Molecular markers in medicinal plant biotechnology: past and present

Plant based medicines have gained popularity worldwide due to their almost negligible side effects. In India, the three traditional medicinal systems, namely homeopathy, Ayurveda and Siddha rely heavily on plants for medicinal formulations. To prevent the indiscriminate collection of these valuable medicinal plants and for their proper authentication and conservation, it is imperative to go for sustained efforts towards proper germplasm cataloguing and devising conservation strategies. For this purpose, molecular markers have a significant role, as they provide information ranging from diversity at nucleotide level (single nucleotide polymorphisms) to gene and allele frequencies (genotype information), the extent and distribution of genetic diversity, and population structure. Over the past twenty years, the molecular marker field has completely transformed the meaning of conservation genetics which has emerged from a theory-based field of population biology to a full-fledged pragmatic discipline. In this review, we have explored the transition and transformation of molecular marker technologies throughout these years.     

分子标记在食（药）用真菌遗传多样性研究中的应用

食（药）用真菌在经济和生态方面都具有重要意义,其遗传多样性研究是资源可持续利用和生物保护学研究的基础,有利于食（药）用真菌种质资源的收集、保存、评价和利用,也有助于其分类学、系统学及进化等的研究。遗传多样性的研究方法很多,分子标记是目前最常用最有效的方法之一。综合分析了分子标记在食（药）用真菌遗传多样性研究中的应用,比较了各种标记的应用范围、优缺点,探讨了分子标记用于食（药）用真菌遗传多样性评价的前景及问题。     

DNA barcoding,microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants

DNA barcoding, microarray technology and next generation sequencing have emerged as promising tools for the elucidation of plant genetic diversity and its conservation. They are proving to be immensely helpful in authenticating the useful medicinal plants for herbal drug preparations. These newer versions of molecular markers utilize short genetic markers in the genome to characterize the organism to a particular species. This has the potential not only to classify the known and yet unknown species but also has a promising future to link the medicinally important plants according to their properties. The newer trends being followed in DNA chips and barcoding pave the way for a future with many different possibilities. Several of these possibilities might be: characterization of unknown species in a considerably less time than usual, identification of newer medicinal properties possessed by the species and also updating the data of the already existing but unnoticed properties. This can assist us to cure many different diseases and will also generate novel opportunities in medicinal drug delivery and targeting.     

湖北河岸带植物中华蚊母树遗传多样性的SRAP分析

利用SRAP分子标记技术,对湖北省河岸带植物中华蚊母树的4个自然居群和1个迁地居群的遗传多样性和遗传结构进行了分析。结果表明,中华蚊母树物种具有较高水平的遗传多样性,7对SRAP引物进行PCR扩增的多态性位点百分率(PPF)为80.43%,每个位点的等位基因数(A)为2,有效等位基因数(Ae)为1.34,总遗传多样性Nei’s基因多样性指数(Hp)为0.215 9,Shannon信息多样性指数(I)为0.350 9。在居群水平上,5个居群总的遗传变异Ht为0.218 8,居群内的遗传变异Hs为0.193 4,居群间的遗传分化系数Gst为0.116 1,表明在总的遗传变异中有88.39%变异存在于居群内,仅11.61%存在于居群间,居群间的基因流Nm为3.807 2,表明居群间有较大程度的基因交流。UPGMA聚类分析和主成分分析显示中华蚊母树主要分为两个居群组,在长江三峡沿岸香溪和乐天溪由于遗传距离比较近聚为一小类再与高家堰聚为一大类,而沿渡河和三峡植物园居群聚为另一大类,表明迁地居群三峡植物园的中华蚊母树与来自巴东沿渡河居群的样本亲缘关系最近,且三峡植物园迁地保护居群基本保育了其遗传多样性总水平。同时在分析讨论了中华蚊母树遗传多样性与其繁育系统、生境及其起源进化的关系的基础上,评价了中华蚊母树的保护策略,并在评价保护成果的基础上,提出了今后进一步保育的策略。结果还表明SRAP标记是分析中华蚊母树遗传多样性和遗传结构非常可靠的一种标记,而且这是使用SRAP标记研究中华蚊母树的首次报道。     

Genetic variation across species' geographical ranges: the central-marginal hypothesis and beyond

There is growing interest in quantifying genetic population structure across the geographical ranges of species to understand why species might exhibit stable range limits and to assess the conservation value of peripheral populations. However, many assertions regarding peripheral populations rest on the long-standing but poorly tested supposition that peripheral populations exhibit low genetic diversity and greater genetic differentiation as a consequence of smaller effective population size and greater geographical isolation relative to geographically central populations. We reviewed 134 studies representing 115 species that tested for declines in within-population genetic diversity and/or increases in among-population differentiation towards range margins using nuclear molecular genetic markers. On average, 64.2% of studies detected the expected decline in diversity, 70.2% of those that tested for it showed increased differentiation and there was a positive association between these trends. In most cases, however, the difference in genetic diversity between central and peripheral population was not large. Although these results were consistent across plants and animals, strong taxonomic and biogeographical biases in the available studies call for a cautious generalization of these results. Despite the large number of studies testing these simple predictions, very few attempted to test possible mechanisms causing reduced peripheral diversity or increased differentiation. Almost no study incorporated a phylogeographical framework to evaluate historical influences on contemporary genetic patterns. Finally, there has been little effort to test whether these geographical trends in putatively neutral variation at marker loci are reflected by quantitative genetic trait variation, which is likely to influence the adaptive potential of populations across the geographical range.     

Genetic Diversity Assessment of Spanish and Some Endangered Tunisian Pea (Pisum sativum L.) Accessions Based on Microsatellite Markers (SSRs)

In the current investigation, 28 accessions of Spanish and Tunisian peas were characterized by eight SSR polymorphic markers to assess their genetic diversity. Many methods have been applied to evaluate these relationships including diversity indices, analysis of molecular variance, cluster analysis, and population structure. The means of diversity indices, the polymorphism information content (PIC), the allelic richness, and the Shannon information index were 0.51, 3.87, and 0.9, respectively. These results revealed a large polymorphism (84.15 %) which produced a higher degree of genetic distance amongst the accessions. The unweighted pair group approach with arithmetic mean divided the collection of these accessions into three major genetic clusters. Therefore, this article has clearly demonstrated the usefulness of the SSR markers that can significantly contribute to the management and conservation of pea germplasm in these countries, as well as to future reproduction.     

Commercialization of wild medicinal plants from Southwest Puebla,Mexico

This paper describes the trade of dry medicinal plants in the southwestern area of the state of Puebla, Mexico. Gatherers who collect medicinal plants represent the poorest economic link in the trade chain. The critical socioeconomic situation of the human population is reflected in the state of the wild plants at the zone, as a result of an increase in their use for economic survival. The stock of medicinal plants handled by regional traders is an indicator of the abundance and diversity of the flora present in the zone, and permits a dynamic exchange of these products with regional traders from outside. Tracing the commercial path of six medicinal plants from the field to the market, only 6.17% of the consumer price, on average, was returned to the collectors. Most of the medicinal plants marketed and used in Mexico are wild plants. This fact, combined with the increasing demand that exists for medicinal plants in this region, create a potential environmental threat. Since this threat is a multifactorial one, involving the complex socioeconomic conditions in which rural workers and their families live, conservation programs for the wild flora (including reassessed gathering methods, production of selected medicinal species and protection of wild populations and market regulations of quality and equity) have to be applied within an organizational framework of the gatherers and peasants in the zone.     

珠江源头入侵种波氏吻虾虎的遗传多样性分析

为了解入侵种波氏吻虾虎Rhinogobius cliffordpopei在珠江源头地区的遗传多样性分布特征及其影响成因,本研究以线粒体细胞色素b(cyt b)基因为分子标记,对珠江源头的9个水库自然种群进行了遗传多样性与遗传分化分析。获得该物种cyt b基因全序列1 141 bp,其中保守位点1 072个,变异位点69个,无插入和缺失位点。96只个体具有5个单倍型,群体单倍型多样性为0.359±0.059,核苷酸多样性为0.021±0.010,表现为低单倍型多样性与高核苷酸多样性的群体遗传特征。以外群子陵吻虾虎R.giurinus、褐吻虾虎R.brunneus和短吻红斑吻虾虎R.rubromaculatus构建的分子系统发育树和网络分支图显示,波氏吻虾虎群体的所有单倍型与外群物种分开,构成一个单系群,并分化为2个明显的系统分支。分子变异分析结果表明,种群间和种群内的遗传变异率分别为62.99%、37.01%,固定指数为0.630(P<0.01),证实波氏吻虾虎群体形成了显著的遗传分化结构。波氏吻虾虎在珠江源头入侵地具有较高的遗传多样性水平与显著的遗传结构,入侵种群可能受到了奠基者事件和遗传瓶颈效应的影响,而多次人为引入和水利大坝的隔离作用可能为该物种扩散分布和积累突变提供了条件。研究结果将为防治波氏吻虾虎的入侵危害及保护土著鱼类物种多样性提供科学指导。     

Conservation of medicinal plants of Western Ghats,India and its sustainable utilization through <Emphasis Type="Italic">in vitro</Emphasis> technology

Climate change, alien species, and use of land for intensive farming and development are causing severe threat to the plant genetic diversity worldwide. Hence, conservation of biodiversity is considered fundamental and also provides the livelihoods to millions of people worldwide. Medicinal plants play a key role in the treatment of a number of diseases, and they are only the source of medicine for majority of people in the developing world. The tropical regions of the world supply the bulk of current global demand for “natural medicine,” albeit with increasing threat to populations in the world and its genetic diversity. India is a major center of origin and diversity of crop and medicinal plants. India poses out 20,000 species of higher plants, one third of it being endemic and 500 species are categorized to have medicinal value. The Western Ghats is one of the major repositories of medicinal plants. It harbors around 4,000 species of higher plants of which 450 species are threatened. Currently, the number of species added to the red list category in this region is increasing, and the valuable genetic resources are being lost at a rapid rate. Demand for medicinal plants is increasing, and this leads to unscrupulous collection from the wild and adulteration of supplies. Providing high-quality planting material for sustainable use and thereby saving the genetic diversity of plants in the wild is important. During the last 25 years of intensive research, Tropical Botanic Garden and Research Institute has developed in vitro protocol for rapid regeneration and establishment of about 40 medicinally important rare and threatened plants of Western Ghats. In situ conservation alone would not be effective in safeguarding these important species. Thus, utilizing the biotechnoligical approach to complement ex situ conservation program is becoming vital. Propagating biotechnology tools in plant conservation program is a prerequisite to succeed in sustainable use and to complement the existing ex situ measures. In addition to propagation, storage of these valuable genetic resources is equally important. In vitro slow growth of 35 species and cryopreservation using embryo/meristem/seed in 20 different species of rare medicinal plants of this region is accomplished. Plants developed in vitro of ten medicinal plants, which have restricted distribution, were reintroduced in the natural habitat as well.     

Characterizing genic and nongenic molecular markers: comparison of microsatellites and SNPs

The implications of transitioning to single nucleotide polymorphism (SNPs) from microsatellite markers (MSs) have been investigated in a number of population genetics studies, but the effect of genomic location on the amount of information each type of marker reveals has not been explored in detail. We developed novel SNP markers flanking 1 kb regions of 13 genic (within gene or <1 kb away from gene) and 13 nongenic (>10 kb from annotated gene) MSs in the threespine stickleback genome to obtain comparable data for both types of markers. We analysed patterns of genetic diversity and divergence on various geographic scales after converting the SNP loci within each genomic region into haplotypes. Marker type (SNP haplotype or MS) and location (genic or nongenic) significantly affected most estimates of population diversity and divergence. Between‐lineage divergence was significantly higher in SNP haplotypes (genic and nongenic), however, within‐lineage divergence was similar between marker types. Most divergence and diversity measures were uncorrelated between markers, except for population differentiation which was correlated between MSs and SNP haplotypes (both genic and nongenic). Broad‐scale population structure and assignment were similarly resolved by both marker types, however, only the MSs were able to delimit fine‐scale population structuring, particularly when genic and nongenic markers were combined. These results demonstrate that estimates of genetic variability and differentiation among populations can be strongly influenced by marker type, their genomic location in relation to genes and by the interaction of these two factors. This highlights the importance of having an awareness of the inherent strengths and limitations associated with different molecular tools to select the most appropriate methods for accurately addressing various ecological and evolutionary questions.     

Conservation genetics: beyond the maintenance of marker diversity

One of the major problems faced by conservation biologists is the allocation of scarce resources to an overwhelmingly large number of species in need of preservation efforts. Both demographic and genetic information have been brought to bear on this problem; however, the role of information obtained from genetic markers has largely been limited to the characterization of gene frequencies and patterns of diversity. While the genetic consequences of rarity may be a contributing factor to endangerment, it is widely recognized that demographic factors often may be more important. Because patterns of genetic marker variation are influenced by the same demographic factors of interest to the conservation biologist, it is possible to extract useful demographic information from genetic marker data. Such an approach may be productive for determining plant mating systems, inbreeding depression, effective population size, and metapopulation structure. In many cases, however, data consisting only of marker frequencies are inadequate for these purposes. Development of genealogical based analytical methods coupled with studies of DNA sequence variation within and among populations is likely to yield the most information on demographic processes from genetic marker data. Indeed, in some cases it may be the only means of obtaining information on the long-term demographic properties that may be most useful for determining the future prospects of a species of interest.     

Array-based high-throughput DNA markers for crop improvement

The last two decades have witnessed a remarkable activity in the development and use of molecular markers both in animal and plant systems. This activity started with low-throughput restriction fragment length polymorphisms and culminated in recent years with single nucleotide polymorphisms (SNPs), which are abundant and uniformly distributed. Although the latter became the markers of choice for many, their discovery needed previous sequence information. However, with the availability of microarrays, SNP platforms have been developed, which allow genotyping of thousands of markers in parallel. Besides SNPs, some other novel marker systems, including single feature polymorphisms, diversity array technology and restriction site-associated DNA markers, have also been developed, where array-based assays have been utilized to provide for the desired ultra-high throughput and low cost. These microarray-based markers are the markers of choice for the future and are already being used for construction of high-density maps, quantitative trait loci (QTL) mapping (including expression QTLs) and genetic diversity analysis with a limited expense in terms of time and money. In this study, we briefly describe the characteristics of these array-based marker systems and review the work that has already been done involving development and use of these markers, not only in simple eukaryotes like yeast, but also in a variety of seed plants with simple or complex genomes.     

鸡的遗传多样性的研究方法及研究进展

遗传多样性是生物学研究中的一个重要领域,研究鸡的遗传多样性,不仅能加强生物多样性的保护。同时对起源进化、分类鉴定及遗传育种等都有重要的意义。本文对目前DNA水平鸡的遗传多样性的研究方法和研究进展进行了详细的阐述。重点介绍了DNA分子标记的特征;概括了在鸡遗传多样性分子标记的方法,包括微卫星分子标记(SSR)、扩增片段长度多态性(AFLP)、随机扩增多态性标记(RAPD)、限制性片段长度多态性标记(RFLP)和单核苷酸多态性标记(SNP)。本文综述了最近有关鸡DNA水平的遗传多样性的研究方法在系统学、遗传结构、生物地理等研究中的应用情况;提出在研究鸡遗传多样性时,可根据研究的目的,选择合适的方法。     

Conservation and documentation of the medicinal plant resources of India

In India, activities in the field of medicinal plants, including conservation of germplasm, have been enhanced significantly during the past couple of decades and a huge volume of data is being generated out of these works. For maintaining the records in a consolidated form, documentation is required to store and manage all information on the related studies. In accordance with the implementation of various plans and programmes, some pioneer organisations started developing databases on medicinal plants. Based on the knowledge on contemporary works, as collected from published literature and websites, this article presents information on current activities in India in two important aspects of the field, namely, (1) conservation of medicinal plants; and (2) management of data generated from such studies. Another important aspect of the article is the announcement of a plant conservation related software, ‘PlantCon’. This digitised database contains data of 40 selected nationally prioritised medicinal plants (list enclosed). The notable difference of ‘PlantCon’ from other databases lies in its conservation-related information which is up-to-date and covers a wide area of Indian geographical sites. The database provides information in a user-friendly manner.     

Molecular tools and analytical approaches for the characterization of farm animal genetic diversity

Genetic studies of livestock populations focus on questions of domestication, within- and among-breed diversity, breed history and adaptive variation. In this review, we describe the use of different molecular markers and methods for data analysis used to address these questions. There is a clear trend towards the use of single nucleotide polymorphisms and whole-genome sequence information, the application of Bayesian or Approximate Bayesian analysis and the use of adaptive next to neutral diversity to support decisions on conservation.     

Genetic diversity of captive forest musk deer (Moschus berezovskii) inferred from the mitochondrial DNA control region

Forest musk deer ( Moschus berezovskii ) were once distributed widely in China. However, wild populations have declined dramatically because of poaching and habitat loss. Captive breeding populations have been established for several decades, but the genetic backgrounds of most captive populations were unclear and the population sizes increased very slowly. To provide useful information for conservation and management of this species, we investigated the genetic diversity and population structure of forest musk deer by analysing a 582-bp fragment of the mitochondrial DNA (mtDNA) control region (CR) in three captive breeding populations in Sichuan Province, China. Ninety-four variable sites and 27 haplotypes were observed in 109 individuals, and the nucleotide and haplotype diversities were relatively high compared with those of other endangered mammals. Of the three investigated populations, the Maerkang population had the highest nucleotide diversity ( π  = 0.0568), haplotype diversity ( h  =   0.836) and average intra-population genetic distance (0.062). The analysis of molecular variance demonstrated that most variation occurred within samples and that there was significant differentiation of the three populations. Estimates of gene flow indicated that there were few genetic exchanges among the three populations. Building pedigree records and increasing gene flow between populations will be helpful for conserving these populations and this species.     

Too much of a good thing? Finding the most informative genetic data set to answer conservation questions

Molecular markers are a useful tool allowing conservation and population managers to shed light on genetic processes affecting threatened populations. However, as technological advancements in molecular techniques continue to evolve, conservationists are frequently faced with new genetic markers, each with nuanced variation in their characteristics as well as advantages and disadvantages for informing various questions. We used a well‐studied population of Tasmanian devils (Sarcophilus harrisii) from Maria Island, Tasmania, to illustrate the issues associated with combining multiple genetic data sets and to help answer a question posed by many population managers: which data set will provide the most precise and accurate estimates of the population processes we are trying to measure? We analysed individual heterozygosity (as internal relatedness, IR) of 96 individuals, calculated using four genetic marker types (putatively neutral microsatellites, major histocompatibility complex‐linked microsatellites, reduced representation sequencing, and candidate region resequencing). We found no correlation in IR values across marker types, suggesting that various genetic markers reflect different aspects of genomic diversity. In addition, some marker types were more informative than others for conservation decision‐making. Reduced representation sequencing provided the highest precision (lowest error) for estimating population‐level genetic diversity, and most closely reflected genome‐wide heterozygosity both theoretically and empirically. Within the conservation context, our results highlight important considerations when choosing a molecular technique for wildlife genetics.     

药用植物内生真菌及活性物质多样性研究进展

药用植物具有丰富的物种多样性,是人类生存与发展的重要自然资源。内生真菌广泛存在于健康植物组织内部,是植物微生态系统的重要组成部分,各种药用植物中蕴藏着非常丰富的内生真菌。通过与药用植物的“协同进化”,某些内生真菌具有了产生与宿主植物相同或相似的生物活性物质的能力。内生真菌产生的各种活性物质,在生物制药、农业生产、工业发酵等方面都表现出美好的应用前景,受到世界各国专家的广泛关注。利用内生真菌发酵实现生物活性物质的工业化生产,可以提高产量、降低产品成本,满足人们日益增长的需求;同时有利于珍稀、濒危药用植物资源的保护,对减少野生药用植物多样性的破坏具有重要意义。本文从药用植物内生真菌物种多样性与产生生物活性物质多样性等方面总结近年最新的研究进展,提出了内生真菌及活性物质研究的未来发展方向。     

Genetic diversity of Eurycoma longifolia inferred from single nucleotide polymorphisms

Eurycoma longifolia Jack. is a treelet that grows in the forests of Southeast Asia and is widely used throughout the region because of its reported medicinal properties. Widespread harvesting of wild-grown trees has led to rapid thinning of natural populations, causing a potential decrease in genetic diversity among E. longifolia. Suitable genetic markers would be very useful for propagation and breeding programs to support conservation of this species, although no such markers currently exist. To meet this need, we have applied a genome complexity reduction strategy to identify a series of single nucleotide polymorphisms (SNPs) within the genomes of several E. longifolia accessions. We have found that the occurrence of these SNPs reflects the geographic origins of individual plants and can distinguish different natural populations. This work demonstrates the rapid development of molecular genetic markers in species for which little or no genomic sequence information is available. The SNP markers that we have developed in this study will also be useful for identifying genetic fingerprints that correlate with other properties of E. longifolia, such as high regenerability or the appearance of bioactive metabolites.     

基于基因组SNPs对东亚家马不同群体遗传多样性的评估

家马地方品种的遗传多样性是动态演变的, 并与其育种模式息息相关; 在过去约300年中, 育种者培育了具突出表型特征的标准化培育品种, 该类型品种对家马地方品种的繁育产生了巨大影响, 这是导致家马地方品种遗传多样性下降及遗传分化的主要因素之一。本研究采集了5个东亚家马地方品种(蒙古马、哈萨克马、河曲马、藏马和西南马)、2个西亚家马品种(阿拉伯马和阿哈尔捷金马)以及2个欧洲家马品种(设特兰矮马和克莱斯黛尔马)的70个样品, 并整合之前所发表的100匹内蒙古地区家马不同群体单核苷酸多态性(SNPs)数据集, 通过全基因组重测序和生物信息学方法分析了东亚家马不同群体的遗传多样性。研究发现东亚家马地方品种具有较丰富的遗传多样性, 相比欧洲和西亚品种产生了显著的遗传分化, 其中蒙古高原群体的遗传多样性最为丰富; 受杂交改良影响, 内蒙古不同群体间产生了一定程度的遗传分化; 河曲马和藏马的遗传背景最为单一, 受引种杂交繁育影响较小。本研究评估了东亚家马地方品种的遗传多样性分布格局和演化特征, 可为建立家马地方种质核心保护群体以及培育新品种提供遗传学理论支持。