BackgroundRed clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover.ResultsIn this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts.ConclusionsWe analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1534-8) contains supplementary material, which is available to authorized users.

14Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted 15 forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and 16 completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. In this study, 17 the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length 18 splice isoforms, and 29,730 novel isoforms from known genes and 2,194 novel isoforms from novel genes were 19 identified. A total of 5,492 alternative splicing events was identified and the majority of alter spliced events in 20 red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8,719 21 including 1,86,517 transcripts have at least one poly(A) site. Furthermore, we identified 4,333 long non-coding 22 RNAs and 3,762 fusion transcripts. Our results show the feasibility of deep sequencing full-length RNA from red 23 clover transcriptome on a single-molecule level. 24 Keywords 25 Full-length transcript; alternative splice; alternative polyadenylation; long non-coding RNA; fusion transcript 26 Abbreviations 27

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The combination of palladium(II) acetate/(R)-xylyl-Phanephos and silver triflate (Pd:Ag = 1:1) was found to be an efficient catalytic system for the asymmetric ring opening reactions of oxabenzonorbornadienes with terminal alkynes. The products were typically afforded in good yields (up to 95% yield) and with high enantioselectivities (up to 99% ee).Asymmetric ring opening reaction of oxa/azabenzonorbornadienes with carbon nucleophiles is one of the most well-received carbon-carbon bond forming reactions for generating stereogenic carbon centers. Notable progress has been achieved over the past few decades. By employing different chiral transition metal catalysts, a number of commonly used organometallic reagents, including organozinc, [1] lithium, [2] Grignard, [3] aluminium [4] and boron reagents, [5] are found to be suitable as nucleophiles in the ring opening reactions. Particularly noteworthy is that some of the products generated from these reactions can be used as key intermediates for the synthesis of natural or bioactive molecules. [6] To date, one of the recent advancements of asymmetric ring opening reactions is the introduction of neutral organic compounds as effective carbon nucleophiles, for instance, terminal alkynes. In 2002, Cheng et al first demonstrated the use of an achiral nickel catalyst in ring opening reactions of oxa/azabenzonorbornadienes with a series of terminal alkynes (also known as alkynylative ring opening reaction). [7] Several years later, an asymmetric version of the alkynylative ring opening reaction of azabenzonorbornadienes with (triisopropylsilyl)acetylene was developed by Hayashi and colleagues by employing a rhodium catalyst possessing (R)-DTBM-Segphos as chiral ligand. The alkynylated ring opening products were generated in high yields and with high enantioselectivities. [8] However, the application of oxabenzonorbornadienes as suitable substrates in this rhodiumcatalyzed asymmetric ring opening reaction has not yet been reported. Recently, Hou and co-workers reported an sp 2 -C,P palladacycle catalyst, for the alkynylative ring opening reaction of oxabenzonorbornadienes. Nevertheless, the attempted study of this enantioselective transformation was not reported. [9] Our group has had a continuing interest in the asymmetric reactions of norbornadiene derivatives for several years. We have established some efficient chiral transition metal catalysts for some of these reactions. [10] Just before Hous report, we also found that the complex of palladium(II) chloride and achiral ligand Xantphos was able to catalyze the alkynylative ring opening reaction of oxabenzonorbornadienes smoothly, but an attempted study for realizing the asymmetric ring opening by switching the ligand from Xantphos to chiral ligands was not successful, since low catalytic efficiency was encountered. [11] In the following study, we fortunately found that the combination of chiral palladium complexes with Lewis acids could form effective cocatalytic systems for the asymmetric ring opening reaction of azab...