Research Progress in Genetics and Molecular Biology of Curcuma L.

Xuejiao HE, Zhicheng YU, Jinshui LIN, Kunxiu CAI, Chaoyang HUANG

Fujian Institute of Tropical Crops, Zhangzhou 363001, China

Abstract There are few studies on the genetic evolution of Curcuma L., and it is easy to have synonyms or homonyms. In order to make better development and utilization of Curcuma L., by consulting the relevant literature, the molecular biology and genetics of Curcuma L. were summarized, and discussed in this paper, in order to lay a foundation for the study of phylogeny and genetic evolution of Curcuma L.

Key words Curcuma L., Genetics, Molecular biology, Research progress

1 Introduction

CurcumaL. as the third largest genus of Zingiberaceae, not only has high ornamental value, but also has high medicinal value. It can also be used as spices and dyes, so it is a plant group with high economic value. The genus is widely distributed in China, India, Indonesia, Malaysia, Thailand and other Southeast Asian countries. There are about 10 species in China, mainly distributed from southeast to southwest China (Fujian, Guangdong, Guangxi, Yunnan, Sichuan, Guizhou.etc.)[1]. Traditional studies believe that it has a good effect in activating blood circulation and removing blood stasis, relieving qi and depression, promoting gallbladder and clearing heart[2]. In recent years, studies have shown that curcumin, the main ingredient ofCurcumaL., has the effects of reducing blood lipid, anti-tumor, anti-oxidation, anti-radiation and so on. When it is used as curry, eating once a week can greatly reduce the incidence of tumor and Alzheimer’s disease[4]. It also plays an important role in pigment (yellow), cosmetics, pesticides and other fields.CurcumaL. plants are widely distributed and different, and flower specimens are difficult to collect, and there are many synonyms in different regions, so it is very important to study its classification and genetic system. In order to make better use and development ofCurcumaL. plants, this paper mainly summarizes the research progress in molecular biology ofCurcumaL. plants, hoping to provide some theoretical reference for the classification, preservation, development and utilization ofCurcumaL. plants.

2 Research progress of molecular biology

In recent years, molecular biology technology has developed vigorously, is widely used in plant classification, identification and plant genetic evolution, and has a broad application prospect in the field of plant research. At present, molecular marker techniques such as RAPD, ISSR, SRAP and RAMP have shown good stability in the study of many species, and have been used in the study of plant genetic relationship, genetic diversity, genetic map and so on.

2.1 RAPDChen Yuhengetal.[5]used RAPD molecular marker technique to study three Turmeric varieties ofCurcumaL., and the results showed thatCurcumasichuaensisandCurcumaphaeocaulisVal. were closely related, and in accordance with their morphological and chemical data, it was considered that the two should be combined. Xiao Xiaoheetal.[6]systematically classifiedCurcumaL., 3 species ofRadixCurcumaeand 6 species of zedoary turmeric by RAPD technique, which provided a new scientific basis for the identification of traditional Chinese medicine ofCurcumaL. Wang Xiaohuietal.[7]established the RAPD-PCR reaction system ofCurcumaphaeocaulisVal. and optimized its conditions, which laid a foundation for the subsequent molecular biology in the classification, identification and genetic evolution ofCurcumaL.

2.2 ISSRTang Xiaochuangetal.[8]established the ISSR-PCR reaction system ofCurcumaphaeocaulisVal. and optimized its conditions, which provided help for the subsequent application of ISSR inCurcumaL. plants. Li Yangyietal.[9]used ISSR molecular markers to analyze the genetic relationship and genetic diversity of 69 materials of 10 species ofCurcumaL. plants in China. The results showed that the materials could be divided into three categories according to ISSR clustering.Curcumasichuaensismay be a cultivated variety ofCurcumaL., and the genetic relationship between species has a certain relationship with geographical environment. For example, the long genetic distance betweenCurcumayunnanensisandcurcumazedoariamay be caused by the differences among populations. The genetic relationship betweenCurcumasichuaensisandCurcumaphaeocaulisVal. was far away, which was similar to the result of RAPD analysis of Xiao Xiaoheetal., but different from that of Chen Yuhengetal., indicating that the genetic relationship between them needs to be further studied.

2.3 RAMPRAMP (Random Amplified Microsatellite Polymorphism) is a molecular marker technique proposed in 1994, which mainly uses a combination of 5’anchored SSR primers and a RAPD primer, and randomly amplifies the microsatellite DNA of the genome, which combines the advantages of RAPD and SSR. Lei Yingxia[11]conducted RAMP molecular marker analysis on 10 species ofCurcumaL. from different populations. The results showed thatCurcumaL. andCurcumasichuaensiswere closely related, and it was possible thatCurcumasichuaensiswas a cultivated variety ofCurcumaL., which was the same as Xiao Xiaohe’s point of view[1]. The genetic relationship betweenCurcumasichuaensisandCurcumaphaeocaulisVal. was relatively close, but they can not be merged, indicating that the genetic relationship betweenCurcumasichuaensisandCurcumaphaeocaulisVal. needs to be further studied. The phylogenetic relationship betweenCurcumayunnanensisandcurcumazedoariawas far away, which was far from the results of Xiao Xiaohe’s RAPD[1], which may be caused by the influence of population or human error. Li Yangyietal.[12]analyzed the genetic variation and genetic relationship of 69 materials of 10 species ofCurcumaL. by RAMP technique. The results showed that 69 materials could be divided into four groups,CurcumaL. andCurcumasichuaensiswere closely related, andCurcumasichuaensismight be a cultivated variety ofCurcumaL. The origin ofCurcumasichuaensismay be Sichuan, and whether it exists as a separate species remains to be further studied.

2.4 SRAPSRAP (Sequence-Related Amplified Polymorphism) technology was put forward in 2001[13], which combines the advantages of RAPD and AFLP. It has high polymorphism, high yield, strong co-dominance, strong repeatability, simple operation, certain versatility of primers and high utilization rate of primers. Based on the above advantages, it has been widely used in plant taxonomy, as well as in comparative genomics, genetic diversity and other research fields[14-16]. Li Minetal.[17]conducted SRAP studies on 9 samples of medicinal materials ofCurcumaL., and considered that SRAP technique could well analyze the interspecific relationship ofCurcumaL. plants. Six varieties ofCurcumaL. could be divided into two groups: one group of inflorescences appeared from leaf sheath, including Radix Curcumae Longae andCurcumasichuaensis, andCurcumasichuaensiswas probably a cultivated variety ofCurcumaL.; a group of inflorescences appeared from the rhizome, includingcurcumazedoaria,Curcumasichuaensis,RadixCurcumaeKwangsiensisandCurcumaphaeocaulisVal., among whichCurcumasichuaensismay be a cultivated variety ofcurcumazedoaria.

2.5 Isozyme analysisIsozyme is the product of gene expression, and specific gene expression will produce specific isozyme zymogram. Isozyme analysis is also widely used in plant genetic relationship analysis, classification and identification, genetic evolution and so on. Through the analysis of POD isozymes, it was found that the zymogram of the sameCurcumaL. plant was not affected by its origin and morphology, and the closely related species had a smaller zymogram distance, so it was speculated that there was no variety ofCurcumaKwangsiensis[21]. Tang Jiayongetal.[22]used PAGE technique to analyze the isozymes of POD and EST to study the genetic relationship among 39 materials of 6 species of medicinal plants ofCurcumaL. The results showed that the genetic relationship between different materials of the same plant was not related to the geographical origin, the genetic relationship betweenCurcumasichuaensisandCurcumaL. was very close, andCurcumasichuaensismay be a cultivated variety ofCurcumaL. By combining the results of the study of four isozymes of SOD, PPO, MDH and COD and the psbA-trnH spacer of chloroplast gene, it is considered that the wild and cultivated species ofCurcumaL. are differentiated to some extent, and thatCurcumasichuaensismay exist as a separate species[23]. Deng Jiabinetal.[24]used PAGE technology to analyze the enzyme bands of SOD, PPO and COD, six species of Curcuma medicinal plants can be distinguished. PPO and COD can also be used to distinguish materials from different populations within species, and cluster analysis can be used to distinguishCurcumaL. fromCurcumasichuaensis. It is also considered that the cultivated type and wild type ofCurcumaL. are differentiated.

2.6 Barcoding technology of DNAWith the development of molecular biology and bioinformatics, DNA barcoding technology is used to identify and classify plants through one or more short DNA sequences[25]. It is convenient, fast and efficient, and has a broad application prospect in the identification, classification and protection of medicinal plants. Shi Linchun[26]amplified and measured 8 candidate sequences of 26 genera and 314 species of Zingiberaceae by PCR, including 3 coding sequences rbcL, matK, rpoB and 5 non-coding sequences trnH-psbA, psbK-psbI, atpB-rbcL, ITS and ITS2. The results showed that ITS and ITS2 sequences were ideal candidate barcoding sequences for identification of medicinal plants of Zingiberaceae. A total of 96 materials of 44 species ofCurcumaL. (from China and Myanmar) and 5 other species were studied by non-coding DNA sequences rbcL, trnH-psbA, matK, trnL-F and coding sequence ITS2. The results showed that ITS2 had a high degree of recognition. If it is combined with other general DNA sequence bar codes,CurcumaL. plants can be distinguished to some extent. The study of Zhong Zhimin[28]also showed that the DNA sequence ITS2 could separate and cluster Zingiberaceae plants. Ren Yaoyaoetal.[29]identified Zingiberaceae plants includingCurcumaL. in Emei Mountain by DNA coding sequence ITS2. The results showed that using ITS2 as DNA bar code can quickly and efficiently identify Zingiberaceae plants and clarify their evolutionary relationship. Cao Huietal.[30]directly sequenced the ribosomal RNA small subunit gene (18s rDNA) and chloroplast lysine tRNA gene (trnK) of 18 species ofCurcumaL. plants by molecular systematics and bioinformatics, and obtained the complete sequence of these two genes. The trnK sequence had obvious interspecific gene bar code. It is considered that the trnK gene sequence has positive significance for the identification ofCurcumaL. plants and their medicinal materials. Sixteen samples ofCurcumaL. were studied by the combination of three DNA bar codes of ITS-LUS D1/D3, rbcL and matK and HPLC fingerprint analysis. The results showed that ITS-LUS D1/D3 bar code combined with HPLC fingerprint analysis can clarify the differences amongCurcumaL. samples, but whether it can be applied to the identification ofCurcumaL. plants remains to be further studied[31]. Zhang Yuxiuetal.[32]evaluated 7 kinds of DNA barcode sequences of 9 samples from three original plants ofRhizomaCurcumae, which were ITS, ITS2, matK, psbA-trnH, trnL-trnF, rpoB and atpB-rbcL, respectively. The results showed that the atpB-rbcL barcode sequence could distinguish the three different original plants ofRhizomaCurcumaeand could be used as a barcode sequence for the identification ofRhizomaCurcumae. The results of Zhang Guifangetal.[33]showed that DNA bar code matK could effectively identify Zingiberaceae plants.

3 Discussion

Molecular marker technology can reflect the differences between different materials to a certain extent, but the current molecular marker methods can not detect the difference of individual bases, so the molecular classification ofCurcumaL. only supports part of the morphological classification. At present, the biggest problem is that there are few groups and gene fragments used in the phylogeny and genetic evolution ofCurcumaL., which leads to the instability of results, so it is necessary to expand the research groups and gene fragments in the future to better construct the phylogenetic tree and genetic evolution map ofCurcumaL.

In the study ofCurcumaL. plants, it was found that most of the DNA barcodes had poor recognition ofCurcumaL. plants. Only a small number of sequences such as ITS2 and trnK have better effect, and when the universal primers such as rbcL are applied toCurcumaL., the sequencing rate is low, the informative locus is low, and the recognition degree is not high. Therefore, the development of specific primers forCurcumaL. plants will become an important direction in the study of genetic relationship ofCurcumaL. plants.

In the future research, we can organically combine morphology and molecular biology, and compare the species reported in China with the species abroad, in order to better reveal the origin and evolution of the species resources of the genus, and provide a basis for the research, development and utilization of the genus. More new research methods can be used, such as simplified genome sequencing, which was applied to the sequencing analysis of Valeriana plants in 2018. It shows that the simplified genome sequencing technology and the reasoning method of using super matrix combined with evolutionary tree are of great significance to the study of species development and genetic evolution. Therefore, more representative gene loci and gene fragments can be obtained by using relatively new sequencing techniques and traditional gene methods in the study of genetic evolution and phylogeny ofCurcumaL., to lay a foundation for the study of phylogeny and genetic evolution ofCurcumaL.