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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

1
Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, National Engineering Laboratory for Tree Breeding, Robinia pseudoacacia Engineering Technology Research Center of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China
2
Shandong Provincial Key Laboratory of Forest Tree Genetic Improvement, Shandong Academy of Forestry, Ji’nan 250014, China
3
He’nan Provincial Key Laboratory of Forest Tree Germplasm Conservation and Breeding, He’nan Academy of Forestry, Zhengzhou 450008, China
4
State-owned Lvcun Forestry Station in Luoning County, Luoyang 471700, China
*
Author to whom correspondence should be addressed.
Forests 2019, 10(8), 644; https://doi.org/10.3390/f10080644
Received: 5 July 2019 / Revised: 24 July 2019 / Accepted: 29 July 2019 / Published: 30 July 2019
(This article belongs to the Section Forest Ecophysiology and Biology)
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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. View Full-Text
Keywords: China; Robinia pseudoacacia L.; EST-SSR; DNA fingerprinting; genetic diversity analysis China; Robinia pseudoacacia L.; EST-SSR; DNA fingerprinting; genetic diversity analysis
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Dong, L.; Sun, Y.; Zhao, K.; Zhang, J.; Zhang, Y.; Li, X.; Xun, S.; Zhang, J.; Wang, S.; Li, Y. 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. Forests 2019, 10, 644.

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