In recent years, SSR molecular markers have been widely used in the study of genetic diversity of species due to the advantages of SSR primers, such as high species specificity and cross-related species transfer, and primer development is the primary condition. At present, only JosphatK. Saina et al.31,15 identified 219 SSR loci based on chloroplast genome of Ailanthus altissima (Mill.) Swingle in 2018.In 2021, 13 EST-SSRs were developed and 10 polymorphic chloroplast microsatellite (cpSSR) markers were constructed based on the transcriptome of Ailanthus altissima (Mill.) Swingle and 219 cpSSR markers. The distribution characteristics of cpSSR and EST-SSR were basically similar, but the difference was that cpSSR did not detect hexanucleotide and compound nucleotide repetition. In this study, 10681 potential microsatellite loci were obtained based on transcriptome sequencing results of tissue-cultured seedlings in young leaves of Ailanthus altissima var. erythrocarpa, which was far lower than that of JosphatK. Saina et al.15 (33084 potential SSR loci), and EST-SSR distribution characteristics were similar. This result may be related to the fact that it is a variety of Ailanthus altissima (Mill.) Swingle.
Most plants use mono-nucleotide, di-nucleotide and tri-nucleotide as dominant repeat motifs, such as Stephanandra incisa20, Bougainvillea cultivars18, Cocos nucifera L.32, Ailanthus altissima (Mill.) Swingle15, Pinus bungeana21, Ulmus pumila L.33, The Ailanthus altissima var. erythrocarpa results of transcriptome analysis showed the same, accounting for 90.12% of total SSR. AG/CT was the most frequent di-nucleotide repeat motifs in most plants, and the homopurine-homopyrimidine extension was often found in the 5' untranslated region, which played an important role in gene expression and nucleic acid metabolism regulation in plants. AAG/CTT is the main nucleotide repeat type in dicotyledons15,20,22,31,34−39. In this study, the di-nucleotide AT/AT repeat type had the highest frequency, which was consistent with the results of pinus bungeana21,40, Bougainvillea cultivars18, and Ailanthus altissima (Mill.) Swingle cpSSR31. The majority of Tri-nucleotide repeats were AGA/TCT, followed by AAG/CTT. Among the 140 pairs of primers screened and synthesized, 106 pairs of primers obtained the amplification bands, and the effective amplification rate reached 75.71%, which was similar to that of Ailanthus altissima (Mill.) Swingle (80%)15 and Cinnamomum chago (70.59%)41. The polymorphism rate of primers was 9.29%, significantly lower than the results of JosphatK.Saina et al.15 (65%), which may be caused by more randomly selected primers, fewer samples, small genetic differences between samples and environmental selection pressure. Therefore, EST-SSR is still the most economical and effective way to develop a large number of primers at the same time using transcriptome data. The chloroplast genome of A. altissima is a circular molecule with a size of 160815 base pairs and has a tetrad structure. The chloroplast genome size was similar to that of Arabidopsis thaliana (L.) Heynh. (120-217kb) and Populus trichocarpa (155-159kb), so it was relatively easy to develop cpSSR markers for A. altissima. Compared with cpSSR, est-ssrs exist in the gene region of nuclear genome and are highly polymorphic15,42. In this study, est-ssrs can be combined with the molecular markers of Ailanthus altissima var. erythrocarpa to study the mutation source points of Ailanthus altissima var. erythrocarpa, providing important research value for the subsequent germplasm resource protection and evolution research of Ailanthus altissima var. erythrocarpa population. Phenotypic traits combined with molecular markers could be used to analyze the correlation of traits, providing guidance for the utilization of germplasm resources and breeding of new varieties.
In this study, 13 pairs of clear polymorphic primers with the expected size were screened, and the average PIC value was 0.603, lower than the average PIC value of A. altissima (0.819)15. PIC > 0.5 indicated that the developed SSR locus had high genetic resolution, which was conducive to population genetic analysis, revealing that the studied Ailanthus altissima var. erythrocarpa had high genetic diversity. The result that PIC value of Ailanthus altissima var. erythrocarpa was lower than that of A. altissima may be related to the small sample size selected in this study, the close provenance and gene differences. Therefore, it is necessary to further study the genetic diversity and structure of this species on the basis of expanding the sample size. The clustering results of genetic relationship and principal component analysis showed that 34 individuals could be completely distinguished and divided into five categories, but the materials of the two sites were indistinguishable. There was no obvious correlation with geographical location, there was strong gene exchange, or it was related to the close proximity of the acquisition site. The range of strain selection could be expanded later. The polymorphism, genetic diversity, population genetic structure and homologous genes between A. altissima and its varieties were analyzed comprehensively and deeply.
DNA fingerprinting technology analyzes the genetic material itself, interferes with immune environmental factors, sample morphology, material source and other factors, and identifies the germplasm genotypes as indicators at the molecular level. It has been widely used in grain, oil, vegetable and fruit crops, flowers, shrubs, arbors and other plants13, 17–18,43.DNA fingerprinting has high simple and stable genetic, variability, multiple loci, can distinguish the same family, belong to different species, different varieties, forms, and even the same strain and subtle variation between individuals DNA, is germplasm genetic relationship, genetic breeding, population genetic structure, ecology and evolution, classification and other valuable genetic marker methods43–45.However, there have been no reports on the fingerprint information of Ailanthus altissima var. erythrocarpa. In this study, four of 13 EST-SSR core primers (P79, P95, P69 and p33) were screened out to distinguish 34 tested Ailanthus altissima var. erythrocarpa materials and construct a unique molecular ID card. The establishment of fingerprinting of Ailanthus altissima var. erythrocarpa is of great significance for its subsequent breeding, which can identify varieties and excellent genes as well as their variation and transmission in offspring, and lay a foundation for the establishment and improvement of fingerprinting database of Ailanthus altissima var. erythrocarpa variety resources, genetic resource management and intellectual property protection. However, in this study, there were few test materials, the sample collection area was narrow, and the fingerprint background of "core germplasm" of Ailanthus altissima var. erythrocarpa was lack of in-depth research, which required further verification of the results' suitability.