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.
Highlights
Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement
DNA fingerprinting and genetic diversity analysis of the 123 main R. pseudoacacia cultivars in China
Following the assembly and splicing of 3095 black locust expressed sequence tags (ESTs) sequences downloaded from the National Center of Biotechnology Information (NCBI) database, a total of 2702 non-redundant unigenes were obtained, of which 180 were contigs and 2522 were singletons
Summary
Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement. This species was widely introduced to Europe and other regions worldwide for its various advantageous characteristics such as rapid growth, resilience, and adaptability [1,2]. It was introduced to China in 1877 and has since become an important tree species that has been planted widely in semi-arid and arid regions [3]. 1970s, research began on black locust cloning and cultivation. More than 100 superior clones and cultivars have been developed in China.
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