Picrorhiza kurroa is a medicinal herb with diverse pharmacological applications due to the presence of iridoid glycosides, picroside-I (P-I), and picroside-II (P-II), among others. Any genetic improvement in this medicinal herb can only be undertaken if the biosynthetic pathway genes are correctly identified. Our previous studies have deciphered biosynthetic pathways for P-I and P-II, however, the occurrence of multiple copies of genes has been a stumbling block in their usage. Therefore, a methodological strategy was designed to identify and prioritize paralogues of pathway genes associated with contents of P-I and P-II. We used differential transcriptomes varying for P-I and P-II contents in different tissues of P. kurroa. All transcripts for a particular pathway gene were identified, clustered based on multiple sequence alignment to notify as a representative of the same gene (≥ 99% sequence identity) or a paralogue of the same gene. Further, individual paralogues were tested for their expression level via qRT-PCR in tissue-specific manner. In total 44 paralogues in 14 key genes have been identified out of which 19 gene paralogues showed the highest expression pattern via qRT-PCR. Overall analysis shortlisted 6 gene paralogues, PKHMGR3, PKPAL2, PKDXPS1, PK4CL2, PKG10H2 and PKIS2 that might be playing role in the biosynthesis of P-I and P-II, however, their functional analysis need to be further validated either through gene silencing or over-expression. The usefulness of this approach can be expanded to other non-model plant species for which transcriptome resources have been generated.

Picrorhiza kurroa是一种具有多种药理应用的药用植物，这是因为存在艾立替木糖苷，picroside-I（PI）和picroside-II（P-II）等。只有正确识别了生物合成途径基因，才能对这种草药进行任何遗传改良。我们以前的研究已经破译了PI和P-II的生物合成途径，但是，基因多拷贝的出现一直是它们使用的绊脚石。因此，设计了一种方法学策略，以鉴定与PI和P-II含量相关的途径基因的旁系同源物并确定其优先级。我们使用了不同的转录组，其在不同的P. kurroa组织中的PI和P-II含量各不相同。识别特定途径基因的所有转录本，并基于多个序列比对进行聚类，以通知代表同一基因（≥99％序列同一性）或同一基因的旁系同源物。此外，以组织特异性方式通过qRT-PCR测试各个旁系同源物的表达水平。总共鉴定出14个关键基因中的44个旁系同源物，其中19个基因旁系同源物通过qRT-PCR显示出最高的表达模式。总体分析入围了6个基因类似物PKHMGR3，PKPAL2，PKDXPS1，PK4CL2，PKG10H2和PKIS2它们可能在PI和P-II的生物合成中发挥作用，但是，它们的功能分析需要通过基因沉默或过表达来进一步验证。该方法的有用性可以扩展到已经为其生成转录组资源的其他非模式植物物种。