3 full race core set version 2

The mRNA was reverse transcribed to cDNA with the common primer named Oligo (dT), and the Gene Specific Primers (GSPs) were generated for the amplifications. A second set of primers that extend from the unknown end of the message back to the known region of the 3’end was provided by the poly (A) tail, while an appended homopolymer tail was used for the 5’end [13 (link)]. Degenerate primers PxTA-F and PxTA-R (Table 1) were designed for the amplification of a partial PxAdoR cDNA. A set of specific primers was synthesized based on the sequence of putative insert for 5'- and 3'-rapid amplification of cDNA ends (RACE). rCai-R1 and rCai-R2 were used for 5'-RACE, and rCai-F1 and rCai-F2 (Table 1) were used for 3'-RACE. The Reverse Transcriptase-Ploymerase Chain Reaction (RT-PCR) and PCR were uesd to amplify the ends of transcripts. RACE was performed by using the 5'-Full RACE Kit and 3'-Full RACE Core Set Version 2.0 (TakaRa, Dalian, China) according to the manufacturer's protocol. RACE products were gel purified and sequenced by Sangon Biotech Company (Shanghai, China).

The homozygous bilocular and multilocular plants in the BC4F5 population were used to amplify the full-length gDNA and cDNA of the target gene. Specific primers (P1, P2, and P3) were designed according to the sequence of Bra015812, the homologous gene of BjLn2 in B. rapa. After the fragments were recovered and compared sequencing with the B. juncea, Cd1, Cd2, Cd3, and Cd4 primers were designed and amplified the fragment of BjLn2 gene. The gDNA and cDNA of the bilocular materials were cloned by G1-F/G6-R and Cd1F/Cd4R, respectively (Table 5). The cDNA of the multilocular material was cloned via primer Cd1F/Cd3R combined a 3′-Full RACE Core Set Version 2.0 (TaKaRa, Dalian, China). Comparative analysis of the candidate genes among the bilocular and multilocular sequences was conducted by Lasergene.

Sequence reads classified into the same virus family or genus with MEGAN 5 were extracted. The accurate loci of each reads and the relative distances between reads of the same virus were determined based on the alignment results obtained with MEGAN 5. We designed specific nested primers based on alignment results for RT-PCR to screen individual samples for the presence and prevalence of pestivirus.
The located reads and contigs were used for reads-based PCR to identify partial genomes in positive individual samples (The primer sequences are available in Supplementary Table 1). Based on partial genomic sequences of each virus, the remaining genomic sequences were determined with genome walking, and 5^′ and 3^′ rapid amplification of cDNA ends (RACE) by using genome walking kit (TaKaRa), 5^′ RACE kit (Invitrogen), and 3^′ full RACE core set, version 2.0 (TaKaRa).