding superior germplasm.


Introduction
Prunus salicina 'Shazikongxinli', belonging to the Rosaceae family of the genus Prunus, is an endemic plum cultivar cultivated in Yanhe County, Southwest China's Guizhou province.It is a native Prunus salicina cultivar that has been cultivated in Yanhe for over 160 years and earned the reputation of "China's Quality Plum."In addition, it has been listed as a National Geographical Indication Product of China.
By 2021, the Shazikongxinli plantation in Yanhe County had expanded to a 30 km 2 area, with an output of approximately 20,000 tons annually (Zhang et al. 2020).Under long-term natural evolution and human selection, the 'Shazikongxinli' gradually formed a cultivar group.At the same time, an extensive area of 'Shazikongxinli' had been planted in Yanhe County in recent years.However, the production of seedlings had been chaotic, and the source of the scion had been heterogeneous, further promoting the diversification of 'Shazikongxinli' cultivars (Wu et al. 2022).Different strains provided germplasm resources for the excavation of high-quality germplasm.But it was unclear whether these differences were simply due to environmental factors and different cultivation techniques and whether the genetic information had been mutated.Therefore, it was necessary to assess the level of genetic variation in the 'Shazikongxinli' population.
Genetic diversity reflects a species' genetic background, breeding potential and utilization value (Liu et al. 2007;Mei et al. 2021).Learning the level of genetic diversity in germplasm is an essential basis for studying the origin and evolution of a species and is also a crucial step for the optimal conservation and breeding of new germplasm (Tao et al. 2014;Urrestarazu et al. 2018).Morphological characters are an important tool for analyzing crop genetic diversity and could estimate the activity of genotypes in given environmental conditions (Rana et al. 2015;Ruchi et al. 2020).For crops such as fruit, morphological markers have tremendous reference value and can visually reflect the merit of the germplasm.Moreover, molecular markers are the primary technique used in genetic variability analysis and are considered more reliable.Several studies were carried out to evaluate the genetic diversity of plums using morphological traits (Zhang and Zhou 1998;Yu et al. 2011) and different types of molecular markers, such as randomly amplified polymorphic DNA (RAPD) (Ben et al. 2015), amplified fragment length polymorphism (AFLP) (Goulao et al. 2001), simple sequence repeats (SSR) (Basilio et al. 2012;Pop et al. 2018;Acuna et al. 2019), inter-simple sequence repeat (ISSR) (Liu et al. 2007;Wu et al. 2019), and single nucleotide polymorphism (SNP) (Wei et al. 2021).Until now, there have been few studies of the genetic variation in Prunus salicina 'Shazikongxinli'.The current research used morphological traits, inter-retrotransposon amplified polymorphism (IRAP), and ISSR to analyze the variation of 'Shazikongxinli' germplasm resources.It aimed to assess the overall genetic diversity among currently cultivated 'Shazikongxinli' to establish theoretical guidance toward breeding superior 'Shazikongxinli' germplasm.

Plant materials
Based on the field survey, 50 healthy adult 'Shazikongxinli' trees were randomly chosen as experimental germplasms from the Prunus salicina 'Shazikongxinli' orchard in September 2020, Nanzhuang Village, China.Eighteen other plum cultivars were also selected as controls at the plum germplasm conservation facility in Baiyi Town, China (Table 1; Fig. 1).Samples of fresh and healthy young blades were collected, frozen in liquid nitrogen, and stored at -80 ℃ for isolation of genomic DNA.

Morphological analysis
Since September 2020, 43 morphological traits (including trait characteristics of flowers, leaves, fruits, etc.) of 68 germplasms were observed following the morphological trait testing standard for plum (GBT19557-8 Guide for testing specificity, consistency, and stability of new plant cultivars-plum).Qualitative traits were scored according to the measurement standard GBT19557-8, and quantitative traits were directly recorded as measured values (Table 2).To avoid experimental error, the same person measured each morphological trait.

Genomic DNA extraction
Genomic DNA from each sample was extracted using the Plant Genome DNA Extraction Kit (Tiangen, Beijing).The DNA quality and concentration were detected using a spectrophotometer (Multiskan Go 1510, Thermo).The integrity of total DNA was detected using 1% agarose gel electrophoresis.Each qualified DNA sample was diluted to approximately 30 ng μL −1 .
Vol.: (0123456789) Molecular marker analysis IRAP: In this work, we developed 65 IRAP primers based on the conserved sequences of the reverse transcriptase of plum reverse transcription transposons Ty1-copia and Ty3-gypsy.By screening, 22 IRAP primers with good repeatability and high polymorphism were obtained for profiling (Table 3).The PCR was performed in a reaction volume of 10 μL containing 1.0 μL DNA (30 ng μL −1 ), 5.0 μL masterMix (Thermo), 1.3 μL primers (10 −5 mol L −1 ), 2.7 μL ddH 2 O. PCR amplification using the following conditions: 4 min at 94 ℃, followed by 40 cycles of 30 s at 94 ℃, 30 s at the specific annealing temperature, and 72 ℃ extension for 1 min, with a final extension of 10 min at 72 ℃.The amplification products were separated on 1.5% agarose gel using the 2000 bp (Tiangen, Beijing) ladder as standard in 1 × TAE buffer.
ISSR: A set of 100 primers were screened, out of which 15 primers were found reproducible and selected for profiling (Table 3).The PCR reaction system was the same as for IRAP.Each reaction was repeated at least twice.

Data analysis
Fifty Prunus salicina 'Shazikongxinli' germplasms were used as the experimental group, and 18 other plum cultivars were used as the control group.The data was analyzed using Microsoft Excel 2019, and the standard deviation and coefficient of variation (CV) of the quantitative traits alongside the Shannon-Weaver diversity index (H') of each qualitative trait were quantified.The Shannon-Weaver diversity index calculation formula was H' = − Σ Pi ln Pi, where Pi was the ratio of the number of materials in the ith level of a trait to the total number of materials (Yao et al. 2022).Genetic distances were calculated using NTSYS-pc2.10 (Rohlf 2000), and clustering analysis was performed by MEGA11 based on the unweighted pair-group method with arithmetic means (UPGMA), optimized the cluster map through iTOL (https:// itol.embl.de/).For molecular markers, the amplification results of each primer in each individual were scored as 0 (absence) and 1 (presence); finally, a binary data matrix of ISSR or IRAP amplification locus was obtained.Genetic diversity parameters included the number of alleles (Na), the number of effective alleles (Ne), Nei's gene diversity (H) (Nei 1973), and Shannon information index (I) (Shannon 1949) were calculated by Popgene32 (Yeh 1997) software.The test germplasms were subjected to a clustering analysis using the NTSYS-pc2.10(Rohlf 2000) and MEGA11 software.The cluster map was optimized through iTOL (https:// itol.embl.de/).

Morphological analysis
The quality traits analysis showed that 28 of the 32 quality traits had variation in the experimental group (Table 4), while 31 traits in the control group had different phenotypes.Moreover, the four traits: calyx pose, petal upper edge shape, seed widest position, and leaf edge tooth type of the 50 'Shazikongxinli' produced only one phenotype and no other variable traits.In the experimental group, the Shannon-Weaver diversity index (H') varies from 0 to 1.199, with an average value of 0.557.There were 18 traits with an index greater than 0.557.Among them, the H' index of new shoots color was the largest,  resulting in four types of variation, with greenishbrown predominating (0.46).In the control group, the Shannon-Weaver diversity index ranged from 0 to 1.391, with an average value of 0.811.There were 16 traits with a diversity index greater than 0.811.The highest polymorphism was observed in flesh color, varied from light yellow (0.21), yellow (0.43), orange (0.07), green (0.07), to red (0.21).In addition, the Shannon-Weaver diversity index was higher in the experimental group than in the control group for seven quality traits, including new shoots color, new shoots length, new shoots internode length, leaf gloss, fruit symmetry, flesh Juice, and viscosity of seed and flesh.
Quantitative trait analysis statistics showed that the variation coefficients of 11 traits in the experimental group ranged from 9.74 to 22.88%, with an average value of 15.57% (Table 5).There were four traits with a coefficient of variation greater than 15.57%, which included: leaf area, weight per fruit, fruit hardness, and seed weight.In the control group, the variation range of the coefficient of variation ranged from 11.50 to 55.39%, with an average value of 26.89%.Four traits, including weight per fruit, fruit hardness, Calyx color 1: Yellow-green, 2: brownish green, 3: green, 4: yellow-red 3 Petal shape 1: Round, 2: obovate, 3: oval, 4: wide oval 4 Corolla size 3: < 1.5 cm, 5: 1.5-2.0cm, 7: > 2.0 cm 5 Petal stacking off condition 3: Petals separate, 5: petals meet, 7: petals overlapping 6 Petal upper edge shape 1: Whole, 2: concave, 3: serrated 7 Stigma position 3: Stigma below stamens, 5: stigma as high as stamens, 7: stigma above stamen 8 New shoots color 1: Tawny, 2: green-brown, 3: green, 4: reddish brown, 5: fuchsia 9 New shoots length 3: < 30 cm, 5: 30-70 cm, 7: > 70 cm 10 New shoots internode length 3: < 1.5 cm, 5: Seed to fruit ratio Average of 10 fruits seed weight, and seed-to-fruit ratio, had coefficients of variation higher than 26.89%.Among both test germplasm groups, the weight per fruit had the highest coefficient of variation, whereas leaf length had the smallest.Also, the experimental group's leaf width and area had greater coefficients of variation than the control group reported.A dendrogram of 68 test germplasms was constructed based on the UPGMA method and grouped germplasm into five clusters (Fig. 2).Cluster 1 included 20 'Shazikongxinli' and two other plum cultivars, which had significantly larger leaves and mainly were oval.Cluster 2 also included 20 'Shazikongxinli' and two different plum cultivars, and the fruit weight of this cluster was significantly more extensive than that of the other 'Shazikongxinli' cluster groups.Cluster 3 included nine 'Shazikongxinli', with the highest soluble solid content and significantly smaller seed weights.In addition, there was only one 'Shazikongxinli' accession in Cluster 4, and this germplasm had a much smaller leaf size and leaf stalk length than the other groups.Taken as a whole, all 50 'Shazikongxinli' plums could be classified into one group, while the 14 plum cultivars in the control group were classified into another group.Cuihong Plum, Red Heart Plum, Fengtang Plum, and Xingxing Plum were clustered with 50 'Shazikongxinli' germplasms, indicating that 'Shazikongxinli' and these four plum cultivars were more similar in phenotypic traits and closer in kinship.

IRAP analysis
In the experimental group, the 22 selected IRAP primers generated 249 reproducible amplified loci, giving 232 polymorphic loci altogether (Table 6).Each primer generated five to twenty obvious loci; the polymorphism ratio ranged from 66.7% to 100%, and the average polymorphism ratio was 90.7%.There were ten primers with a polymorphism ratio of 100%, and the lowest was Ty1-1 (66.7%).Meanwhile, 22 IRAP primers amplified 261 loci in 18 plum cultivars of the control group, with an average of 11.86 loci per primer, and there were 249 polymorphic loci for an average polymorphic ratio of 93.3%.
Popgene32 software was used to analyze the number of alleles observed (Na), the number of effective alleles (Ne), Nei's gene diversity (H), and the Shannon information index (I) (Table 6).The Fifty 'Shazikongxinli' used in the test had averages of Na and Ne of 1.907 and 1.409, respectively, and the range of Nei's gene diversity (H) was 0.139 (Ty3-9)-0.369(Ty3-2) with a mean value of 0.255; The Shannon information index (I) ranged from 0.244 (Ty3-9) to 0.547 (Ty3-2) and had a mean value of 0.397.Likewise, the mean values of Na and Ne for the other 18 plum cultivars in the control group were 1.933 and 1.477, respectively.The H value ranged from 0.172 (Ty1-8) to 0.436 (Ty1-9), with a mean value of 0.292.Besides, the range of the I value was 0.278 (Ty1-8)-0.626(Ty1-9), and the mean value was 0.449.Overall, all of the experimental groups' genetic indices were lower compared to those of the control group.This was in accordance with the contention that there would be less diversity within a cultivar than between cultivars.
The genetic relationships of the tested germplasms were unraveled using the Neighbor-joining method based on Jaccard similarity coefficients computed with IRAP markers.The average genetic similarity coefficient among the 50 'Shazikongxinli' germplasms was 0.510, while the average coefficient between the 50 'Shazikongxinli' and 18 other plum cultivars was 0.411.In addition, the average similarity coefficient among the 18 plum cultivars was 0.418.Two main clusters, namely, I and II, were obtained from the dendrogram taking 0.46 as a threshold (Fig. 3).All 50 test 'Shazikongxinli' were clustered together (Cluster I), and two other plum cultivars were also clustered into I (Red Heart plum and Xingxing plum).This indicated that these two plums were more closely related to 'Shazikongxinli' fro IRAP marker results.

ISSR analysis
Out of 100 ISSR primers, 15 were selected for further analysis based on unambiguous and reproducible loci.Fifteen ISSR primers amplified 141 loci in 50 'Shazikongxinli', including 105 polymorphic loci (Table 7).On average, each primer amplified 9.4 loci with 7.0 polymorphic loci.The average polymorphism ratio was 73.5%, ranging from 50 to 100%.And there are three primers with a polymorphism ratio of 100%.Further, 15 ISSR primers amplified 155 loci, including 136 polymorphic loci, in the 18 plum cultivars used as the control group.On average, each primer amplified 10.3 loci, of which 9.1 were polymorphic.The polymorphism ratios of the 15 ISSR primers ranged from 62.5 to 100%, with an average polymorphism ratio of 86.4%.In the experimental group, the average number of alleles observed per primer (Na) was 1.735, ranging from 1.500 to 2.000, the number of effective alleles (Ne) was 1.137 (1.032 to 1.252), the Nei's gene diversity (H) was 0.100 (0.031 to 0.158), and the Shannon information index (I) was 0.176 (0.073 to 0.252).For the other 18 plum cultivars, the mean values of Na and Ne were 1.864 (1.625 to 2.000) and 1.366 (1.219 to 1.546), respectively.The H value was 0.236 (0.159 to 0.328), and the I value was 0.375 (0.274 to 0.491) (Table 7).NTSYS-pc2.10calculated the genetic similarity coefficients of 68 test plum germplasms.The results showed that the average genetic similarity coefficient among the 50 'Shazikongxinli' germplasms was 0.823, while the average coefficient between the 50 'Shazikongxinli' and 18 plum cultivars was 0.502.In addition, the average similarity coefficient among the 18 other plum cultivars was 0.558.A dendrogram drawn from the similarity coefficients based on a neighbor-joining method was constructed, and the total samples were classified into two clusters taking 0.70 as a threshold; cluster one was named I, while cluster two was named II (Fig. 4).For the ISSR marker, Cluster I consisted of 50 germplasms; all were 'Shazikongxinli', while Cluster II contained 18 other plum cultivars.Nearly all of the 'Shazikongxinli' samples were clustered independently in the IRAP and ISSR markers, showing that the cluster analysis results were accurate and trustworthy.

Discussion
In order for species to be conserved and managed sustainably, it was essential to comprehend genetic diversity (Lynch et al. 1999).Understanding the level of genetic variation and kinship in the germplasm resources of 'Shazikongxinli' could help select diverse parental combinations with maximum genetic variability efficiently and breed superior progeny (Liu et al. 2011;Guan et al. 2020).The present research estimated the genetic diversity among 68 plum germplasms using morphological markers and two molecular markers (IPAR and ISSR).The combined use of these three marker datasets allowed an accurate assessment of the level of genetic diversity in 'Shazikongxinli' germplasm resources.The Shannon-Weaver diversity index (H') and coefficient of variation were essential indicators of the diversity of germplasm resources for traits   (Yao et al. 2022;Hu et al. 2022).In the current study, the mean H' and CV values of 43 traits in 50 'Shazikongxinli' were smaller than the control group.Nonetheless, 50 'Shazikongxinli' exhibited higher levels of variation in some morphological traits like new shoots length, leaf area, weight per fruit, fruit hardness, and seed weight.This may be caused by the degradation of the caste of local cultivars during long-term cultivation and the mixing of different strains produced during seedling breeding.Leaf and fruit traits were considered reliable in evaluating genetic relationships and taxonomic studies (Almajali et al. 2012).In the present study, both experimental groups of materials exhibited significant variation in morphological traits comprising weight per fruit, leaf area, seed weight, and fruit hardness.This result was similar to the conclusions of Yu et al. (2011) and Li et al. (2011) in Chinese plum germplasm resources, indicating that Chinese plums were rich in polymorphism in leaves and fruits.The variation level of 'Shazikongxinli' in fruit traits was high, which was not conducive to the development of 'Shazikongxinli' to a certain extent.
The uneven quality of the fruit tended to damage the brand reputation of 'Shazikongxinli'.On the other hand, it also provided a source of germplasm samples for breeding new 'Shazikongxinli' cultivars with different shapes, colors, and hardness (Wang et al. 2015).
The genetic diversity parameters were an essential indicator for measuring the variation of genetic information (Wu et al. 2019;Brenda et al. 2022).Both molecular markers in this study showed high levels of polymorphism in the test germplasms.By comparing the results of the two molecular markers (IRAP and ISSR) in the experimental group and the control group, it was found that the polymorphism ratio, Na, Ne, H, and I values of 18 plum germplasms were higher than those of 50 'Shazikongxinli' in the experimental group.This was consistent with the expectation that the level of genetic diversity within a single cultivar was less than that of multiple cultivars, and although the number of test samples in the experimental group was much larger than that of the control group, the plum cultivars in the control group were different cultivars formed through long-term mutation or hybridization and thus had richer genetic information (Kazija et al. 2014;Sehic et al. 2015).In this study, several genetic diversity indices of the 50 'Shazikongxinli' tested were also relatively high, especially for the IRAP markers.This indicated that the current genetic information within the 'Shazikongxinli' population had been relatively abundant and also showed that the IRAP primers developed in this study had high variation detection ability.The findings of this study also revealed that IRAP markers reported DNA polymorphisms at a higher level than ISSR.This could be due to the different nature of the genomic region coverage and loci number of these two techniques, which reveal different characteristics of genetic variation (Karim et al. 2017).The IRAP marker was developed based on the random insertion of retrotransposon to generate polymorphisms (Kalendar et al. 1999).Retrotransposons could be activated under special conditions, such as adversity, to generate new insertional mutations.Thus, compared with SSR, ISSR, and other markers for interspecific genealogy studies, IRAP markers were more suitable for intraspecific variation detection and analysis of genetic variation and kinship at the individual level within a species (Kalendar and Alan 2014).
Using phylogenetic analysis, the morphological markers and two molecular markers had similar clustering results, and both were able to cluster the 68 test germplasms into two groups.All Fifty 'Shazikongxinli' germplasms were clustered together, while most of the other plum cultivars in the control group were clustered into another category, suggesting that these marker systems were effective in studying genetic diversity in the germplasm resources of 'Shazikongxinli'.Furthermore, in the clustering results for both morphological and IRAP markers, both Red Heart plum and Xingxing plum were clustered together with 50 'Shazikongxinli', indicating that these two plum cultivars could be more closely related to 'Shazikongxinli'.Moreover, according to the clustering results, the genetic background of the 'Shazikongxinli' gourd was inferred narrow in the present study (Ruchi et al. 2020).Hence, it became crucial to broaden the genetic base of the 'Shazikongxinli' gourd to breed superior germplasm.This could be achieved by the introgression of genes from other cultivars or wild species with enormous variability (Martinez et al. 2003;Wang et al. 2015).

Conclusion
In brief, the investigation revealed a degree of genetic variation in the 'Shazikongxinli' population, providing a fundamental theoretical basis for selecting and breeding excellent accessions to the 'Shazikongxinli' germplasm.In addition, morphological markers, ISSR, and IRAP markers showed high levels of DNA polymorphism in plums and could be used to assess the genetic diversity and relationships.The combined use of several marker technologies will be more useful in the integrated evaluation of the genetic diversity of 'Shazikongxinli' from different genetic marker regions.Meanwhile, IRAP markers were more suitable than ISSR markers for detecting intra-cultivar or individual-level variability due to the nature of the markers.

Fig. 3
Fig. 3 Dendrogram generated of IRAP marker based on Neighbor-joining method

Fig. 4
Fig. 4 Dendrogram generated of ISSR marker based on Neighbor-joining method

Table 1
Detailed information on plum germplasms used in this investigation Vol:. (1234567890)

Table 2
The characterization and taxonomic values of 32 morphological traits of plum Stick to the petals, 2: separate from petals, 3: curved toward the pedicel 2

Table 3
List of IRAP and ISSR primer sequences.The information includes primer names, annealing sequences 5′-3′, and temperature (Tm)

Table 5
Statistical results of 11 quantitative traits of 68 test germplasms SD Standard deviation, CV Coefficient of variation

Table 6
Polymorphism information of 22 IRAP primers AB amplified bands, PB polymorphic bands, P% polymorphism%, Na number of alleles observed, Ne number of effective alleles, H Nei's gene diversity, I Shannon information index

Table 7
Polymorphism information of 15 ISSR primers AB amplified bands, PB polymorphic bands, P% polymorphism%, Na number of alleles observed, Ne number of effective alleles, H Nei's gene diversity, I Shannon information index Vol.: (0123456789)