Development and Application of EST-SSR Markers for DNA Fingerprinting and Genetic Diversity Analysis of the Main Cultivars of Black Locust (Robinia pseudoacacia L.) in China

Abstract

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement. More EST-SSR (Expressed sequence tag simple sequence repeat) markers of black locust can be used as a complement and improvement of Genomic-SSR markers for the identification of the function of gene and the construction of genetic map. Additionally, currently there is no simple method for identifying black locust cultivars. In this study, we obtained 2702 unigenes from 3095 expressed sequence tags (ESTs) from the National Center of Biotechnology Information (NCBI) database to identify simple sequence repeats (SSRs) in R. pseudoacacia samples. A total of 170 SSR loci were found to be distributed in 162 non-redundant sequences with a frequency of 6.29%. Dinucleotide repeats were the most predominant types among microsatellites (62.35%), followed by tri-nucleotide repeats (25.88%); the remaining SSRs accounted for less than 12%. The repeat motifs AG/TC (29.25%) and CT/GA (29.25%) were the most abundant among dinucleotides, and AAT/TTA (15.91%) was the most common among tri-nucleotides. A total of 62 primer pairs were designed to screen polymorphic and stable SSR loci. The resulting 25 EST-SSR markers capable of amplifying polymorphic, stable, and repeatable products. Eight newly developed EST-SSR markers and four published SSR markers were selected for DNA fingerprinting and genetic diversity analysis of the 123 main R. pseudoacacia cultivars in China. The 12 SSR loci amplified 102 alleles, with an average number of alleles per locus of 8.5 (range 4-15). The average polymorphism information content at the 12 SSR loci for the 123 cultivars was 0.670 (range 0.427-0.881). The 123 cultivars clustered into six main groups based on similarity coefficients, with most cultivars in one subgroup. Fingerprinting was performed using eight SSR markers; 110 black locust cultivars were distinguished. The results of this study increase the availability of EST-SSR markers in black locust and make it a simple method for checking the collection, the certification, and the correct attribution of clones and cultivars.

Efficient Fingerprinting of the Tetraploid Salix psammophila Using SSR Markers

“…Normally, a single pair of markers cannot differentiate each of the sampled individuals [28]. In this study, 22 SSR markers were highly polymorphic in 261 accessions of S. psammophila with an average number of alleles (A) of 10.41 and average number of four allele genotypes (G) for each locus of 68.36, which was much greater than the values shown in a few former studies of diploid plants (Robinia pseudoacacia L. [29] and Vigna radiata L. [30]. ), implying that the abundance of genotypes in tetraploids is helpful to efficiently reconstruct the fingerprints.…”

“…PIC plays a crucial role in the assessment of availability of SSR markers which reflect polymorphism of markers. The average value of PIC was 0.63, which was higher than related estimates in other plant samples [29][30][31]. The higher polymorphism for tetraploid plants utilized to score alleles benefits from the clear resolution of the capillary DNA fragments [32], and the TP-M13-SSR not only avoided amplicons overlapping but also provided an economic and effort saving method for microsatellite genotyping [18].…”

“…In this study, nine-marker combination (G+I+J+N+O+Q+S+T+U) could distinguish the 261 accessions from one another, which was much less than the number of markers shown in a few earlier studies of diploid plants. For example, Zhao et al [30] constructed the fingerprints based on eight SSR markers, which could identify 151 mung bean varieties; a total of 110 cultivars of Chinese black locust were distinguished by eight pairs of EST-SSR and SSR primers [29].…”

Development and Characterization of Simple Sequence Repeat Markers for, and Genetic Diversity Analysis of Liquidambar formosana

“…The SSR locus frequency was 1/7.3 kb, lower than that of Camellia sinensis (1/4.99 kb) [40] but much higher than those of Pinus dabeshanensis (1/23.08 kb) [41] and Larix principis-rupprechtii (1/26.8 kb) [42]. The predominant repeat type was the dinucleotide (52.56%), as was true for Robinia pseudoacacia L. [43], Populus tomentosa [44], and Hevea brasiliensis [45], but not Persea americana Mill [46] and Dalbergia odorifera T. Chen. [47].…”

Evaluation of genetic diversity and population structure of Fragaria nilgerrensis using EST-SSR markers

“…However, SSR markers have become an particularly important tool because of their co-dominant, polymorphic and cross-transferable nature. Compared with SSR, EST-SSR (Expressed Sequence Tag -Simple Sequences Repeat) has the advantage of transferability among plant species, and is widely used in plant genetic mapping (Dong et al, 2019;Yang et al, 2019;Zheng et al, 2019), genetic diversity evaluation (Ge et al, 2019;Ohbayashi et al, 2019;Sharma et al, 2020), and species identification (Li et al, 2014). Its location in the transcriptional region suggests polymorphism may be directly related to some functional genes (Zhang et al, 2015).…”