The western part of South America is a centre of diversity for tomatoes, but genetic diversity studies are lacking for parts of that region, including Bolivia. We used 11 simple sequence repeat (SSR) markers (including seven novel markers) to evaluate genetic diversity and population structure of 28 accessions , and to compare their genetic variation against phenotypic traits, geographical origin and altitude. In total, 33 alleles were detected across all loci, with 2–5 alleles per locus. The top three informative SSRs were SLM6-11, LE20592 and TomSatX11-1, with polymorphism information content (PIC) of 0.65, 0.55 and 0.49, respectively. The genetic diversity of Bolivian tomatoes was low, as shown by mean expected heterozygosity (He) of 0.07. Analysis of molecular variance (AMOVA) revealed that 77.3% of the total variation was due to variation between accessions. Significant genetic differentiation was found for geographical origin, cultivation status, fruit shape, fruit size and growth type, each explaining 16–23% of the total variation. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree and principal coordinate analysis (PCoA) scatter plot both revealed differentiation between accessions with determinate flowers and accessions with indeterminate flowers, regardless of cultivation status. The genetic profiles of the accessions suggest that the Bolivian tomato gene pool comprises both strictly self-pollinating and open-pollinating genotypes. S. lycopersicum L.) [S. lycopersicum [Bolivia, Chile, Ecuador, the Galapagos Islands and Peru together constitute the centre of origin and distribution of tomatoes (icum L.) , althougicum L.) . Wild toicum L.)  and may icum L.) . In Boliicum L.)  and publicum L.) ,7. Some icum L.)  and a gepersicum . A wide persicum ,9,10. HoRecent advances in genomics and molecular technologies have enabled the characterisation of genetic diversity and its effective use in commercial breeding . The genCurrent knowledge on the genetic variation in Bolivian core germplasm is insufficient. Further research is needed to understand the genetic relationship between Bolivian tomato germplasm and the domestication process, and also to unveil desirable quality and agronomic traits . The aimThe cultivated and wild tomato germplasm obtained from different sources for this study are referred to hereafter as “accessions” for the sake of simplicity. The 28 tomato accessions analysed represented cultivars, advanced lines, landraces and wild populations . Three hSeeds of the 28 accessions were planted in 4.5-L plastic trays filled with nutrient-rich soil (0.08 L per plug) in a greenhouse at the Department of Plant Breeding, Swedish University of Agricultural Sciences (SLU). The emerging seedlings were grown under day/night temperature of ±24 °C/19 °C, 16 h of light and 60% relative humidity until sampling of leaf tissue for DNA extraction at one month after planting. Young leaf tissue was sampled separately from 10 individual plants of all accessions except one, which was represented by nine plants . The fresh leaf tissue taken from each plant was placed in a separate 2-mL Eppendorf tube containing two glass beads with diameter 3 mm, and immediately dipped in liquid nitrogen. The frozen samples were stored at −80 °C until DNA extraction.g for 3 min. A 200 μL subsample of the supernatant of each sample was transferred to a 96-well plate and DNA extraction was performed with a QIAamp DNA kit, using a QIAcube HT extraction robot . The integrity of the extracted DNA was evaluated by agarose gel electrophoresis (1.5% w/v) and the quantity was determined using a NanoDrop spectrophotometer . All extracted DNA samples were kept at −20 °C until polymerase chain reaction (PCR) analysis, while the working solutions (5 ng/μL) were kept at 4 °C for up to 24 h. The frozen leaf samples were homogenised for 1 min at 30 Hz in a Mixer Mill . Then 400 μL of CTAB-based buffer  were added to each sample, followed by incubation for 15 min at 56 °C and centrifugation  at 9000× S. lycopersicum cultivar ‘Heinz 1706’ ) was used to identify SSRs in the tomato genome, which were then utilised as new genomic analysis resources for tomato. First, genomic regions of 400 bp to 1200 bp, representing the 12 tomato chromosomes, were randomly sampled. These sequences were searched for identification of dinucleotide and trinucleotide repeat motifs, using WebSat, internet-based software developed for SSR identification [The genome of the fication ,20, targTen genotypes were selected from the different tomato accessions for a first testing of the newly designed primer-pairs in amplifying the target SSR loci under optimised PCR conditions (described below). Twelve of the primer-pairs amplified extra fragments in addition to the target loci, and were therefore excluded, while the remaining primer-pairs amplified only their target loci. To confirm that they matched the target sequences, the amplified products of the 12 primer-pairs were purified and sequenced. Thereafter, the PCR products were purified using E.Z.N.A. Cycle Pure Kit V-spin  and 2 μL of 10 μM sequencing primer and 15 μL of 1 ng/μL purified PCR product for each sample were mixed and sent to Eurofins Genomics Sequencing GmbH , where Sanger sequencing was conducted. Each amplified product was sequenced with both the forward and reverse primers used for the PCR. The DNA sequences of the PCR products were then aligned with their corresponding reference sequences using CLUSTAL X version , which cIn parallel with the development of the new SSR markers, 40 primer-pairs previously reported to amplify polymorphic SSR loci ,23,24,254)2SO4 and 20 mM MgCl2), 0.3 μL dNTPs (25 mM), 7.5 μL of each forward and reverse primer (10 μM), 0.2 μL DreamTaq DNA Polymerase (5 U/μL), and 5 μL of 5 ng/μL DNA template. A negative control reaction, replacing the DNA template with sterile Millipore water, was also included. The 16 selected primer-pairs were used to amplify the target loci of 279 individual genotypes from the 28 tomato accessions. The 5′-end of the forward primers was labelled with either 6-FAM or HEX fluorescent dye , for detection of amplified products during capillary electrophoresis. A GCTTCT hexamer was added to the 3′-end of the reverse primers (PIG tailing) to prevent the Taq polymerase from adding non-template sequences to the PCR products, as described in Ballard et al. . The PCRThe PCR analysis was carried out using a Bio-Rad thermal cycler S1000  with the following cycling parameters: initial denaturation for 3 min at 95 °C, followed by 35 cycles of denaturation for 30 s at 94 °C, annealing for 40 s at 3–5 °C below the primer’s melting temperature and primer extension for 40 s at 72 °C, and a final primer extension for 20 min at 72 °C. After each PCR run, electrophoresis was performed using 1.5% agarose containing GelRed on randomly selected amplified products using a 50 bp Gene Ruler ladder  as size standard, followed by scanning with a BioDoc-It Imaging System . This led to exclusion of four of the 12 newly developed SSRs, because a significant number of samples failed to be amplified. Hence, the amplified products of 12 SSR loci were used in capillary electrophoresis, as described below. The PCR products of the 12 SSR loci were multiplexed into four panels following the criteria described in Geleta et al. , except The capillary electrophoresis step was followed by peak identification using GeneMarker version 2.7.0  software with the default settings . Each peST) and fixation index (F). Polymorphism information content (PIC) of each locus was calculated as described in Botstein et al. [Various genetic diversity parameters were estimated for each locus across all accessions and for each accession across all loci. POPGENE version 1.32  was usedST and average pairwise differences between and within accessions were used to generate graphs using a series of R scripts within Rcmd, a console version of the R statistical package, by triggering the command button added to Arlequin version 3.5.2.2 toolbar.Analysis of molecular variance (AMOVA) was conducted to determine the variance within and between accessions and the variance at higher hierarchal level, using Arlequin version 3.5.2.2 . MatriceNei’s unbiased genetic distance between the 28 accessions was calculated using GeneAlEx 6.41 and then used as input data for Unweighted Pair Group Method with Arithmetic Mean (UPGMA)-based cluster analysis using MEGA7 software, where the optimal tree with the sum of branch length −2.13486148 is shown . The genIS, 0.62, 1.0 and 0.87 for FIT, and 0.72, 0.90 and 0.80 for FST. Estimated genetic differentiation (GST) at locus level varied from 0.67 (for SLR20) to 0.89 (for TomSatX11-1), with a mean of 0.77, and was highly significant at all loci (p = 0.001)  .The genetic diversity of each accession was estimated using several parameters . AccessiAmong the 28 accessions, six did not have polymorphic loci (PPL = 0), as indicated above, whereas less than 10% of the loci were polymorphic in six other accessions (PPL < 0.1) . The advThe genetic diversity of each accession was estimated by Shannon’s I and He (gene diversity). In addition to the six accessions that did not show genetic variation (see above), 14 accessions had very low genetic variation, with I and He values below 0.14 and 0.10, respectively . Four acST = 0.773, p < 0.001), while 22.7% was attributable to variation within accessions, of which 7.1% was accounted for by variation among individuals within accessions and 15.6% by variation within individuals. AMOVA was performed using 1000 permutations at both the accession and higher hierarchical levels . The resp < 0.001). Cultivated and wild accessions also differed significantly, with cultivation status accounting for 17.9% of the total variation (p < 0.001) ); altitude (<500 m above sea level (masl), 950–1200 masl, 1450–1750 masl, and 1850–-2250 masl); cultivation status (cultivated and wild); fruit shape ; fruit colour (red and yellow); fruit size ; and growth type . Accessions from La Paz differed significantly from those of the other regions in Bolivia, and region of origin accounted for 21.7% of the total variation revealed by the markers used (< 0.001) . In addi< 0.001) . ST analysis of the 28 accessions revealed significant differentiation (p < 0.05) among the vast majority (94%) of the pairs of accessions (p < 0.05)  . ‘Rio Grsk in ST . ST  further revealed the genetic relationship between the 28 tomato accessions . The firAnalysis of admixture model-based population structure using the STRUCTURE and STRUCTURESELECTOR programs revealed that three genetic clusters (K) was the optimal number, according to the method of Evanno et al.  Supplem. This suS. lycopersicum var. ceraciforme and S. neorickii [Our analysis of 28 tomato accessions revealed a low level of diversity persisting in exanimated Bolivian tomatoes, despite the fact that Bolivia is part of the centre of diversity  or origieorickii . This noS. lycopersicum cultivars and three wild Solanum species [Use of SSR loci previously employed to study the genetic diversity of tomatoes revealed a considerably lower number of alleles for the Bolivian tomatoes investigated here than reported for tomatoes from other countries. For instance, only three alleles were detected at the SLR20 locus among the 28 tomato varieties studied here, whereas Korir et al. (2014) reported five alleles among 42 tomato varieties from China and Kenya at this locus . Further species , but in ST and GST values at each SSR locus. Highly significant differentiation between tomato accessions has been reported previously for local landraces from southern Italy and contemporary tomato varieties [Among the 11 SSR loci studied, SLM6-11 was the most informative, with a PIC value of 0.65, corresponding to the value reported in a previous study . Hence, arieties , and forarieties .Tomato is predominantly a self-pollinating species ,50, and For the hybrid group, Ho exceeded He except for ‘HT-23’ and ‘HT-37’, with both having higher He than Ho and high positive fixation index (F). Steps taken following hybridisation could explain the differences seen in F and He/Ho ratio. One possibility is that ‘HT-23’ and ‘HT-37’ have undergone a series of self-pollination events after the hybridisation event, resulting in homozygosity at the majority of their loci and positive F values. In contrast, the two Israeli commercial cultivars (‘Lia’ and ‘Shanty’) and the Bolivian hybrid ‘HT-36’ are most likely F1 hybrids, since they possess maximum levels of heterozygosity (F = −1), while ‘Huichol’, a cultivar from Thailand, and the Bolivian hybrid ‘HT-25’ may have been reproduced through open pollination after the hybridisation event took place. The 20 Bolivian accessions can be classified into three subgroups based on their expected heterozygosity, which measures the genetic diversity within the accessions: those with He = 0 (inbred), those with He = 0.01–0.08 (extremely low diversity), and those with He = 0.11 = 0.21 (low to medium diversity). Shannon’s I values can also be used to discern these subgroups. However, in terms of phenotypic characteristics and geographical region of origin, each of these groups was found to be generally diverse. For example, the six inbred accessions differ in fruit colour and shape, as well as altitude and geographical region of origin of the germplasm. That group also comprised both cultivated and wild types. Additionally, some of those with very small fruits appeared to be inbred, while others had He values as high as 0.21. Hence, neither geography nor phenotypic characteristics sufficiently explained the genetic variation within the accessions.ST analyses, while they show some differences in their phenotypic characteristics.The results of the present analysis indicated differences in reproductive mechanisms among both cultivated and wild Bolivian tomatoes. The accessions BOL-8223-HT, BOL-8281-HT, BOL-8284-HT, BOL-8328-HT, BOL-8330-HT and BOL-8340-HT are genetically uniform, and are characterised by very small red fruits except for BOL-8223-HT . Their lack of within-accession genetic variation may indicate that they have cleistogamous flowers that prevent pollen movement, resulting in strict inbreeding. Crossbreeding such inbred accessions can be advantageous, since they will produce genetically suitable F1 hybrids that may be superior to their parents in terms of desirable traits. It is possible, for example, to crossbreed BOL-8223-HT (cultivated) with BOL-8330-HT (wild), since they are genetically distinct, as revealed by our cluster, PCoA and pairwise FThe accessions BOL-8225-HT, BOL-8282-HT, BOL-8290-HT, BOL-8316-HT, BOL-8322-HT) and ‘HT-23’ have similar characteristics to the above group except that they have genetic variation within accessions. However, the high fixation index values obtained here (F = 1) indicate that these accessions are strictly self-pollinating types, a reproductive mechanism determined in previous studies to be dominant in tomatoes . In viewThe two most genetically diverse Bolivian accessions, BOL-8288-HT  and BOL-8348-HT , are both wild accessions bearing small round fruits. They differ in fruit colour  and flowering habit  and in the altitude and geographical location of the sampling site. Further characterisation may lead to identification of genotypes with desirable characteristics that can be incorporated into elite cultivars through crossbreeding. The present study clearly indicated that none of the specific geographical regions or altitude ranges within Bolivia can be considered a hotspot for the genetic diversity of Bolivian tomatoes.S. lycopersicum accessions revealed significant variation both between accessions (77.3%) and within accessions (22.7%), which is in line with the characteristics of species that are predominantly self-pollinating. A previous study [S. cheesmaniae and S. galapagense) also revealed much higher variation between accessions  than within accessions. In fact, these two wild species are strict inbreeders, unlike some open-pollinating S. lycopersicum accessions analysed in the present study. However, higher variation within accessions  has previously been reported for S. lycopersicum and S. pimpinellifolium, both being self-compatible [S. peruvianum, only 32.2% of the total variation is between accessions [The analysis of molecular variance (AMOVA) results for the 28 us study  that evampatible . In contcessions . A possiS. chesmaniae, but four times higher than that of S. galapagense from different regions of the Galapagos Islands [Based on the highly significant genetic variation found between the accessions and the significant differentiation between over 90% of accession pairs, crossbreeding between genotypes bearing desirable agronomic and fruit characteristics may prove to be the most effective approach for cultivar development. It should be noted that in the present study, hierarchical AMOVA revealed significant differences between groups based on a variety of factors, such as region of origin, domestication/breeding status, fruit shape, fruit size and flowering habit. Around 20% of the total genetic variation differentiated accession groups from La Paz versus other regions in Bolivia, pointing to the importance of isolation by distance and geographical barriers in population differentiation. This significant divergence can also be explained partly by the fact that most of the accessions from La Paz are wild populations with small fruits and semi-determinate flowers, while most of the accessions from other regions are cultivated types. This differentiation level is comparable with that of  Islands . On the Although cultivated Bolivian tomatoes differed significantly from their wild counterpart, that differentiation explained only 18% of the total variation. The majority of the cultivated tomatoes still bear small fruits, similar to wild types, indicating that little has been accomplished in terms of selection-based improvement in fruit size. Significant variations were observed between fruit shape groups, fruit size groups and flowering habit groups, accounting for 16.8%, 22.5% and 22.0% of the total variation, respectively. These results are not surprising, since the commercial cultivars and advanced breeding lines have predominantly cylindrical and round fruits, while the majority of the wild accessions have somewhat flattened fruits. Additionally, commercial cultivars and advanced breeding lines have larger fruits and determinate flowers, while wild and landrace accessions have small fruits and indeterminate/semi-determinate flowers.Since determinate growth habit and larger fruits are desired characteristics in tomato cultivars, resulting in synchronous maturity that facilitates harvesting and higher fruit yields, these traits have been the target of domestication and breeding programmes ,58. TherThere was significant genetic differentiation between cultivated and wild accessions, with the exception of BOL-8282-HT and BOL-8284-HT, as visualised by the heatmaps of pairwise comparisons, UPGMA tree, PCoA scatter plot and STRUCTURE graph. Thus, use of wild accessions in crossbreeding with cultivated accessions of tomatoes provides the potential to develop cultivars that have favourable fruit characteristics and are well-adapted to Bolivian agroecosystems. It should be noted, however, that there are higher genetic similarities between the Bolivian cultivated tomatoes and the foreign commercial cultivars evaluated here than between Bolivian cultivated and wild tomatoes. This is an excellent example of how domestication has shaped crop evolution through the selection of germplasm for multiple desirable traits that are generally categorised as “domestication syndrome” traits . FurtherAmong the cultivated accessions, BOL-8284-HT was the most genetically distinct, exhibiting an indeterminate flowering habit and very small, slightly flattened red fruits. BOL-8335-HT and BOL-8349-HT were the most genetically distinct wild accessions, both exhibiting a semi-determinate flowering habit and producing very small red fruits. Consequently, these accessions could be valuable for crossbreeding to facilitate the development of superior genotypes through genetic recombination for further breeding.It is noteworthy that some closely clustered accessions, e.g., BOL-8328-HT vs. BOL-8330-HT, ‘Rio Grande’ vs. ‘HT-23’ and ‘HT-36’ vs. ‘HT-37’, have similar fruit characteristics and flowering habits, suggesting the presence of genetically similar accessions that could be considered “duplicates” in the Bolivian ex situ conserved tomato germplasm. In contrast, some closely clustered accessions (high genetic similarity) such as BOL-8316-HT and ‘HT-37’ exhibited different fruit characteristics and flowering habit. Thus, grouping the accessions based on their phenotypic characteristics, followed by genotypic characterisation based upon single nucleotide polymorphisms (SNPs) and SSRs, would allow the creation of a core collection comprising distinct accessions.In previous population genetics studies, different approaches have been used to determine how populations are genetically structured ,62,63,64S. neorickii, S. chmielews, S. lycopersicum ceraciforme and S. lycopersicum spp. [S lycopersicum L. were included. The best-represented region was La Paz, which had a significant number of accessions in the core collection. Nonetheless, this study provides valuable novel information on genetic diversity and genetic structure for potential use in breeding programmes. Most of the alleles found in the cultivated accessions derived from two of the three genetic populations identified. Alleles of these genetic populations appear to be widely distributed in non-Bolivian and Bolivian accessions, since they are well represented in these accessions. The accessions that formed cluster 1 in the UPGMA tree, shown as sky blue and green clusters in the PCoA plot in The commercial cultivars and most of the Bolivian advanced breeding lines displayed highly similar population structures to wild accessions collected from La Paz region, implying that the core germplasm collected in La Paz plays a significant role in tomato improvement programmes in Bolivia. To our knowledge, only one previous study has examined the genetic diversity of Bolivian core germplasm, using 31 accessions of cum spp. . That stThe number of alleles detected in this SSR-based study on Bolivian tomato accessions ranged from two to five, indicating low allelic diversity of examined Bolivian accessions. TomSatX11-1 proved to be the most informative of the newly developed SSR markers in this study and should be prioritised for population genetics analysis of tomatoes, together with other highly informative markers such as SLM6-11 and LE20592. While Bolivia lies within the centre of diversity and origin of tomatoes, explored Bolivian tomatoes have generally low genetic variation within each accession. However, there is highly significant genetic differentiation between the accessions, explaining approximately 75% of the total genetic variation. There is also significant genetic variation between wild and cultivated tomatoes and between tomatoes with different geographical origin, fruit shape, fruit size and flowering habits. However, there is no significant genetic difference between tomatoes from different altitude ranges or tomatoes with different fruit colours. The genetic differentiation between tomatoes with determinate and indeterminate flowers may predate tomato domestication. Tomatoes have genetically determined mechanisms that contribute to either cleistogamous flowers, generating genetically uniform genotypes, here represented by six accessions , or to flowers that allow open pollination, here represented by three accessions . The two most genetically diverse Bolivian accessions in the present study were BOL-8288-HT  and BOL-8348-HT . There is limited gene flow both within and between cultivated types and wild populations, resulting in genetic admixture. Crossbreeding of genotypes of genetically distinct cultivated accessions, such as BOL-8284-HT and BOL-8316-HT, could lead to the development of superior cultivars through genetic recombination.


Camellia oleifera clustered into one class, Camellia vietnamensis and Camellia gauchowensis clustered into one class, and Camellia crapnelliana and Camellia chekiangoleosa clustered into another class. It could be speculated that the genetic relationship of C. vietnamensis and C. gauchowensis is quite close. SSR markers could reflect the genetic relationship among oil-tea camellia germplasm resources, and the results of this study could provide comprehensive information on the conservation, collection, and breeding of oil-tea camellia germplasms.Oil-tea camellia trees, the collective term for a class of economically valuable woody oil crops in China, have attracted extensive attention because of their rich nutritional and pharmaceutical value. This study aimed to analyze the genetic relationship and genetic diversity of oil-tea camellia species using polymorphic SSR markers. One-hundred and forty samples of five species were tested for genetic diversity using twenty-four SSR markers. In this study, a total of 385 alleles were identified using 24 SSR markers, and the average number of alleles per locus was 16.0417. The average Shannon’s information index (I) was 0.1890, and the percentages of polymorphic loci (P) of oil-tea camellia trees were 7.79−79.48%, indicating that oil-tea camellia trees have low diversity. Analysis of molecular variance (AMOVA) showed that the majority of genetic variation (77%) was within populations, and a small fraction (23%) occurred among populations. Principal coordinate analysis (PCoA) results indicated that the first two principal axes explained 7.30% (PC1) and 6.68% (PC2) of the total variance, respectively. Both UPGMA and PCoA divided the 140 accessions into three groups.  Camellia chekiangoleosa, Camellia oleifera, Camellia crapnelliana, Camellia vietnamensis Huang, etc. [Oil-tea camellia trees is the collective term for a group of plants of high economic value. There are approximately 50 species of these trees, and they belong to the family Theaceae . Oil-teang, etc. . Neverthng, etc. . Thus, iC. vietnamensis is a species of oil-tea tree from Hainan Island, the southernmost city in China with a unique geographical location and superior climate [C. vietnamensis from Hainan Island, which is considered an independent and traditional plant resource according to the long-term isolation from the mainland [C. oleifera, which is widely grown in mainland China. It is more suitable for a tropical climate, has a large amount of genetic variation, and has higher contents of active ingredients in the oil [Due to its extensive planting under different ambient conditions in China, oil-tea camellia trees have formed species with different growth habits, morphological characteristics, and degrees of oil quality . C. viet climate , and som climate . C. vietmainland ,10, is s the oil .Camellia species [Camellia sinensis [C. chekiangoleosa [C. oleifera [Camellia japonica [Camellia fascicularis [Camellia meiocarpa and C. oleifera by SSR markers [C. oleifera had abundant genetic variation [Camellia species from Hainan was identified by the chloroplast genome sequences and SSR analysis [Simple sequence repeats (SSRs), also called short tandem repeats (STRs) or microsatellites, are widely distributed in the genomes of animals and plants . The ran species , such assinensis , C. chekngoleosa , C. oleioleifera , Camellijaponica , and Camicularis . Huang f markers . Combiniariation . An unidanalysis . As a coCamellia species have mainly involved SSRs [C. vietnamensis and other Camellia species. Therefore, in this study, we collected SSR molecular markers from 140 oil-tea camellia samples, followed by the non-hierarchical analysis of molecular variance (AMOVA), the unweighted pair group method with arithmetic (UPGMA), principal coordinates analysis (PCoA), and population structure analysis, in the hope of providing some data basis and theoretical basis for the delineation of the relatives, resource system, and population structure of oil-tea camellia species.To date, molecular marker studies in ved SSRs ,17, RAPDved SSRs ,21), ISSved SSRs ,23, and C. oleifera, 17 C. vietnamensis species, and 2 C. chekiangoleosa. The seeds included 13 C. oleifera, 3 C. chekiangoleosa, 5 C. crapnelliana, and 5 Camellia gauchowensis specimens. Details of the samples are shown in A collection of 140 oil-tea camellia accessions was used in this study, which were divided into 5 groups , includiw/v) agarose gel electrophoresis and the Agilent 2100 Bioanalyzer (USA). Good quality DNA was used directly for SSR analysis or stored at –20 °C for further use.Sample DNA was extracted by the TIANGEN genomic DNA extraction kit . DNA quality and concentration were then checked by 1% (C. vietnamensis (NCBI accession number: PRJNA825399) [−1) of forward and reverse primers, 7.5 µL of 2 × Taq PCR master mix , 1 µL (50 ng·µL−1) of template DNA, and 4.5 µL of ddH2O. The PCR program was as follows: 96 °C, 3 min; 96 °C for 30 s, 50–60 °C for 30 s, and 72 °C for 1 min, and these three procedures were cycled 30 times; 72 °C, 10 min. Two microliters of amplified PCR products were used in 2% (w/v) agarose gel electrophoresis to check whether the amplified fragment size and concentration were in the normal ranges at each locus with reference to the DNA marker alignment. Then, 1.0 µL of the fluorescent PCR product was diluted 30 fold with ultrapure water and prepared for machine detection. The diluted PCR products were separated by capillary electrophoresis by the ABI 3730XL DNA Analyzer , and data were handled by Gene Marker v.2.2.0 software .Ninety-six pairs of SSR primers were selected for pre-screening based on the transcriptome data of A825399) , in whicA825399)  with minAccording to the PCR results, a binary matrix was formed in which the presence of the product was marked as 1 and the absence of the product as 0. The results of the 1/0 data matrix were utilized to analyze the genetic diversity of oil-tea camellia trees. Based on the number of alleles, the level of discrimination of each SSR marker was assessed by calculating the percentage of polymorphic loci (P), Nei’s genetic diversity (h), Shannon diversity index (I) , gene diAccording to the DICE coefficient , Nei’s gThe 140 accessions from five oil-tea camellia species were analyzed by SSR markers. The alleles detected by 24 pairs of primers at the polymorphic sites ranged from 6 to 31. A total of 385 alleles were generated by amplification, resulting in an average of 16.0417 alleles per locus . The meaThe alleles at each locus in each sample were coded into a fingerprint in the form of a 0/1 matrix based on bands amplified using 24 pairs of primers. Fingerprinting gives a visual representation of the differences for each sample . As coulsI within species reached 0.134. S1 had the highest genetic variability (0.214), and S4 had the lowest value (0.041). When computed at the individual level, the mean I was 0.1890. The results indicate that the genetic differences among different groups were small and the genetic diversity was not very rich.The detailed information of each genetic locus of each species is shown in Fst) among five groups showed moderate genetic differentiation (Fst = 0.231).Nei’s genetic distance (D) is a measure of genetic difference among biological populations and can be measured in terms of quality traits and also with quantitative traits. The estimation of genetic distance is important for exploring the origins of cultivars, analyzing the relationships among populations, mapping phylogenetic trees and predicting heterosis, and guiding parental selection. The range of genetic identity among species was 0.8616–0.9719, calculated from 285 amplified fragments. As shown in C. oleifera was clustered into one subclade, C. vietnamensis and C. crapnelliana were clustered into one subclade, and C. chekiangoleosa and C. gauchowensis were clustered into one subclade. In addition, some C. oleifera and C. vietnamensis were clustered into one subclade. Furthermore, two-dimensional PCoA revealed four distinct clusters on the basis of Nei’s genetic distance among individuals (C. oleifera) samples were clustered together, S2 (C. vietnamensis) and S5 (C. gauchowensis) samples were clustered together, S3 (C. chekiangoleosa) samples were clustered together, and S4 (C. crapnelliana) samples were clustered together.Based on Nei’s genetic distances among individuals and groups, the clustering analysis among individuals was accomplished using the aboot method of the R package poppr, by selecting Nei’s distance and bootstrapping 1000 times. Cluster analysis among populations was subjected to UPGMA trees drawn using the phylip software. According to the genetic distances, a phylogenetic tree was built . As can ividuals . PCoA anIn linkage disequilibrium, there is a shift between the probability that a haplotype will appear and the probability that it will be randomly combined. The extent of this offset determines the extent of linkage disequilibrium. The degree of linkage disequilibrium was characterized by the square of the R value, which, when equal to 0, indicates complete linkage equilibrium—independent inheritance. When the R-squared equals 1, it indicates complete linkage disequilibrium. All 24 SSR loci were in linkage disequilibrium with each other; the a maximum R-squared was 1, and a minimum R-squared was 0.0099 .C. oleifera samples were clustered in one population; a small proportion of C. oleifera samples were clustered separately; C. vietnamensis, C. gauchowensis, C. crapnelliana, and C. chekiangoleosa were another cluster. The results of population structure analysis were similar to those of UPGMA analysis (The results of STRUCTURE showed a clear maximum for Ln(PD)-derived delta K (∆K) at K = 3 A,B, and analysis A.Ip of oil-tea camellia was 0.041–0.214, and the range of p was 7.79–79.48% , showing moderate genetic diversity. When the polymorphism information content (PIC) was less than 0.25, SSR primers showed little polymorphism; when 0.25 < PIC < 0.5, moderate polymorphism; when PIC > 0.5, high polymorphism [C. pubipetala, and the results showed that the I value was 0.4100; the PPB (percentage of polymorphic bands) was 80.43% [C. japonica, and the range of alleles was 1–12. The mean was 4.46 alleles per locus. The range of PIC was 0.15–0.86, and the average was 0.59 [p in this study was 34.49%, which is similar to the above results. The ranges of Ne, h, and Ip of 24 markers in this study were 1.041–1.197, 0.027–0.128, and 0.041–0.214, respectively. The differences are larger when compared with the results of Dong et al., who used 16 SSR marker pairs for 54 oil-tea trees  for genetic diversity analysis. The ranges of Ne, h, and I were 1.17–1.70, 0.14–0.40, and 0.26–0.59, respectively [In this research, the genetic diversity of five oil-tea camellia species was analyzed by using SSR markers. The range of alleles in SSR was 6–31. A total of 385 alleles were found. An average of 16.0417 alleles were found for each SSR-primer pair. At the species level, the range of s 80.43% . AlthougC. vietnamensis was clustered with C. gauchowensis, which is similar to the findings of Qi et al. [C. vietnamensis collected from Hainan Province showed high similarity to those of C. gauchowensis, whose provenance was Gaozhou in Guangdong Province [C. vietnamensis and C. gauchowensis were found to be clustered together by cpDNA sequences and SSR marker analysis [C. vietnamensis and C. gauchowensis to each other was quite near. Dai et al. analyzed the chloroplast genome trnH-psbA and matK sequences of 101 different kinds of oil-tea camellia seedlings by DNA barcoding technology [C. vietnamensis was clustered into one branch and C. chekiangoleosa was clustered into another, and the clustering results in this study agree strongly with these results. The findings suggest that C. vietnamensis in Hainan has a relatively close relative. Additionally, the phenomenon of self-incompatibility might occur in close relatives, which might be one of the reasons for the low seed-setting rate of C. vietnamensis in Hainan. Accurate genetic relationships among germplasm accessions are important for variety development, evolutionary studies, and resource conservation ,36. Threi et al.  and Cheni et al. . They foProvince , and C. analysis , so it cchnology . They foC. oleifera samples were clustered one population; a small proportion of C. oleifera were in another cluster; and C. vietnamensis, C. gauchowensis, C. crapnelliana, and C. chekiangoleosa were one cluster. There were small fractions of C. oleifera samples that clustered with other Camellia species. It was indicated that plants of the same group came not only from the same region but also from different regions. Possibly, species with different genetic backgrounds may cluster together. This indicates that the kinship of germplasm is extremely complex. The reasons for this phenomenon might be as follows. First, occasional genetic mutations and long-term natural selection have made finding the relatives of oil-tea camellia more complicated. Second, the effect of genetic drift was greater during the natural differentiation of oil-tea camellia than those of natural environmental factors, leading to the failure to divide by geographic region when clustering. Using indirect measures, the gene flow among populations was estimated by the value of Nm [For a more accurate analysis of the genetic structure of oil-tea camellia, STRUCTURE was used for further analysis, and the results indicated that the 140 accessions were classified into three clusters. Among them, most of ue of Nm . The Nm ue of Nm ,28. ThirCamellia plants. He et al. used nine pairs of SSR primers to analyze 150 accessions of C. oleifera, and the results indicated that the genetic diversity level of C. oleifera is high [C. fascicularis with fourteen pairs of primers for SSR markers [C. fascicularis were roughly divided into three clusters, and the genetic variation within populations accounted for 49.95% of variation. To sum up, the results of this study indicate that the genetic variation of oil-tea camellia samples was mainly found within populations, and inbreeding occurred within the population, such as with C. vietnamensis. The degree of gene exchange among species was low.The results of genetic structure analysis indicate that the genetic variation of oil-tea camellia samples mainly appeared within species, accounting for 77% of the total variation, leaving only a small portion (23%) occurring among species. That might result from habitat fragmentation and geographical barriers. Some experts have also obtained similar results with other  is high . In addi markers . The resCamellia accessions. Based on genetic distances and clustering by UPGMA, the 140 accessions could be classified into three clusters. Most individuals from S1 grouped together, samples from S2 and S5 grouped together, and samples from S3 and S4 formed the same branch. In addition, some individuals from S1 and S2 were clustered together, which relates to the results of the PCoA. The Bayesian-model-based genetic structure analysis indicated that the studied accessions belonged to three populations. Among them, most of the C. oleifera samples were clustered into one population; a small proportion of C. oleifera were in another cluster; C. vietnamensis, C. gauchowensis, C. crapnelliana, and C. chekiangoleosa were one cluster. Taken together, the findings should be instructive for oil-tea camellia species’ introduction, breeding, germplasm preservation, and new-variety development, and provide a theoretical foundation for the classification and identification of oil-tea camellia species in southern China and the research on relatives.In this research, 24 pairs of SSR primers were selected to analyze the genetic relationship and population structure of 140 oil-tea camellia accessions using fluorescence detection by capillary electrophoresis. The results indicate that genetic diversity was abundant among the 140


Sechium edule (Jacq.) Sw. (Cucurbitaceae) is a species native to Mexico and Central America. The collection, characterization, and evaluation of accessions maintained in genebanks is essential for the conservation of this species. However, there are no specific varietal descriptors that differ from those used in a phenetic approach and are adapted to international registration guidelines to help distinguish, improve, cluster, and protect intraspecific variants of common use and those obtained by breeding. Therefore, 65 morphological descriptors  were evaluated in 133 accessions obtained from Mexico, Guatemala, and Costa Rica located in the National Germplasm Bank of S. edule in Mexico. These characteristics were observed to be phenetically stable for five generations under the same agroclimatic conditions. In addition, an analysis of amplified fragment length polymorphism (AFLP) was applied to 133 samples from a set of 245 accessions. According to the multivariate analysis, 26 of the 65 descriptors evaluated  enabled differentiation of varieties of S. edule. The AFLP analysis showed a high level of polymorphism and genetic distance between cultivated accessions and their corresponding wild ancestor. The variations in S. edule suggest that the morphological characteristics have differentiated from an essentially derived initial edible variety , but unlike other cucurbits, there is no evidence of the ancestral edible for Sechium since the seed is unorthodox and there are no relicts. Sechium, the differentiation process is related to adaptive specialization to the environment with a continuous gradient in characteristic values. Thus, traits that effectively reflect genetic differences between individuals, populations, and species are needed to accurately discern phylogenetic relationships.The characterization and intellectual protection of vegetables, with the aim of ex situ conservation, basic research, and genetic improvement programs, are established using a series of morphological, chemical, genetic, and physiological descriptors that fulfill the requirements of the distinction, uniformity, and stability (DUS) test, by applying the standards of the International Union for the Protection of New Varieties of Plants (UPOV), and, recently, using digital phenotyping to discriminate varietal groups ,2. AccorSechium edule (Jacq.) Sw.) (Cucurbitaceae) is a species originally from Middle America that has been cultivated for human consumption and transferred to different agroclimatic environments. S. edule is a perennial ascending shrub, presenting tendrils and tuberous roots. The leaves are simple with a long petiole and lobulated or angular palmate shapes. The flowers are unisexual, axillar, staminate, and pistillate in the same knot. The fruit is pendulous, large, obovoid or piriform, with longitudinal depressions. The fruit surface is soft, light or dark green, shiny, and can be boldly or finely pubescent, with a variable number of spines, and there is a single seed [Chayote  has integrated different accessions of S. edule from Mexico, Guatemala, and Costa Rica with the highest variability in order to establish the chayote Germplasm National Bank (BANGESe) in Veracruz, Mexico, where distinctive morphological descriptors were designed for validation The purpose of the research is to formalize the differentiation of genotypes using secondary characteristics, primarily morphological, that demonstrate heritability and stability in their generation, identifying the greatest genetic reciprocity with such secondary variables to facilitate their differentiation and use. Genetic diversity studies based on molecular genetic markers are widely used to distinguish genotypes of S. edule because, unlike many morphological traits, they are not subject to environmental influences [The Interdisciplinary Group of Research in fluences ,7. Genetfluences . Howeverfluences .S. edule, opening the possibility of creating new primers for genetic studies of this species.The resulting morphological and biochemical characterization studies are of great importance to efficiently describe, classify, and manage accessions of plant species. Generally, the center of origin of a species date from a great genetic and morphological selection. Regarding this, Cadena et al.  characteMolecular markers can be used to efficiently distinguish between closely related individuals of chayote. Machida-Hirano et al.  reportedAmplified restriction fragment length polymorphism (AFLP), a molecular technique used to investigate population genetics, genome mapping, relationships between molecular polymorphism, etc., can be used to analyze genetic relationships among accessions . This isZea mays, Cucurbita spp., and Phaseolus vulgaris, have been classified intraspecifically as distinctive races and ecotypes based on secondary, stable, and heritable morphological characteristics, which has facilitated their identification, conservation, and enabled new uses. The aim of this study was to evaluate the morphological characteristics and level of polymorphism of biological variants of S. edule and validate distinctive varietal descriptors adapted to international registration guidelines to help distinguish, improve, group, and register intraspecific varieties of common use and those obtained by plant breeding.Many neotropical species, such as The analysis of correspondence demonstrated five principal dimensions (DIM) for the qualitative variables that explained 70.43% of the variability , and their contributions were explained in three main dimensions . VariablThe analysis of hierarchical conglomerates denoted the distribution of evaluated accessions based on the qualitative characteristics and attempted to determine their relative importance as explanatory variables. In this sense, the semi-partial correlations defined the role played by each characteristic, aside from their apparent prominence in the phenotype, by means of grouping and assigned them a causality. The above mentioned is relevant because of its statistical value. The outstanding variables were demonstrated to be characteristics that may be influenced by the environment; however, they continued to be important even though they could be quantitatively transformed , since they acquired continuity and stability in their values. Another important aspect of these variables was their relative value in the statistical load of the component, which allowed the establishment of relationships with other evaluated parameters, for example, pubescence, venation order, shape, furrows, or ribs, thus increasing the degree of continuity despite environmental influence. These variables showed unalterable characteristics that were homogeneous and uniform from generation to generation.The values obtained from the annual measurement of morphological characteristics were lower since the standard error was very low. These results were due to measurements being taken in the month of June (beginning of flowering) and ending in late July in order to reduce crossbreeding and obtain self-pollinated samples. Some varieties began to bear fruit before emitting lateral vines (precocity), making it easier to obtain samples without interbreeding.Regarding the quantitative variables, principal component analysis (PiCo) demonstrated that the first six components explained up to 78.6% of the variability of the accessions . The values obtained for Pearson’s correlation coefficients (PC) between two quantitative random variables was independent of their measurement scale, as shown in S. edule (Jacq.) Sw.S. edule, 228 AFLP-type molecular markers were used to analyze kinship relationships between the different cultivated and wild varietal groups. Cluster analysis was run in Rstudio using the factoextra and FactoMineR packages. The distance matrix of the AFLP markers was constructed using the ade4 package and Dice coefficients. The optimal number of groups was calculated using the NbClust package, which performed a consensus of 30 indices, resulting in a majority rule, thus determining eight groups as ideal to describe the distance matrix. Group I highlighted varietal complexes with small fruit, such as albus minor, nigrum conus, and nigrum minor. Group II contained 75% of the amarus sylvestris accessions. Group III included all of the wild accessions of S. edule. Group IV was made up of various varietal complexes, making an in-depth discussion difficult. Groups V and VI were characterized by individuals with smooth fruit, such as virens levis and albus levis. Group VII combined the genotypes of the varietal complexes nigrum xalapensis and nigrum minor, although a subgroup with individuals of the albus levis varietal group was observed. Group VIII was very diverse with elements of different varietal complexes, as in groups III and IV. It was possible to make cuts in the dendrogram to create more groups with concordance, which was supported in the consulted literature and based on the varietal complexes of S. edule  clustered close to the varietal groups virens levis, nigrum xalapensis, and spiny green chayote. In general, the varietal complexes of S. edule tended to cluster in the center of the graph. However, the nigrum levis, albus minor, and nigrum conus varietal groups were located very close to each other and closest to the wild type materials because they shared common morphological characteristics   .S. edule were evident  are most preferred, followed by var. virens (light green) and finally var. albus with yellow skin. This indicates an important relationship between the conservation and support of genetic resources of S. edule and the morphological distinctions that mark the preference of the consumer, which strongly impacts domestication or reproductive isolation.The ecological and cultural conditions of traditional agriculture have helped to preserve the genetic diversity of several economic species and influence the response of current cultivated forms to processes of empirical selection in different regions. In the case of Zingiber officinale (0.347) [Molecular characterization identified 91 loci with 96.7% polymorphism. The number of effective alleles was 1.18 per locus (capable of being passed on to the next generation). The expected heterozygosity had an average value of 0.127 ± 0.013, indicating moderate levels of genetic diversity compared to other species that used the same markers, such as  (0.347) .var. amarus sylvestris has dark green fruit and a strongly bitter flavor, while the groups nigrum and virens have fruit with a neutral flavor, and albus has slightly sweet fruit [Groups of plants strongly manipulated by humans can reflect different life histories because of agroclimatic variables, reproductive isolation, and domestication processes. In this study, genetically differentiated groups were observed, some more than others, with evidence of subgrouping. Cadena-Iñiguez et al.  noted thet fruit ,27.S. edule, the soft seed coat does not allow it to be preserved [Diversity arises by speciation, forming one or more species from an ancestor . Reprodureserved , and theS. edule were evaluated to promote the development of breeding programs. The morphological characteristics were related to the fruit  and sugar, ascorbic acid, and phenol content. To complement this study, 28 RAPD-type molecular markers and 30 ISSR were added. The RAPD analysis showed a polymorphism of 88.38% and some of the chayote varieties that shared common morphological characteristics (light green and dark green fruit) also shared the same genomic fragments. Referring to the ISSR analysis, a polymorphism of 62.5% was observed. The fruit with light green and dark green tones showed the highest ascorbic acid and phenol content. In northeastern India, the morphological and genetic characteristics of 74 local varieties of Principal component analysis indicated that 77.74% of the total variation was explained by the first three components. The first main component was supported by the morphological characteristics of the fruit , while the second component was characterized by the chemical characteristics of the fruit . Light green chayote varieties showed the highest average rates of polymorphism for ISSR and RAPD markers. The cluster analysis revealed the absence of a relationship between clusters and geographical distances of the chayote varieties. The results obtained, in terms of chemical characteristics, were similar to those reported by Cadena-Iñiguez et al. .In 36 accessions of chayote from India, 18 quantitative and qualitative morphological characteristics of fruits were evaluated. The same study was complemented with 12 DAMD-type molecular markers. Principal component analysis showed that 49.68% of the total variation was explained by the first two components. The main characteristics that contributed to this variation were weight, shape, length, and width of the fruit, as well as length and density of the loin. These characteristics were distinctive of the chayote varietal complexes described by Cadena et al. . MoleculS. edule, which was 154,550 bp in size and contained a total of 122 genes . The guanine-cytokine content was 37.2%. The results of sequencing this genome demonstrated high phylogenetic affinity with other chloroplast genomes of species belonging to the Cucurbitaceae family, mainly Cucumis spp. and Cucurbita spp.Cui et al.  sequenceOur study bears similarities to other research on genetic and morphological diversity conducted in India ,11. The S. edule suggest that the plants have conserved the expression of essential characteristics resulting from the initial genotype or from the subsequent combination of genotypes of different varieties [var. amarus sylvestris and the wild type (ancestor). The high level of genetic polymorphism, attributed to adaptive specialization, reproductive isolation, conservation in backyards, intercrossings, constant selection, etc., has not erased the initial characteristics, except for the bitter taste, the loss of which was attributed to cultivation, domestication, and adjustments in the mevalonic acid pathway that modified the cucurbitacin and pigment content, thus facilitating adaptation to new environments [The morphological variations observed in arieties . Figure ronments ,11.Sechium edule (BANGESe) is in Veracruz, Mexico (19°08′48″ N and 97°57′00″ W). The vegetation type is mountain cloud forest , with an annual mean temperature of 19–22 °C, relative humidity of 85–90 %, and annual mean precipitation of 2250 mm. The soils are vitric and luvisolic, rich in organic matter, low in calcium, and high in iron, manganese, and zinc nutrients, with moderate fertility, thick texture, and fragments of volcanic glass (pH 4.3–6.5) [The Germplasm National Bank of 4.3–6.5) .The plants used in this study have been included in BANGESe since 2005 and are native to Costa Rica, Guatemala, and the Mexican states of Chiapas, Oaxaca, Puebla, Morelos, Hidalgo, Michoacán, Nayarit, San Luis Potosí, State of Mexico, and Veracruz . Most ofS. edule, the number of individuals per accession [Sechium germplasm bank for accumulating evidence of characteristic stability. This was important since the original provenance of each accession differed.Because previous biochemical, morphological, and anatomical studies showed stability of the distinctive features of ccession  was reduThe sample size for this analysis (n = 133) started from a population of 245 accessions in the germplasm bank. The agroclimatic conditions for all accessions were the same (2.0 ha of collection surface) and their essential characteristics were recorded for for five generations. Young and healthy leaves of the 133 chayote plant accessions were extracted to obtain the DNA using the CTAB (cetyltrimethylammonium bromide) method with some modifications . ThroughThe products from PCR were corroborated on agarose gel (0.8%) for 60 min at 80 V, and later diluted before the pre-amplification reaction was carried out. The products of the amplification were separated by polyacrylamide gel electrophoresis (6%) at 200 V for 3 h, the gels were stained with silver nitrate, and then a 1:10 dilution with formamide was carried out for the PCR-selective products, which were evaluated by capillary electrophoresis using a 3500 XL Genetic Analyzer . For the principal coordinate analysis, the percentage of polymorphism per band and percentage of variation explained were determined using AFLP-SURV 1.0  softwareThe genetic distances were scored according to the presence (1) or absence (0) of 228 AFLP markers and the Dice coefficients . ConstruS. edule suggest that the morphological characteristics have been differentiating from an essentially derived ancestral variant. For this reason, we suggest that the characteristics evaluated in the current study should be adopted as the main varietal descriptors in the technical guide of this phytogenic resource for food and agriculture. These descriptors will update the initial guide (phenetics) for the distinction, registration, and legal protection of varieties, both those of common use and those obtained by breeding. They will also contribute to varietal separation and differentiation for ex situ conservation purposes, basic research, and bioprospective studies in a format suitable for international use.Twenty-six varietal descriptors allowed differentiation between the evaluated chayote accessions. The main characteristics that enabled distinction were considered qualitative and quantitative; however, the evaluations under field conditions over five years demonstrated that the features were stable and unalterable from generation to generation. The visual distinction manifested mainly in fruit variables, such as color, shape, size, flavor, and presence/absence of spines. There was a high level of genetic polymorphism between cultivated accessions and their wild ancestor. The polymorphic variations in


Malus Mill genotype is highly heterozygous, and many theoretical problems such as genetic relationship and evolution process among germplasm are difficult to be solved by traditional analysis methods. The development of SCoT(start codon targeted polymorphism) molecular markers suitable for apples is of great significance for studying the origin, evolution, genetic relationship and genetic diversity of Malus Mill germplasm resources. In this paper, the genetic relationship and evolution of 15 materials were analyzed by SCoT molecular marker. The results showed that the gene differentiation coefficient values of four Malus Mill plants at the species level were 0.423, 0.439, 0.428 and 0.460, respectively, which indicated that there was obvious genetic differentiation among the populations of these four Malus Mill plants, but there were some differences among the populations of different Malus Mill plants. The gene differentiation coefficient of coextensive populations with different geographical distribution varied from 0.177 to 0.086 (average 0.138), which indicated that the genetic similarity of species in coextensive composite populations was high and there was a close genetic relationship among species. This indicates that SCoT molecular markers can be effectively used in the analysis of intraspecific genetic relationship and identification of intraspecific strains of Malus Mill plants. Malus Mill is a genus of Rosaceae with high economic value. Malus Mill plants such as M.hallianaKoehne, M.baccata (L.)Borkh, M.hupehensis (Pamp.)Rehd, M.micromalus are important garden trees.Many species are used as rootstocks in apple production –3. The tGenetic marker is an easily recognizable manifestation of biological genotype. With the understanding of gene from phenomenon to essence, genetic marker has gradually developed from simple morphological marker, cytological marker and biochemical marker to DNA molecular marker which can directly reflect genetic polymorphism at the DNA level .Scot  markers with unique advantages have been widely used in Apple research , 12.HoweSCoT marker can not only obtain the target genes closely related to traits, but also track traits. It has the advantages of simple operation, high polymorphism, abundant genetic information, low cost and strong universality of primers, among which the most prominent ones are high polymorphism detection efficiency and abundant genetic information. At present, it has been widely used in the research of genetic diversity, population genetic structure, germplasm identification, gene differential expression and molecular genetic map of plant germplasm resources. Literature  ConstrucGenetic diversity refers to the sum total of genetic variation among different groups or individuals within a group, which is the basis and important component of biodiversity. The evaluation of genetic diversity of plant natural populations and artificially cultivated strains is the basis of effective protection, development and utilization of germplasm resources. Literature  100 pairMalus Mill, as a classified “difficult genus”, is characterized by diverse plant forms, complex variation of characters, and the intersection of characters among many species is difficult to classify. Due to the widespread interspecific hybridization, many species have complicated classification problems, which are difficult to identify. Literature  holds thGene mapping is an important condition for obtaining excellent genes. When new genes appear in genetic breeding work, it is necessary to locate them on specific chromosomes, and measure their arrangement order and distance on chromosomes, so as to quickly and accurately locate genes. The key of gene mapping is to determine the parent combination between the populations used for mapping. Molecular markers play an important role in the germplasm resources of fruit trees. Molecular markers have many advantages, such as greatly improving the efficiency of animal and plant varieties, helping to develop and identify new varieties, and reducing breeding costs through marker-assisted breeding. Developing more new molecular markers is the new direction of molecular marker technology development in the future, and it is also the effort direction of researchers. In-depth research on fruit tree germplasm resources by molecular markers is beneficial to all fields, which has become an irreversible trend.The materials used are M.hupehensis (Pamp.)Rehd.var. Mengshanens, M. Hupehensis (Pamp.) Rehd, M.hupehensis Rehd. var.taiensis, M.toringo De Vriese, M.toringoides(Rehd.) Hughes, M.sikkimensis (Wenz.)Koehne, Riwacrabapple, M.rockii Rehder. The collected Malus Mill fruits are mashed and washed with water to separate the seeds from the pulp, dried to remove impurities, and the complete and full seeds are selected and stored in a refrigerator at 4°C for later use.The treated seeds were put into a refrigerator at 4°C to break dormancy at low temperature, and after 21 days, the seeds were put into an incubator at 28°C. Cross experiments were conducted on the time of breaking dormancy and the optimum temperature for seed germination after breaking dormancy. It was found that the dormancy of seeds could be basically broken in about 21 days, and 28°C was the optimum temperature for seed germination, and the time of breaking dormancy of different species was slightly different.ProFlex™ PCR System PCR thermal cycler, MultifugeX1R high-speed freezing centrifuge, Gel Doc™ EZ gel imaging system, JY600C electrophoresis tank, Thermo Scientific Nano-Drop 2000 Spectrophotometers spectrophotometer.Among the published SCoT primers of Malus Mill plants, the primers with high polymorphism and good stability were selected for the experiment. Among the SCoT-labeled primers, three fluorescent markers FAM , hex (hexachlorofluoroscein) and Tamra (carboxy tetramethylrhodamine) were added to the 5' end of each pair of primers.Put an appropriate amount of apple leaves in a mortar, add a small amount of quartz sand, grind into powder, and subpackage into 1.5 mLeppendorf tubesCells were lysed for 30 min~60 min in the warm bath of 65°C constant temperature water bath pot, during which the centrifugal tube was turned upside down for 2 ~ 3 times‐1Na Ac and 2 ~ 2.5 times the volume of frozen absolute ethanol, and mix carefullySuck the supernatant, add 1/10 times the volume of 3mm · LPut it in the refrigerator at-20°C for 30 min, gently pick out the flocculent precipitate in the tube, and put it into a new centrifuge tube .μL dissolved DNA in EP tube, add 1 μL RNase, and keep at 37°C for 1 h or -4°C for one nightDry DNA at room temperature, add 1 × TE 5 The extracted DNA was stored in a refrigerator at -20°CThe total DNA of apple leaves was extracted by improved CTAB (cetyltrimethylamine bromide) :Put an μg · μL‐1) = OD260×50× dilution multiple×1000.The extracted DNA was detected by 1% agarose gel electrophoresis. The DNA with high brightness, clear main band and no dispersion band was diluted and put into the ultraviolet spectrophotometer for detection. The ratio of OD260/280 was between 1.7 and 1.8, and the purity was high. The DNA concentration was calculated by probe labeling, and the concentration of DNA sample  for 30 min, and then images are collected and saved by gel imaging system.After the PCR amplification procedure is completed, 5 ~ 8 Image Lab(BIO-RAD) is used to analyze the film, and the number of DNA bands amplified by each treatment is counted. After manual correction, D2000 DNA Marker is used as the reference standard molecular weight to predict the molecular weight of amplified bands, and the molecular weight is regarded as the same position within the range of 5 BP. Then, statistics are made according to the size and site of amplified fragments of each sample. The number of bands at the same position is “1”, and the number of bands with no bands or weak bands that are difficult to distinguish at the same position is “0”.Then, the similarity coefficient is calculated by the Qualitative date program in NTSYS-pc2.10e data software, and the similarity coefficient matrix is obtained. The SHAN program and UPGMA method are used for cluster analysis, and then the cluster diagram is generated by Treeplot module.The successful preparation of DNA and the avoidance of partial degradation are the basis and key to the success of SCoT, because the quality of the template, that is, whether it contains polysaccharides, polyphenols, protein, quinones and pigments, will affect the future PCR amplification reaction. In this experiment, the improved CTAB method was used to extract apple genomic DNA, and the OD260/OD280 values of the tested materials were all between 1.8 and 2.0. 0.8% agarose gel electrophoresis showed that the main band was clear, without degradation, and the purity was high, which met the requirements of SSR analysis.The most amplified bands of 9 SCoT primers were SCoT12, with 1,344 bands, and the least was SCoT9, with 688 bands. The average number of amplified bands per primer was 948.The polymorphism ratio of 9 primers ranged from 72.5% to 100%, among which SCoT33 had the highest polymorphism ratio and SCoT12 had the lowest polymorphism ratio, and the average polymorphism ratio (Np) of 9 SCoT primers was 85.34%. The polymorphism ratio of each primer is shown in Malus Mill plant seeds have dormancy phenomenon, so it is the most important step to explore the conditions for breaking seed dormancy to obtain experimental materials. The fifteen Malus Mill plant experimental materials studied in this experiment include eleven wild species materials (M.hupehensis (Pamp.) Rehd. var.Mengshanens), M.hupehensis Rehd. var.taiensis, M.hupehensis (Pamp.)Rehd, M.baccata (L.)Borkh, M.rockii Rehder, M.dumeri (Bois.)Chev, M.toringoDe Vriese, M.toringoides(Rehd.) Hughes, M.sikkimensis (Wenz.)Koehne, Riwacrabapple, M. ombrophila Hand.-Mazz) and four cultivar materials .According to the degree of chromosomal evolution, most of Malus Mill plants in this study are metaphase chromosomes and near metaphase chromosomes, with a karyotype of 2B, which belongs to a relatively primitive type. The karyotype of M.ombrophilaHand-Mazz is 1A, the most primitive, and that of M.toringo De Vriese is 3B, the most evolutionary.Karyotype parameters of fifteen Malus Mill taxa are shown in According to the number of hybrid chromosomes, the chromosome homology between them is lower than that of M.toringo De Vriese and M.toringoides(Rehd.) Hughes, that is, the genetic relationship between them is not high. In the next research, the screening range of parents of M.hupehensis Rehd. var.taiensis should be expanded.According to Nei's gene diversity analysis, the gene differentiation coefficient values of four Malus Mill plant species at the level of M. baccata(L.) Borkh, M.rockii Rehder, M.doumeri (Bois.)Chev. and M. Toringo de Vriese were 0.423, 0.439 and 0.428, respectively. 0.460, indicating that there is obvious genetic differentiation among the populations of these four Malus Mill plants, but there are some differences in the degree of population differentiation among different Malus Mill plants. That is, the main source of intraspecific genetic variation of four Malus Mill plants is intra-population variation among individuals, and the intraspecific genetic differentiation has reached a very significant level (P <0.001). As shown in In order to further explore the relationship between genetic distance and geographical distance, Mantel tests were carried out on the genetic distance and geographical distance of four tested species . The resCluster results of 15 materials by principal coordinate analysis .It was found that 15 test materials were divided into three categories. Three species of Pyrus are the first category; M. doumeri (Bois.) of Malus mill Chev, grassland Begonia and narrow leaf Begonia are the second category, among which grassland Begonia and narrow leaf Begonia are more closely related. The third category is divided into four sub groups. The first category includes Hawthorn Begonia and m. ombrophila hand- Mazz and Dianchi Begonia, M. ombrophila hand- Mazz is closely related to Begonia Dianchi;Category 2 includes M. sikkimensis (Wenz.) Koehne, M.toringoides(Rehd.) Hughes and begonia; The fourth category includes: M. halliana Koehne, Xinjiang wild apple, betel seed, catalpa seed, flat edge Begonia, Chinese apple, Oriental apple, forest apple and brown Begonia.The analysis of population structure of 15 tested materials by using Structure 2.3.1 software showed that the number of allele frequency characteristic types K in the samples showed a continuous increasing trend. When K =3, from the distribution of the maximum covariant Q value of 15 materials, the covariant Q of 13 materials is ≥0.6, accounting for 69.7% of the tested materials, indicating that the genetic relationship among these materials is single .The composite ratio and efficacy index can evaluate the efficiency of marker polymorphic sites and the efficiency of site polymorphism . Among tAmong the two markers, the effective recombination ratio and efficacy index of ScoT are higher than those of EST-SSR, and SCoT marker has higher polymorphism detection efficiency. The marker index can comprehensively reflect the expected heterozygosity and effective recombination ratio of the average locus. The SCoT marker is 2.337, which is higher than the EST-SSR marker of 1.199.In order to study whether there is mutual influence of population genetic structure among the sympatrically distributed species of Malus, this experiment analyzed the genetic structure of apple species within four sympatrically distributed populations of Malus, namely populations A, B, C and D .The results showed that the gene differentiation coefficient of coextensive populations with different geographical distribution varied from 0.177 to 0.086 (average 0.138), which indicated that the genetic similarity of species in coextensive composite populations was high and there was a close genetic relationship among species. The average gene flow among individuals in the same domain distribution population is 3.362, which indicates that the frequency of gene exchange among several species of Glycyrrhiza uralensis Fisch. distributed in the same domain is higher.However, the degree of interspecific differentiation in coextensive populations in different regions is relatively different, and the degree of genetic differentiation among species in group A is the highest, which indicates that there are differences in the degree of gene exchange among species in coextensive populations in different regions. The more frequent the gene exchange, the higher the genetic similarity among species. It shows that there are differences in the degree of inter-species gene exchange among coextensive populations in different regions. The more frequent the gene exchange, the higher the genetic similarity among species.Malus Mill plants have natural hybridization ; The exiAmong the designed 35 pairs of primers, 16 pairs can amplify polymorphic products. The reason why some primers cannot amplify products may be : The priThe results of population structure analysis are also consistent with the results of cluster analysis and principal coordinate analysis. SCoT, IRAP and SSR all divide apple species into three groups when K =3, one of which is Malus Mill plant materials in North China and Northwest China, the other is Malus Mill plant materials in East China and South China, and the last is Malus Mill plant materials in Southwest China. From their structural analysis chart, it can be found that most apple varieties have gene exchange, and Malus Mill plants with similar regions are more likely to have gene exchange, which indicates that whether Malus Mill plant populations are hybridized or not is related to their regional distribution.The results are the same as those obtained by AMOVA analysis  that onlAccording to the analysis of gene differentiation coefficient, the genetic similarity among the four coextensive populations is high, and the gene flow among the coextensive populations is higher than that among the intraspecific populations. In addition, the analysis of the genetic distance among the coextensive composite populations  shows thKaryotype analysis and genome in situ hybridization both showed that Malus Mill's plant classification system still had some imperfections.M.hupehensis Rehd. var.taiensis and M.hupehensis (Pamp.)Rehd.var. mengshanens are both variants of M. hupehensis (Pamp.) rehd. The number of chromosomes with hybridization signals is less than that of M.toringo De Vriese and M.toringoides(Rehd.) Hughes, The number and proportion of chromosomes with hybridization signals in M.sikkimensis (Wenz.)Koehne, M.toringoides(Rehd.) Hughes and M.toringo De Vriese are also higher than those in M. M.hupehensis Rehd. var.taiensis, so it is necessary to further study the genetic relationship among Malus Mill plants.In this experiment, the results obtained by cluster analysis, principal coordinate analysis and population structure analysis are roughly the same, and all three methods effectively show the genetic relationship and genetic diversity among Malus Mill materials. However, there are obvious differences in the classification order of groups and lines, which may be related to the fact that Malus Mill plants have both wild species and cultivated species, and there are many interspecific hybrids. There may be some differences between genetic relationship analysis at molecular level and morphological genetic relationship analysis based on some characters, and there may also be differences between different molecular marker techniques.Literature  studies To sum up, Malus Mill plants in its neighborhood and coextensive distribution area, the transitional hybridization zone formed by interspecies hybridization, on the one hand, provides a source of variation for species evolution; On the other hand, Malus Mill plants in the hybridization zone, as an interspecific transition group, become the medium of gene flow among Malus Mill plants, and accelerate the gene exchange among Malus Mill plants. It is speculated that in the evolution process of Malus Mill plant species, due to the imperfect reproductive isolation mechanism, there has been a certain inter-species gene exchange. The evolution and formation of Malus Mill plants are related to the parent species with the same domain and domain distribution, which is more in line with the neighborhood and same domain model .In this study, SCoT molecular markers were used to study the genetic relationship among Malus populations, and the influence of interspecific hybridization infiltration on species formation and differentiation was discussed, and the formation and evolution trend of hybrid Malus plants was analyzed. The main conclusions are as follows:SCoT marker has a high polymorphism and a large amount of information, which can reveal the level of genetic diversity and genetic relationship between Malus Mill plants and within species. It is an effective molecular marker for studying the genetic structure of Malus Mill natural population, interspecific gene infiltration and so on.It is proved that M.sikkimensis (Wenz.)Koehne and M.toringoides(Rehd.) Hughes are closely related to M.baccata (L.)Borkh group. M.hupehensis Rehd. var.taiensis and M.hupehensis (Pamp.)Rehd.var. mengshanens are the same varieties of M. hupehensis (Pamp.) rehd. Although the chromosome homology is not high, their morphology is similar. Riwacrabapple is quite different from M.sikkimensis (Wenz.)Koehne, which supports its independent seed formation; Support the viewpoint of M.hupehensis (Pamp.)Rehd's multi-point origin.The diversity level of Malus plants is higher than that of other apple species distributed in the same domain, and it has certain advantages in quantity and adaptability, which promotes gene exchange among Malus plants.The classification system and genetic relationship of Malus Mill plants are becoming more and more clear, but the intra-genus classification system needs further improvement, and the inter-species and intra-species relationships need further experiments to prove that the classification of Malus Mill plants still needs further study.


Caryopteris mongholica Bunge is a rare broad-leaved shrub distributed in the desert and arid regions of Mongol and North China. Due to land reclamation, natural habitat deterioration and anthropogenic activities in recent years, the wild resources have sharply reduced. To effectively protect and rationally use it, we investigated the genetic diversity and population structure from 18 populations across the range of C. mongholica in China by reduced representation sequencing technology.Ho), expected heterozygosity (He), and average nucleotide diversity (π) were 0.43, 0.35 and 0.135, respectively. Furthermore, the NM17 population exhibited higher genetic diversity than other populations. The phylogenetic tree, principal component analysis (PCA) and structure analysis showed the sampled individuals clustered into two main groups. The NM03 population, with individuals clustered in both groups, may be a transitional population located between the two groups. In addition, most genetic variation existed within populations (90.97%) compared to that among the populations (9.03%). Interestingly, geographic and environmental distances were almost equally important to the observed genetic differences. Redundancy analysis (RDA) identified optical radiation (OR), minimum temperature (MIT) and mean annual precipitation (MAP) related variables as the most important environment factors influencing genetic variation, and the importance of MIT was also confirmed in the latent factor mixed models (LFMM).We found the overall average values of observed heterozygosity (C. mongholica. These genetic features provided vital information for conserving and sustainably developing the C. mongholica genetic resources.The results of this study facilitate research on the genetic diversity of The online version contains supplementary material available at 10.1186/s12870-022-03681-y. Caryopteris mongholica Bunge  is an endangered shrub mainly distributed in the arid and semi-arid areas of Mongolia and North China . Accordcrossing . As the yopteris , C. mong species , 5. Recengholica , 7. Due C. mongholica has high morphological and genetic diversity, owing to its wide geographic distribution and the survival in a broad range of environmental conditions   C. monghSingle nucleotide polymorphisms (SNPs) are the most abundant and universal sequence variations in all genomes, which makes them very useful markers for genetic analysis . With thC. mongholica genetic diversity has relied on traditional molecular marker (inter-simple sequence repeat). However, this marker has its own limitations in resolving the geographical differentiation of population, especially when using few genetic markers. Here, we identified SNPs of C. mongholica from 18 natural populations in Northwest China using GBS to address the above drawbacks. We aimed to answer the following questions with the detected SNPs: (1) what is the level of genetic diversity and population structure of C. mongholica in these different populations? (2) does geographic and environmental distance affect the genetic diversity of C. mongholica? (3) what is the relative importance of environmental variables to the genetic diversity of C. mongholica? According to the results, at the last, we provided a reference for the protection and rational utilization of the wild C. mongholica resources.Early study relating to Ho, He, and π ranged from 0.35(NM14) to 0.73(NM17), 0.28(NM14) to 0.70(NM17), 0.126(NX07) to 0.151(NM17), respectively, with the means of 0.43, 0.35, and 0.135, respectively , with a stable GC content ranging from 34.38% to 38.33%. Sequencing generated 126 billion paired-end reads from 125 individuals, of which 99.99% reads passed initial quality filters  showed two groups corresponding to the 18 populations of ica Fig. . The Helica Fig. .Fig. 3PrK = 2) were most likely, as determined by the Admixture software  and two individuals (NM03-09 and NM03-13) from the NM03, and a large second group with all the remaining populations and individuals  from the NM08 population formed a new cluster with these three populations. In addition, the previous large group branched, now comprising 50 individuals, respectively.The structure clustering analysis estimated that two genetic groups , and the remaining 14 populations as group B (Daqing Mountain-Grassland group).At the species level, the hierarchical analysis of molecular variance (AMOVA) results showed that the majority of the observed genetic variation could be attributed to differences within populations (90.97%) rather than to the variation among populations (9.03%) Table .Table 2AFST/(1-FST) and geographic distance . A significant correlation between pairwise The variance inflation factor (VIF) was less than 10 for all variables included in the RDA. The first axis of RDA accounted for 87.54%. the second axis of RDA accounted for 82.83%, while the third accounted for 77.28% of the environmental variables’ relations , for which no complete reference genome exists. Therefore, studies of C. mongholica genomics using a genome resequencing-based approach that requires a complete reference genome are limited. SNPs are currently markers of choice for several types of genetic diversity studies in C. mongholica. Using the GBS methodology on genomic the DNA of C. mongholica, we developed the first genomic-SNP markers for this important drought-tolerant shrub species.Recently, genomic resources have allowed us to study genetic mechanism of evolution in more detail. Many researchers have been studying the genetic information of iversity , 17. C. C. mongholica was found to have highest He value (0.70), whereas the NM14 had the lowest (0.28)  than among them (21.02%). Pollen spread and seed dispersal are the most important determinants of gene flow. In nature, C. mongholica is mainly pollinated by wind and insects [C. mongholica usually depends on seed propagation, and long-distance seed dispersal must depend on seed propagation, thus, on frugivorous animals [C. mongholica populations is likely small and that the frequent gene flow between them might be caused by wind-dispersed pollen or pollinators.Our results revealed that higher genetic variations in the us study . Wu char insects , and the insects . In the ong them 1.02%. Po animals . TherefoC. mongholica individuals into two groups. This division is consistent with their geographical origins and genetic backgrounds [C. mongholica may have evolved from ancestors initially distributed in the Hengduan mountain region of western Sichuan and northwestern Yunnan and gradually expanded to the Loess Plateau and Inner-Mongolia Plateau [C. mongholica is complicated, especially when migrating from the Hengduan mountains region in southwest of the Loess Plateau in northwest China, which explains why the genetic relationship of some C. mongholica individuals did not well correlate with the geographical origins in this study. For example, “NM02-18” and “NM03-25” were from Dalad Banner Nei Mongol and Otog Banner Nei Mongol, clustered in the same clade and displayed relatively close genetic distance . Geological events [C. mongholica.Our assignment test results based on the ML tree separate kgrounds . C. mong Plateau . The migl events –30 and sl events . In addiK = 2, which was supported by the results of ML tree and PCA  and the Daqing Mountain-Grassland group (group B). Figure C. mongholica populations from the northeast side of sampling sites. They were located in relatively wide geographical locations, including grasslands and plains, which may have promoted gene flow among them. In addition, of seven individuals from the NM03 location were used as plant material, two  showed a membership coefficient of more than 50% and clustered in group A, the other five individuals were grouped in the group B  and 13º of longitude (~ 103 – 116ºE) across Northwest and North China  agarose gel. The purity and concentration of DNA in each sample were determined using the NanoPhotometer spectrophotometer  and Qubit DNA Assay Kit in Qubit 2.0 Flurometer , respectively.In this study, all of ina Fig. : Fig. S3MspI , T4 DNA ligase (NEB), ATP (NEB), and MspI Y adapter N-containing barcode. Restriction-ligation reactions were heat-inactivated at 65℃ and digested with the additional restriction enzyme EcoRI (NEB) at 37℃. The restricted ligation samples were purified using Agencourt AMPure XP (Beckman). The purified samples were PCR amplified with Phusion Master Mix (NEB) universal primer and index primer to add the index. The PCR products were purified, pooled, and electrophoresed on 2% agarose gel. Fragments between 265 and 315 bps were selected and purified. Pair-end sequencing was performed on the selected tags using the Illumina PE150 platform.GBS libraries were constructed in accordance with the modified protocol . GenomicC. mongholica, under these circumstances, de novo generation of a GBS reference was constructed following [Adapter sequences and low-quality bases  from the tail of each read were removed using Trimmomatic v0.36 . Becauseollowing . We seleollowing , and theollowing  to prediK ranged from 2 to 3. To avoid overestimating the number of potential clusters caused by the presence of IBD, as is often found in continuous populations, we further used conStruct [K), Admixture software [K value. The genetic structure of each identified population was assessed by calculating nucleotide diversity per base pair (π), observed heterozygosity (Ho) and expected heterozygosity (He) were determined for each population identified. The Stack software [π and inbreeding coefficient (FIS). Arlequin software [Ho and He. DnaSP software was used to calculate the pairwise FST/(1-FST) and gene flow (Nm) from Hudson et al. [The genetic structure was characterized using phylogenetic tree construction, PCA, and population structure analysis. The ML phylogeny tree was inferred based on the filtered SNPs using RAxML  to deteronStruct  to identsoftware  was usedsoftware  was usedsoftware  was usedn et al. .AMOVA was conducted to quantify genetic variation at two different hierarchical levels: among population within the germplasm group and within populations. Genetic variations were further tested by assigning populations to genetic clusters identified by population structure analyses. The analyses were conducted in Arlequin , and theA) the correlation between environmental and geographic distance, (B) IBD, and (C) IBE. According to the geographic coordinates of the different C. mongholica collection sites, the climate data for each sample site were obtained from the WorldCLIM global high-resolution climate database (http://www.worldclim.org/). A total of 17 bioclimatic variables were used to calculate IBE  and geographic distance and environmental distance with significance determined using 999 permutations in the R package vegan [To investigate the role of geographic and environmental factors in shaping the spatial genetic differentiation, we calculated: in the R package vegan . RDA invAdditional file 1: Table S1. List of 125 C. mongholica individuals tested in this study. Table S2. Summary of GBS data. Table S3. Environmental parameters used in this study, and the mean (±standard deviation) values of 18 locations. Table S4. 17 bioclimatic variables in the 18 locations of C. mongholicaAdditional file 2: Fig S1. Plot of ADMIXTURE cross-validation error. Fig S2. Cross-validation results comparing the non-spatial and spatial models. Fig S3. Pink and viole-blue flowers of C. mongholica. Fig S4. Manhattan plots representing the distribution of significance values -log10 obtained by the genetic-environment association approach LFMM for 17 bioclimatic variables.