| Paulownia is a genus of scrophulariaeae, mostly deciduous trees, but evergreen trees in the tropical rainforest. Paulownia is an important fast-growing timber species native to China and one of greening tree species. The development of paulownia production has important value in the national economy. There are serious problems in Genus Paulownia that excellent genetic resources are easy to be lost, some wild resources are endangered(due to less population and promotion of new varieties), commodity timber species are confused, material quality is inconsistent. DNA barcode technology is one of the better solutions.DNA barcode is a kind of technology which based on the principles of DNA molecular evolution, using standard DNA fragments and the principle and method of modern molecular phylogeny of traditional species for rapid accurate and automatic identification of biology with the latest technology. Similar to barcodes in the supermarket, which are used to scan to distinguish the tens of thousands of different kinds of goods. Genus Paulownia are studied in the research. The problem on the identification of closely related species of Genus Paulownia will be solved by using DNA barcode technology. It may provide value reference for identification of closely related species and selection of good material parent of Genus Paulownia. At the same time, filtering and establishment of Genus Paulownia DNA barcodes will be carried out, in order to lay the foundation of Paulownia visual digital database.The Chloroplast DNA rps16, TrnK, trn L-F, rps2, rbcL region sequences, and nuclear DNA ITS2, ETS region sequences, and mitochondrial DNA nad1 intron2 region sequences from 12 Species and 2 varieties including 51 individuals in Genus Paulownia were obtained by using direct sequencing method. Assessment on success rate of amplification and sequencing were performed to evaluate universality of eight DNA barcodes in Genus Paulownia. Characteristic of the sequence was also analyzed. Ultimately, five key DNA barcodes as rps16, TrnK, ITS2, ETS, TrnL-F were used in the study on DNA barcode identification of Genus Paulownia. Results were as follow:(1) Universality evaluation of eight DNA barcodes in Genus Paulownia were: Success rate of amplification and sequencing are two important indicator in evaluation of DNA barcode sequences. Success rate of amplification all were 100%, and success rate of sequencing were 69.8%~100%. Success rate of amplification and sequencing showed good generality in Genus Paulownia. They were taken as candidate DNA barcode sequences to carried on the next experiment.(2) Analysis of characteristics of 8 DNA barcodes were: Sequence lengths of TrnK region were 239 bp ~ 1828 bp after aligned, No. of total variable loci detected from the aligned sequence were 2~84, and No. of variable loci(substitution site) were 2~83. And the average GC content were 33.14%~72.19%. Paulownia elongata were identified by inserting A at 1660 bp site within Trn K region. Paulownia lamprophylla were identified by inserting A at 188 bp site within ITS2 region sequence. Paulownia catalpifolia were distinguished by characteristics of base C missing at 191 bp site(Câ†'-), of base substitution at 238 bp site(Gâ†'A) and at 304 bp site(Aâ†'C), and at 320 bp and 323 bp(Gâ†'C) within ETS region sequence.To sum up, numbers of total variable loci detected from rps16, TrnK, ITS2, ETS sequence region were high, which illustrated rapid evolutionary rate took place in Genus Paulownia. These four markers were suggested to study the systematics and taxonomy on Genus Paulownia.(3) The screening and identification of DNA barcodes of Genus Paulownia were: Effectiveness of rps16, Trn K, ITS2, ETS DNA barcodes were measured by calculating specifications of the inter-specific and Intra-specific variation, and barcoding gap analysis and the identification efficiency till we screened the right candidate DNA barcode for Paulownia identification. Following results were:The mean value of All inter-specific distance was 0.027%, the mean value of Theta prime was 0.031%, the mean value Minimum inter-specific distance was Zero, the mean value of All intra-specific distance was 0.021%, the mean value of Theta was 0.025%, the mean value of Coalescent depth was 0.052%. Barcoding gap diagram depicted barcoding gap among rps16 sequence. Species identification efficiency was 13.33% by BLAST l. rps16 sequence was regarded as DNA barcode for Genus Paulownia.The mean value of All inter-specific distance was 0.091%, the mean value of Theta prime was 0.090%, the mean value Minimum inter-specific distance was 0.012%, the mean value of All intra-specific distance was 0.048%, the mean value of Theta was 0.052%, the mean value of Coalescent depth was 0.063%. Barcoding gap diagram depicted barcoding gap among TrnK sequence. Species identification efficiency was 21.42% by BLAST l. Trn K region was regarded as DNA barcode for Genus Paulownia.The mean value of All inter-specific distance was 2.132%, the mean value of Theta prime was 2.096%, the mean value Minimum inter-specific distance was 0.186%,, the mean value of All intra-specific distance was 0.902%, the mean value of Theta was 0.822%, the mean valueof Coalescent depth was 1.354%. Barcoding gap diagram depicted barcoding gap among ITS2 sequence. Species identification efficiency was 50% by BLAST l. ITS2 region was regarded as DNA barcode for Genus Paulownia.Paulownia recurva coded as PT1569 XS, was identified by Four Stem-loops Firstly. There were very similar structure with Five Stem-loops except a nice distinction between Paulownia kawakamii and Paulownia taiwaniana. Compared to ITS2 secondary structure of Paulownia kawakamii, there was one more inner ring on the longest bases of the structure of Paulownia taiwaniana. This fully proves that analysis of ITS2 secondary structure, is of great significance for Paulownia identification.The mean value of All inter-specific distance was 3.555%, the mean value of Theta prime was 3.906%, the mean value Minimum inter-specific distance was 0.544%, the mean value of All intra-specific distance was 1.697%, the mean value of Theta was 1.542%, the mean value of Coalescent depth was 2.895%. Barcoding gap diagram depicted barcoding gap among ETS sequence. Species identification efficiency was 28.57% by BLAST l. ETS region was recommend as standard barcode for Genus Paulownia.The mean value of All inter-specific distance was 0.142%, the mean value of Theta prime was 0.132%, the mean value Minimum inter-specific distance was 0.008%, the mean value of All intra-specific distance was 0.096%, the mean value of Theta was 0.065%, the mean value of Coalescent depth was 0.127%. Barcoding gap diagram depicted no significant barcoding gap among TrnL-F sequence. Trn L-F was not considered as DNA barcode for Genus Paulownia.In conclusion, analysis of comprehensive index of ideal barcode revealed that rps16, TrnK, ITS2, ETS can be recommended as barcodes for Genus Paulownia. 9 species and 2 varieties were identified by alignment of TrnK+ITS2+ETS compound fragment sequence. Furthermore, the combined fragment of TrnK and ITS2 be used in combination with ETS can be regard as DNA barcode for Genus Paulownia. |
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