Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.)

Background

Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.

Results

The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.

Conclusions

The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.     

镉在莲各器官中累积规律的研究

莲子是我国重要的特产优质资源,但莲等水生植物容易吸收积累重金属.为了探究莲对重金属镉的吸收积累特点,利用盆栽外施不同浓度的镉(0,1.0 mg·kg-1,2.5 mg·kg-1)培养两个莲子品种,检测分析镉对莲子生物量的影响及莲各器官中镉的含量.结果表明:随着外施镉浓度的增加,两种莲子生物量下降,各器官中镉含量增加,且...     

Identification of copy number variation and population analysis of the sacred lotus (Nelumbo nucifera)

ABSTRACT

The sacred lotus (Nelumbo nucifera) is widely cultured in East Asia for its horticultural, agricultural, and medicinal values. Although many molecular markers had been used to extrapolate population genetics of the sacred lotus, a study of large variations, such as copy number variation (CNV), are absent up to now. In this study, we applied whole-genome re-sequencing to 24 lotus accessions, and use read depth information to genotype and filter original CNV call. Totally 448 duplications and 4,267 deletions were identified in the final CNV set. Further analysis of population structure revealed that the population structure patterns revealed by CNV and SNP are largely consistent with each other. Our result indicated that deep sequencing followed by genotyping is a quick and straightforward way to mine out CNV from the population, and the CNV along with SNP could enable us to better comprehend the biology of the plant.     

Isolation and characterization of microsatellite markers in the sacred lotus (Nelumbo nucifera Gaertn.)

The sacred lotus (Nelumbo nucifera Gaertn.) is an aquatic plant of economic and ornamental importance in China. From an (AG)_n‐enriched genomic library, 24 microsatellites were isolated and identified by using the (fast isolation by the AFLP of sequences containing repeats) FIASCO protocol. Eleven loci showed polymorphism with two to six alleles per locus. These markers yielded 42 alleles in a survey of 32 accessions of the sacred lotus. Eleven effective primer pairs of simple sequence repeats were designed and will be used as genetic markers to evaluate the fine‐scale population structure of the sacred lotus in the future.     

Genetic diversity and differentiation of lotus (Nelumbo nucifera) accessions assessed by simple sequence repeats

Nelumbo nucifera (lotus) is a perennial aquatic crop of substantial economical and ecological importance. Currently, the evaluation of the genetic variation of lotus germplasm accessions using codominant simple sequence repeat (SSR) markers is significant, and it is essential for understanding the population structure of N. nucifera. Here we report the genetic diversity and differentiation of 92 N. nucifera accessions (82 cultivated varieties and 10 wild lotus) using 50 polymorphic SSR markers. A total of 195 alleles were detected, with an average of 3.9 alleles/locus. The mean polymorphic information content (PIC) and the mean expected heterozygosity were 0.43 and 0.50, respectively. The genetic relationships among accessions were estimated using an unweighted pair‐group method with arithmetic average (UPGMA) cluster and principal coordinate analysis (PCA). Both methods revealed that the lotus accessions from China and those from its adjacent Asian countries formed a single cluster, respectively. The cultivated varieties were correlated with their major characteristics in cultivation (the seed, rhizome and flower type) rather than their geographic distribution. On the basis of the Bayesian model‐based analyses, two genetically distinct groups (the seed lotus group and the rhizome lotus group) were generated, with a strong differentiation between them (F_ST = 0.57). The seed lotus group exhibited higher genetic diversity than did the rhizome lotus group. The results herein indicated that the current levels of genetic diversity and differentiation between the lotuses have been greatly influenced by artificial selection.     

Whole genome re‐sequencing reveals evolutionary patterns of sacred lotus (Nelumbo nucifera)

Sacred lotus (Nelumbo nucifera or lotus) is an important aquatic plant in horticulture and ecosystems. As a foundation for exploring genomic variation and evolution among different germplasms, we re‐sequenced 19 individuals from three cultivated temperate lotus subgroups (rhizome, seed and flower lotus), one wild temperate lotus subgroup (wild lotus), one tropical lotus group (Thai lotus) and an outgroup (Nelumbo lutea). Through genetic diversity and polymorphism analysis by non‐missing SNP sites widely distributed in the whole genome, we confirmed that wild and Thai lotus exhibited greater differentiation with a higher genomic diversity compared to cultivated lotus. Rhizome lotus had the lowest genomic diversity and a closer relationship to wild lotus, whereas the genomes of seed and flower lotus were admixed. Genes in energy metabolism process and plant immunity evolved rapidly in lotus, reflecting local adaptation. We established that candidate genes in genomic regions with significant differentiation associated with temperate and tropical lotus divergence always exhibited highly divergent expression pattern. Together, this study comprehensive and credible interpretates important patterns of genetic diversity and relationships, gene evolution, and genomic signature from ecotypic differentiation of sacred lotus.     

Genetic diversity and inferred ancestry of Asian lotus (Nelumbo nucifera) germplasms in Thailand and Vietnam

Tropical lotus (Nelumbo) is an important and unique ecological type of lotus germplasm. Understanding the genetic relationship and diversity of the tropical lotus is necessary for its sustainable conservation and utilization. Using 42 EST-SSR (expressed sequence tag-simple sequence repeats) and 30 SRAP (sequence-related amplified polymorphism) markers, we assessed the genetic diversity and inferred the ancestry of representative tropical lotus from Thailand and Vietnam. In total, 164 and 41 polymorphic bands were detected in 69 accessions by 36 EST-SSR and seven SRAP makers, respectively. Higher genetic diversity was revealed in Thai lotus than in Vietnamese lotus. A Neighbor-Joining tree of five main clusters was constructed using combined EST-SSR and SRAP markers. Cluster I included 17 accessions of Thai lotus; cluster II contained three Thai accessions and 11 accessions from southern Vietnam; and cluster III was constituted by 13 accessions of seed lotus. Consistent with the results from the Neighbor-Joining tree, the genetic structure analysis showed that the genetic background of most Thai and Vietnamese lotus was pure, as artificial breeding has been rare in both countries. Furthermore, these analyses indicate that Thai and Vietnamese lotus germplasms belong to two different gene pools or populations. Most lotus accessions are genetically related to geographical distribution patterns in Thailand or Vietnam. Our findings showed that the origin or genetic relationships of some unidentified lotus sources can be evaluated by comparing morphological characteristics and the data of molecular markers. In addition, these findings provide reliable information for the targeted conservation of tropical lotus and parent selection in breeding novel cultivars of lotus.     

莲胚芽暗萌发过程中的光合系统发育研究

被子植物在黑暗中萌发生长不能合成叶绿素和建成光合系统,但是将莲（Nelumbo nucifera Geartn.)胚芽置于黑暗中萌发生长时,却可以清楚地观察到它的光合系统进行发育;首先,在原位低温荧光光谱上,LHCⅡ的荧光发射逐步红移变成典型的PSⅡ荧光发射,同时随着萌蝗延长,PSⅠ的荧光发射也从无到有,逐渐增强;其次,对暗萌发10d莲苗的叶绿体进行部分变性凝胶电泳分析也得到了PSⅠ的叶绿素蛋白复合物条带。通过Western blots的蛋白免疫检测,在暗萌发莲苗中也证实了LHCⅠ组分中有Lhca1的存在;最后,对暗萌发莲苗叶绿体的PSⅡ和PSⅠ电子传递活性测量结果表明,在暗中发育形成的PSⅡ和PSⅠ核心都是有光化学活性的。章讨论了莲胚芽暗萌发过程中进行光合系统发育的原因。     

Effective pollinators of Asian sacred lotus (Nelumbo nucifera): contemporary pollinators may not reflect the historical pollination syndrome

Background and Aims

If stabilizing selection by pollinators is a prerequisite for pollinator-mediated floral evolution, spatiotemporal variation in the pollinator assemblage may confuse the plant–pollinator interaction in a given species. Here, effective pollinators in a living fossil plant Nelumbo nucifera (Nelumbonaceae) were examined to test whether beetles are major pollinators as predicted by its pollination syndrome.

Methods

Pollinators of N. nucifera were investigated in 11 wild populations and one cultivated population, and pollination experiments were conducted to examine the pollinating role of two major pollinators (bees and beetles) in three populations.

Key Results

Lotus flowers are protogynous, bowl shaped and without nectar. The fragrant flowers can be self-heating during anthesis and produce around 1 million pollen grains per flower. It was found that bees and flies were the most frequent flower visitors in wild populations, contributing on average 87·9 and 49·4 % of seed set in Mishan and Lantian, respectively. Beetles were only found in one wild population and in the cultivated population, but the pollinator exclusion experiments showed that beetles were effective pollinators of Asian sacred lotus.

Conclusions

This study indicated that in their pollinating role, beetles, probable pollinators for this thermoregulating plant, had been replaced by some generalist insects in the wild. This finding implies that contemporary pollinators may not reflect the pollination syndrome.     

Short photoperiod induces dormancy in Lotus (Nelumbo nucifera)

BACKGROUND AND AIMS: Lotus (Nelumbo nucifera) has been cultivated as an ornamental and food plant in Japan for more than 1000 years. As large areas are required for its cultivation (approximately 2 m2 per plant), physiological research, such as into the effect of environmental factors on dormancy, has not been well studied until recently. In this paper, seedlings were used to examine environmental factors affecting dormancy induction. METHODS: In a first experiment, seeds were sown from 6 April to 6 October at 2-month intervals, and cultivated for 2 months in an unheated greenhouse. In a second experiment, seeds were prepared for germination on 16 November and 16 May and the seedlings were grown at 25 or 30 degrees C under natural daylength in phytotron growth rooms. After 1 month, the seedlings were cultivated at 20, 25 or 30 degrees C for a further month. The number of leaves and rhizome branches on the main stem were counted, and growth of rhizomes on the main stem was calculated using a rhizome enlargement index (= maximum internode diameter/internode length) after 2 months of culture in both experiments. KEY RESULTS: Rhizomes elongated without enlargement when the seeds were sown in April and June. Sowing the seeds in August and October resulted in rhizome enlargement from the tenth and fifth internodes, respectively. Rhizomes enlarged in the November-sowing but elongated in the May-sowing irrespective of temperature treatments under natural daylength in the phytotron rooms. The seedlings cultivated from May at 25-30 degrees C for 2 months had more leaves, and more rhizome branches and nodes than those cultivated from November. CONCLUSIONS: Short days led to induced dormancy in lotus.     

Nutrient uptake and release in ponds under long-term and short-term lotus (Nelumbo nucifera) cultivation: Influence of compost application

Uptake and release of nutrients from ponds used for lotus cultivation were measured in ponds under short-term (1 yr) cultivation with compost application (pond I) and under long-term (20 yr) cultivation without compost application (pond II). Total inflow loads of TN (irrigation water, rainfall and compost) during lotus cultivation period in ponds I and II were 72.3 and 34.3 kg ha^?1 182 day^?1, respectively. TN removal rates in ponds I and II were 77.3 and 49.8% of total inflow load, respectively. Major removal mechanisms of TN were attributed to microbial processes and uptake by lotus. The total outflow loads (infiltration and runoff) of TN during the lotus cultivation period were 13.9 kg ha^?1 182 day^?1 (19.2% of total inflow TN load) for pond I, and 11.3 kg ha^?1 182 day^?1 (32.9% of total inflow TN load) for pond II. For TP the total inflow loads (irrigation water, rainfall and compost) during lotus cultivation in ponds I and II were 80.8 and 1.9 kg ha^?1 182 day^?1, respectively. TP removal rates in ponds I and II were 84.9 and ?274.1% of total input, respectively. Phosphorus removal was attributed to lotus uptake and soil adsorption. The total outflow loads (infiltration and runoff) of TP during lotus cultivation period were 10.1 kg ha^?1 182 day^?1 (12.5% of total inflow TP load) for pond I, and 6.6 kg ha^?1 182 day^?1 (355.6% of total inflow TP load) for pond II. TN and TP in runoff from pond I (with compost) was higher than that in pond II (without compost), showing that TN and TP in runoff were strongly influenced by compost addition. Therefore, in order to satisfy established water-quality standards, the amount of compost used in lotus cultivation should be evaluated.     

Photosystem Development in Dark-grown Lotus (Nelumbo nucifera) Seedlings (in English)

It is well known that no chlorophyll synthesis and photosystem biogenesis have been detected in dark-grown angiosperm seedlings. However, in this report, we showed that both PSⅡ and PSⅠ could be formed in dark-grown lotus ( Nelumbo nucifera Gaertn.) seedlings. Lots of evidence were given: First, during the dark-grown period, the single fluorescence emission peak at 679 nm in lotus embryo red-shifted and transformed into the normal PSⅡ fluorescence emission; Simultaneously, PSⅠ fluorescence emission at 730 nm appeared and increased obviously; Second, with partial denaturing SDS-PAGE method, PSⅠ chlorophyll-protein complex could be clearly separated from 10 days dark-grown lotus seedlings; Third, the existence of Lhca1 was also proved by Western blots. Moreover, measurements of electron transfer rate demonstrated that both PSⅡand PSⅠ core in dark-grown lotus seedlings were photochemically active.     

莲花期调控研究进展

莲(Nelumbo)是我国十大传统名花中唯一的水生花卉,具有很高的观赏价值及经济价值。花期是影响莲观赏价值的重要性状,花期调控对其生产和应用有着极其重要的意义。莲一般6-8月开花,但是通过花期调控,可以实现一年四季均能开花。本文针对莲花期的定义和特点、花期调控的基本原理、分子机制和调控技术等方面进行系统地综述,并对莲花期调控的前景进行展望,以期为莲花期调控的深入研究和产业发展提供参考。     

不同质量浓度多效唑对碗莲品种'火花'生长和 叶片部分生理指标的影响

研究了不同质量浓度(0~50 mg·L-1)多效唑对碗莲(Nelumbo nucifera Gaertn.)品种'火花'('Huohua')生长的影响及多效唑处理过程中叶片部分生理指标的变化,并进行了隶属函数值分析和相关性分析.结果表明:质量浓度10~50 mg·L-1多效唑处理抑制碗莲品种'火花'生长.随着多效唑质量浓度提高,立叶数、立叶长、立叶宽、立叶高、花径、花高和开花总数总体上呈逐渐降低的趋势,且总体上与对照组(质量浓度0 mg·L-1多效唑)差异显著.随着处理时间延长,质量浓度10~50 mg·L-1多效唑处理组叶片中丙二醛(MDA)含量、过氧化物酶(POD)活性和多酚氧化酶(PPO)活性的变化趋势各异;质量浓度10~50 mg·L-1多效唑处理组叶片中可溶性蛋白质含量和超氧化物歧化酶(SOD)活性,以及质量浓度10、20和30 mg·L-1多效唑处理组叶片中过氧化氢酶(CAT)活性总体上呈先升高后降低的变化趋势,而质量浓度40和50 mg·L-1多效唑处理组叶片中CAT活性则呈逐渐降低的变化趋势.相关性分析结果表明:碗莲品种'火花'生长指标的隶属函数值间以及叶片中可溶性蛋白质含量和POD活性的隶属函数值与生长指标的隶属函数值间的相关性总体在0.05或0.01水平上显著.研究结果显示:质量浓度10、20和30 mg·L-1多效唑处理可有效控制碗莲品种'火花'生长,提高其观赏性;且其生长指标以及叶片中可溶性蛋白质含量和POD活性可以作为判断碗莲品种'火花'矮化效果的可靠、简易指标.     

Effects of lotus root (the edible rhizome of Nelumbo nucifera) on the deveolopment of non-alcoholic fatty liver disease in obese diabetic db/db mice

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that would ameliorate NAFLD is therefore of interest. Lotus root, the edible rhizome of Nelumbo nucifera, contains a high level of polyphenolic compounds, and several health-promoting properties of lotus root have been reported. The present study examines whether dietary lotus root powder can protect db/db mice from hepatic injury. After 3 weeks of feeding, the hepatomegaly, hepatic triglyceride accumulation, and elevated hepatic injury markers in the serum were markedly alleviated in the Lotus diet-fed db/db mice relative to the control mice. These effects were partly attributable to suppression of the lipogenic enzyme activities and mRNA expression by the Lotus diet. The serum levels of adiponectin, which has been reported to have a protective effect against NAFLD, were significantly higher in the Lotus group than in the Control group of the db/db mice. Moreover, the hepatic expression of such inflammatory genes as tumor necrosis factor-alpha and monocyte chemoattractant protein-1 were markedly suppressed by the Lotus diet. We speculate that the development and progression of NAFLD were prevented by suppressing the expression of lipogenic and inflammatory genes as a result of the higher serum adioponectin level in the Lotus diet-fed db/db mice.     

Alkaloid constituents from flower buds and leaves of sacred lotus (Nelumbo nucifera,Nymphaeaceae) with melanogenesis inhibitory activity in B16 melanoma cells

Methanolic extracts from the flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) were found to show inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extracts, a new alkaloid, N-methylasimilobine N-oxide, was isolated together with eleven benzylisoquinoline alkaloids. The absolute stereostructure of the new alkaloid was determined from chemical and physicochemical evidence. Among the constituents isolated, nuciferine, N-methylasimilobine, (?)-lirinidine, and 2-hydroxy-1-methoxy-6a,7-dehydroaporphine showed potent inhibition of melanogenesis. Comparison of the inhibitory activities of synthetic related alkaloids facilitated characterization of the structure-activity relationships of aporphine- and benzylisoquinoline-type alkaloids. In addition, 3–30 μM nuciferine and N-methylasimilobine inhibited the expression of tyrosinase mRNA, 3–30 μM N-methylasimilobine inhibited the expression of TRP-1 mRNA, and 10–30 μM nuciferine inhibited the expression of TRP-2 mRNA.     

中国莲（Nelumbo nucifera）幼苗抗氧化系统对砷胁迫的响应

为了研究中国莲（Nelumbo nucifera）抗氧化系统对砷胁迫的响应,研究比较了两种不同价态无机砷As（Ⅲ）和砷As（V）对中国莲幼苗可溶性蛋白含量、丙二醛（MDA）含量以及抗氧化系统的影响。结果表明,中国莲幼苗中MDA和可溶性蛋白质含量随砷浓度的增加呈现先升高后降低的趋势。中国莲幼苗的MDA和蛋白质含量受As（V）的影响不如As（11]）敏感。抗氧化系统酶中,超氧化物歧化酶（SOD）对砷处理最敏感,当As（11I）浓度在2．5pmol／L和As（V）浓度在100μmol／L时,SOD酶活性显著高于对照组。过氧化物酶（POD）在As（Ⅲ）处理浓度为10umol／L时就出现显著上升,相对而言,过氧化氢酶（CAT）对As（V）比较敏感。实验结果表明,随着浓度的增加,砷对幼苗产生的氧化胁迫导致SOD、CAT和POD三种酶活性有所增加,以配合清除细胞内的活性氧自由基（ROS）,维持细胞代谢的稳定。本研究为进一步研究砷胁迫下莲的生理和生长变化、以及莲的培育和移植提供了部分基础数据。