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.     

Stomata actively regulate internal aeration of the sacred lotus Nelumbo nucifera

The sacred lotus Nelumbo nucifera (Gaertn.) possesses a complex system of gas canals that channel pressurized air from its leaves, down through its petioles and rhizomes, before venting this air back to the atmosphere through large stomata found in the centre of every lotus leaf. These central plate stomata (CPS) lie over a gas canal junction that connects with two‐thirds of the gas canals within the leaf blade and with the larger of two discrete pairs of gas canals within the petiole that join with those in the rhizome. It is hypothesized that the lotus actively regulates the pressure, direction and rate of airflow within its gas canals by opening and closing these stomata. Impression casting the CPS reveal that they are open in the morning, close at midday and reopen in the afternoon. The periodic closure of the CPS during the day coincides with a temporary reversal in airflow direction within the petiolar gas canals. Experiments show that the conductance of the CPS decreases in response to increasing light level. This behaviour ventilates the rhizome and possibly directs benthic CO₂ towards photosynthesis in the leaves. These results demonstrate a novel function for stomata: the active regulation of convective airflow.     

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.     

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.     

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.     

Improving Nelumbo nucifera genome assemblies using high‐resolution genetic maps and BioNano genome mapping reveals ancient chromosome rearrangements

Genetic and physical maps are powerful tools to anchor fragmented draft genome assemblies generated from next‐generation sequencing. Currently, two draft assemblies of Nelumbo nucifera, the genomes of ‘China Antique’ and ‘Chinese Tai‐zi’, have been released. However, there is presently no information on how the sequences are assembled into chromosomes in N. nucifera. The lack of physical maps and inadequate resolution of available genetic maps hindered the assembly of N. nucifera chromosomes. Here, a linkage map of N. nucifera containing 2371 bin markers [217 577 single nucleotide polymorphisms (SNPs)] was constructed using restriction‐site associated DNA sequencing data of 181 F₂ individuals and validated by adding 197 simple sequence repeat (SSR) markers. Additionally, a BioNano optical map covering 86.20% of the ‘Chinese Tai‐zi’ genome was constructed. The draft assembly of ‘Chinese Tai‐zi’ was improved based on the BioNano optical map, showing an increase of the scaffold N50 from 0.989 to 1.48 Mb. Using a combination of multiple maps, 97.9% of the scaffolds in the ‘Chinese Tai‐zi’ draft assembly and 97.6% of the scaffolds in the ‘China Antique’ draft assembly were anchored into pseudo‐chromosomes, and the centromere regions along the pseudo‐chromosomes were identified. An evolutionary scenario was proposed to reach the modern N. nucifera karyotype from the seven ancestral eudicot chromosomes. The present study provides the highest‐resolution linkage map, the optical map and chromosome level genome assemblies for N. nucifera, which are valuable for the breeding and cultivation of N. nucifera and future studies of comparative and evolutionary genomics in angiosperms.     

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.     

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.     

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...     

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.     

Whole genome sequencing reveals the genomic diversity,taxonomic classification,and evolutionary relationships of the genus Nocardia

Nocardia is a complex and diverse genus of aerobic actinomycetes that cause complex clinical presentations, which are difficult to diagnose due to being misunderstood. To date, the genetic diversity, evolution, and taxonomic structure of the genus Nocardia are still unclear. In this study, we investigated the pan-genome of 86 Nocardia type strains to clarify their genetic diversity. Our study revealed an open pan-genome for Nocardia containing 265,836 gene families, with about 99.7% of the pan-genome being variable. Horizontal gene transfer appears to have been an important evolutionary driver of genetic diversity shaping the Nocardia genome and may have caused historical taxonomic confusion from other taxa (primarily Rhodococcus, Skermania, Aldersonia, and Mycobacterium). Based on single-copy gene families, we established a high-accuracy phylogenomic approach for Nocardia using 229 genome sequences. Furthermore, we found 28 potentially new species and reclassified 16 strains. Finally, by comparing the topology between a phylogenomic tree and 384 phylogenetic trees (from 384 single-copy genes from the core genome), we identified a novel locus for inferring the phylogeny of this genus. The dapb1 gene, which encodes dipeptidyl aminopeptidase BI, was far superior to commonly used markers for Nocardia and yielded a topology almost identical to that of genome-based phylogeny. In conclusion, the present study provides insights into the genetic diversity, contributes a robust framework for the taxonomic classification, and elucidates the evolutionary relationships of Nocardia. This framework should facilitate the development of rapid tests for the species identification of highly variable species and has given new insight into the behavior of this genus.     

Whole genome sequencing of Ethiopian highlanders reveals conserved hypoxia tolerance genes

Background

Although it has long been proposed that genetic factors contribute to adaptation to high altitude, such factors remain largely unverified. Recent advances in high-throughput sequencing have made it feasible to analyze genome-wide patterns of genetic variation in human populations. Since traditionally such studies surveyed only a small fraction of the genome, interpretation of the results was limited.

Results

We report here the results of the first whole genome resequencing-based analysis identifying genes that likely modulate high altitude adaptation in native Ethiopians residing at 3,500 m above sea level on Bale Plateau or Chennek field in Ethiopia. Using cross-population tests of selection, we identify regions with a significant loss of diversity, indicative of a selective sweep. We focus on a 208 kbp gene-rich region on chromosome 19, which is significant in both of the Ethiopian subpopulations sampled. This region contains eight protein-coding genes and spans 135 SNPs. To elucidate its potential role in hypoxia tolerance, we experimentally tested whether individual genes from the region affect hypoxia tolerance in Drosophila. Three genes significantly impact survival rates in low oxygen: cic, an ortholog of human CIC, Hsl, an ortholog of human LIPE, and Paf-AHα, an ortholog of human PAFAH1B3.

Conclusions

Our study reveals evolutionarily conserved genes that modulate hypoxia tolerance. In addition, we show that many of our results would likely be unattainable using data from exome sequencing or microarray studies. This highlights the importance of whole genome sequencing for investigating adaptation by natural selection.     

抗赖氨酸加苏氨酸莲体细胞变异体的筛选

对籽莲红花建莲（Ｎｅｌｕｍｂｏｎｕｃｉｆｅｒａｃｖ．Ｈｏｎｇｈｕａｊｉａｎｌｉａｎ）和白心湘莲（Ｎ．ｎｕｃｉｆｅｒａｃｖ．Ｂａｉｘｉｎｘｉａｎｇｌｉａｎ）杂交而成的幼胚的子叶、胚轴及幼胚叶（芽）形成的愈伤组织,施以赖氨酸加苏氨酸胁迫培养３０天,从中筛选出抗性愈伤组织并形成再生植株。低浓度的赖氨酸加苏氨酸促进愈伤组织的生长,而高浓度的赖氨酸加苏氨酸则抑制愈伤组织生长,直至具有致死作用,这种致死作用是因为高浓度的赖氨酸加苏氨酸抑制了天冬氨酸合成途径中的天冬氨酸激酶和高丝氨酸脱氢酶造成的,细胞发生变异后对赖氨酸和苏氨酸产生了抗性,即与天冬氨酸合成途径有关的氨基酸增加。抗性愈伤组织与未胁迫的愈伤组织的氨基酸测定表明,在总计１７种氨基酸中,抗性愈伤组织有１４种氨基酸含量超过原始愈伤组织,１种持平,２种不及原始愈伤组织。再生抗性植株的莲籽氨基酸测定显示,在１７种氨基酸中,有１２种超过母本红花建莲,１５种超过父本白心湘莲。     

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。     

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.     

花莲种质资源形态性状多样性研究(英文)

对从国家种质资源圃中选取的40份花莲种质资源的19个形态性状进行了观察。结果发现花型、花瓣数、雌蕊是否瓣化、雌蕊瓣化程度和根茎颜色与花蕾形状呈显著正相关,其中花型、花瓣数和根茎颜色与花蕾形状呈极显著高度相关。通径分析表明现蕾期对花型有显著正向效应(0.552),其次是花瓣数(0.516)和根茎颜色(0.368)。与花型呈显著负相关的性状有花瓣长(-0.801)、现花期(-0.558)和叶宽(-0.451)。主成分分析显示前四个成分累计贡献率达65.68%。聚类分析将所有材料分为四组。     

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.