## 3 Discussion  
Shikonin extracted from _L. erythtrorhizon_ has a wide spectrum of medical and commercial values, and its biosynthesis is jointly regulated by various enzymes that are members of multiple gene families, especially HMGR in the upstream MVA pathway, and PGT in the downstream pathway to link the upstream MVA and phenylpropanol pathways [22; 52]. HMGR, which catalyzes the formation of MVA from HMG-CoA, is the first key rate-limiting enzyme in the metabolic synthesis of shikonin and terpenoids [29; 52]. In this study, we successfully identified 11 _LerHMGRs_ from _L. erythtrorhizon_ and 113 _HMGR_ family genes from the whole genome sequence of the other 35 representative plants, and their characteristics, phylogenetic relationships, duplications and losses, and expansion events were systematically studied. It complements _HMGR_ gene family analysis in some species recently sequenced [32]. More importantly, through expression patterns analysis and in vitro enzyme activity, we hypothesized that _LerHMGR1_ and _LerHMGR2_ play key rate-determining roles in shikonin biosynthesis. Our analysis revealed that more _HMGR_ family genes were detected in higher plants than in lower plants, particularly in _P. tabuliformis_, _L. erythtrorhizon_, and _O. europaea_, which may be due to the large number of transposons in Gymnosperms [37] and the multiple WGD or WGT events in Angiosperms [53], leading to the duplication of more _HMGR_ genes to adapt to the ecological environment. The analysis of _LerHMGR_'s evolutionary status and expansion events demonstrated the importance of WGD, segmental duplication, and tandem duplication in the evolution of the shikonin/alkanmin pathway in _L. erythtrorhizon_, which is consistent with other studies of specialized metabolisms in plants [54; 55; 56]. We identified a total of 11 _LerHMGR_ family members from the _L. erythtrorhizon_ genome published by [PERSON] et al. [42], which is contradictory to the number of _LerHMGR_ family members we identified from the genome published by [PERSON] et al. in 2020 [22]. Blast analysis results for two _HMGR_ group sequences showed that the eight _LerHMGR_s identified from the genome by [PERSON] et al. belong to the eleven _LerHMGR_ family members identified from the genome by [PERSON] et al. (Table S6). Additionally, ten _LerHMGR_s sequences were further confirmed via amplification using specific primers. The 433 bp sequence fragment of _HMGR_ previously cloned from _L. erythtrorhizon_[36] is part of _LerHMGR1_ identified in this study. However, _LerHMGR11_ could not be amplified using specific primers, and its expression levels in _L. erythtrorhizon_ roots, stems, and leaves, as well as hairy roots in a B5 light culture and M9 dark culture, were not detected when analyzed by qPCR, which may be due to the low expression level of _LerHMGR11_. HMGR is mostly found in plants as a gene family [55] and its importance may stem from the fact that it can maintain beneficial metabolite production, that their functional redundancy allows plants to survive adversity or stress, or that each member is in charge of a different final compound synthesis [57]. We investigated the expression patterns of _LerHMGRs_ in the tissues and their responses to M9 medium in the darkness (Figure 4). With the exception of _LerHMGR2_, which showed a significant increase in hairy roots/callus cultured in M9 in the darkness compared to B5 in the light, as well as a root-specific expression where shikonin was biosynthesized, _LerHMGR1_ also showed an up-regulatedexpression in the M9 dark culture and root, while the expression levels and induction amplitude of other homologous genes were lower or even had no difference in the M9 dark culture. These findings suggest that _LerHMGR2_ and _LerHMGR1_ may play a major and redundant role in shikonin biosynthesis. Additionally, we noticed that other _LerHMGRs_, such as _LerHMGR3-LerHMGR10_, have an obvious level of expression in leaves, stems, or roots. This implies that the _LerHMGRs_ members may have distinct roles in plant growth.