Assessment of phylogenetic relationships and genetic diversity of Sagittaria trifolia using phenotypic traits and SNP markers.

The aquatic perennial herb Sagittaria trifolia L. commonly known as arrowhead, has been utilized in China both as a culinary vegetable and in traditional medicines. Characterizing the phylogenetic relationships and genetic diversity of arrowheads is crucial for improved management, conservation, and efficient utilization of the germplasm resources associated with this species. Herein, we presented the phenotypic traits and genome-wide DNA marker-based analyses of 111 arrowhead accessions, most of which were from China. Cluster analysis revealed that arrowhead could be categorized into two clusters based on 11 phenotypic traits, with Cluster 1 comprising two subclusters. All accessions were clustered into three sub-clusters based primarily on leaf shape and tuber weight. A set of 759,237 high-quality single-nucleotide polymorphisms was identified and used to assess the phylogenetic relationships. Population structure and phylogenetic tree analyses suggested that the accessions could be classified into two major groups, Group I was further subdivided into two subgroups, aligning with the clusters identified through morphological classification. By employing Sagittaria lichuanensis as an outgroup, the rooted tree revealed that the evolutionary relationships within the three groups followed a progression from Group I-1 to Group I-2 and finally to Group II. The landraces were clustered into one group along with the remaining wild accessions. The level of genetic diversity for Group I (π = 0.26) was slightly lower than that which was estimated for Group II (π = 0.29). The lowest pairwise differentiation levels (Fst, 0.008) were obtained from the comparison between groups I-2 and II, indicating that the two groups were the most closely related. This study provides novel insights into germplasm classification, evolutionary relationships, genomics and arrowhead breeding.

Influence of Blanching on the Gene Expression Profile of Phenylpropanoid, Flavonoid and Vitamin Biosynthesis, and Their Accumulation in Oenanthe javanica.

Field blanching is a process used in agriculture to obtain sweet, delicious, and tender stems of water dropwort by obstructing sunlight. The nutritional and transcriptomic profiling of blanched water dropwort has been investigated in our previous studies. However, the effect of blanching on the production of secondary metabolites and different vitamins in water dropwort has not been investigated at the transcriptomic level. This study explored the transcriptomic variations in the phenylpropanoid biosynthesis, flavonoid biosynthesis, and different vitamin biosynthesis pathways under different blanching periods in the water dropwort stems (pre-blanching, mid-blanching, post-blanching, and control). The results show that polyphenol and flavonoid contents decreased; however, the contents of vitamins (A, B1, B2, and C) and antioxidant activity increased significantly after blanching. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of blanched water dropwort showed the downregulation of many important genes involved in phenylpropanoid and flavonoid biosynthesis pathways, and the downregulation of these genes might be the reason for the reduction in polyphenol and flavonoid contents. We also examined and highlighted the genes involved in the higher vitamin content, antioxidant activity, pale color, tenderness, and sweetness of the blanched stem of water dropwort. In conclusion, the present study explored the role of phenylpropanoid and vitamin biosynthesis, and it will provide a basis for future investigation and application in the blanch cultivation of water dropwort.

Comparative Transcriptomic Analysis Provides Novel Insights into the Blanched Stem of Oenanthe javanica.

In the agricultural field, blanching is a technique used to obtain tender, sweet, and delicious water dropwort stems by blocking sunlight. The physiological and nutritional parameters of blanched water dropwort have been previously investigated. However, the molecular mechanism of blanching remains unclear. In the present study, we investigated transcriptomic variations for different blanching periods in the stem of water dropwort (pre, mid, post-blanching, and control). The results showed that many genes in pathways, such as photosynthesis, carbon fixation, and phytohormone signal transduction as well as transcription factors (TFs) were significantly dysregulated. Blanched stems of water dropwort showed the higher number of downregulated genes in pathways, such as photosynthesis, antenna protein, carbon fixation in photosynthetic organisms, and porphyrin and chlorophyll metabolism, which ultimately affect the photosynthesis in water dropwort. The genes of hormone signal transduction pathways (ethylene, jasmonic acid, brassinosteroid, and indole-3-acetic acid) showed upregulation in the post-blanched water dropwort plants. Overall, a higher number of genes coding for TFs, such as ERF, BHLH, MYB, zinc-finger, bZIP, and WRKY were overexpressed in blanched samples in comparison with the control. These genes and pathways participate in inducing the length, developmental processes, pale color, and stress tolerance of the blanched stem. Overall, the genes responsive to blanching, which were identified in this study, provide an effective foundation for further studies on the molecular mechanisms of blanching and photosynthesis regulations in water dropwort and other species.

Corrigendum: Phenotypic, Nutritional, and Antioxidant Characterization of Blanched Oenanthe javanica for Preferable Cultivar.

[This corrects the article DOI: 10.3389/fpls.2021.639639.].

Effect of Salt Stress on Growth, Physiological Parameters, and Ionic Concentration of Water Dropwort (Oenanthe javanica) Cultivars.

Salt stress is an important environmental limiting factor. Water dropwort (Oenanthe javanica) is an important vegetable in East Asia; however, its phenotypic and physiological response is poorly explored. For this purpose, 48 cultivars of water dropwort were grown hydroponically and treated with 0, 50, 100, and 200 mm NaCl for 14 days. Than their phenotypic responses were evaluated, afterward, physiological studies were carried out in selected sensitive and tolerant cultivars. In the present study, the potential tolerant (V11E0022) and sensitive (V11E0135) cultivars were selected by screening 48 cultivars based on their phenotype under four different levels of salt concentrations (0, 50, 100, and 200 mm). The results depicted that plant height, number of branches and leaves were less effected in V11E0022, and most severe reduction was observed in V11E0135 in comparison with others. Than the changes in biomass, ion contents, accumulation of reactive oxygen species, and activities of antioxidant enzymes and non-enzymatic antioxidants were determined in the leaves and roots of the selected cultivars. The potential tolerant cultivar (V11E0022) showed less reduction of water content and demonstrated low levels of Na+ uptake, malondialdehyde, and hydrogen peroxide (H2O2) in both leaves and roots. Moreover, the tolerant cultivar (V11E0022) showed high antioxidant activities of ascorbate peroxidase (APX), superoxide dismutase, peroxidase, catalase (CAT), reduced glutathione (GSH), and high accumulation of proline and soluble sugars compared to the sensitive cultivar (V11E0135). These results suggest the potential tolerance of V11E0022 cultivar against salt stress with low detrimental effects and a good antioxidant defense system. The observations also suggest good antioxidant capacity of water dropwort against salt stress. The findings of the present study also suggest that the number of branches and leaves, GSH, proline, soluble sugars, APX, and CAT could serve as the efficient markers for understanding the defense mechanisms of water dropwort under the conditions of salt stress.

Uncovering phylogenetic relationships and genetic diversity of water dropwort using phenotypic traits and SNP markers.

The water dropworts Oenanthe linearis Wall. ex DC. and O. javanica (Blume) DC. are aquatic perennial herbs that have been used in China as vegetables and traditional medicines. However, their phylogenetic relationships and genetic diversity are poorly understood. Here, we presented the phenotypic traits and genome-wide DNA marker-based analysis of 158 water dropwort accessions representing both species. The analysis revealed that Oenanthe linearis was readily segregated into linear-leaf and deep-cleft leaf water dropworts according to their leaf shapes at flowering. Oenanthe javanica was classified by clustering analysis into two clusters based mainly on the morphological characteristics of their ultimate segments (leaflets). A set of 11 493 high-quality single-nucleotide polymorphisms was identified and used to construct a phylogenetic tree. There was strong discrimination between O. linearis and O. javanica, which was consistent with their phenotype diversification. The population structure and phylogenetic tree analyses suggested that the O. linearis accessions formed two major groups, corresponding to the linear-leaf and deep-cleft leaf types. The most obvious phenotypic differences between them were fully expressed at the reproductive growth stage. A single-nucleotide polymorphism-based analysis revealed that the O. javanica accessions could be categorized into groups I andII. However, this finding did not entirely align with the clusters revealed by morphological classification. Landraces were clustered into one group along with the remaining wild accessions. Hence, water dropwort domestication was short in duration. The level of genetic diversity for O. linearis (π = 0.1902) was slightly lower than that which was estimated for O. javanica (π = 0.2174). There was a low level of genetic differentiation between O. linearis and O. javanica (Fst = 0.0471). The mean genetic diversity among accessions ranged from 0.1818 for the linear-leaf types to 0.2318 for the groupII accessions. The phenotypic traits and the single-nucleotide polymorphism markers identified here lay empirical foundation for future genomic studies on water dropwort.

Phenotypic, Nutritional, and Antioxidant Characterization of Blanched Oenanthe javanica for Preferable Cultivar.

Blanching is a technique used in blocking sunlight for the production of tender, sweet, and delicious stems in the field. This technique is also used in water dropwort (Oenanthe javanica), an important vegetable in East Asia. In China, the steamed stems of water dropwort are prepared with boiled rice. However, the effect of blanching on the nutritional level and antioxidant capacity of water dropwort has not been explored yet. The current study aims to determine the nutrient contents and antioxidant capacities of five cultivars and select the best cultivar. They were mainly compared in terms of phenotypic, physiological, nutritional, and antioxidant levels after blanch cultivation. Results indicate that blanching significantly influenced the phenotype, physiology, and nutritional level of water dropwort in all cultivars. Although few parameters decreased with blanching, starch, sugars, vitamins, minerals, and antioxidant activities increased significantly in the blanched stems in mid- and post-blanching periods. The most noticeable changes were detected in post-blanching samples. Furthermore, the best cultivar (V11E0012) was identified among them. Therefore, blanched water dropwort could be consumed for achieving more nutraceuticals and antioxidants, and cultivar V11E0012 could be recommend for blanching cultivation.

Genetic diversity and population structure of eddoe taro in China using genome-wide SNP markers.

Taro (Colocasia esculenta) is an important root and tuber crop cultivated worldwide. There are two main types of taro that vary in morphology of corm and cormel, 'dasheen' and 'eddoe'. The eddoe type (Colocasia esculenta var. antiquorium) is predominantly distributed throughout China. Characterizing the genetic diversity present in the germplasm bank of taro is fundamental to better manage, conserve and utilize the genetic resources of this species. In this study, the genetic diversity of 234 taro accessions from 16 provinces of China was assessed using 132,869 single nucleotide polymorphism (SNP) markers identified by specific length amplified fragment-sequencing (SLAF-seq). Population structure and principal component analysis permitted the accessions to be categorized into eight groups. The genetic diversity and population differentiation of the eight groups were evaluated using the characterized SNPs. Analysis of molecular variance showed that the variation among eight inferred groups was higher than that within groups, while a relatively small variance was found among the two morphological types and 16 collection regions. Further, a core germplasm set comprising 41 taro accessions that maintained the genetic diversity of the entire collection was developed based on the genotype. This research is expected to be valuable for genetic characterization, germplasm conservation, and breeding of taro.

Resequencing of 296 cultivated and wild lotus accessions unravels its evolution and breeding history.

Lotus (family: Nelumbonaceae) are perennial aquatic plants that represent one of the most ancient basal dicots. In the present study, we resequenced 296 lotus accessions from various geographical locations and germplasms to explore their genomic diversity and population structure. This germplasm set consisted of four accessions of American wild lotus and 292 accessions of Asian lotus, which were divided into four subgroups: wild, rhizome, flower and seed. Total single nucleotide polymorphisms (SNPs) suggested that the wild lotus had the highest variant number (7 191 010). Population structure and genome diversity analysis indicated that the American wild lotus demonstrated a distant genetic relationship with the Asian lotus. Furthermore, the seed and rhizome lotus groups had not originated from a single source but rather had a more complex multisource origin. Besides that, the seed lotus showed higher genetic diversity, which might have been due to the gene flow from the flower lotus to seed lotus by artificial crossing, and the rhizome lotus showed a much lower genetic diversity than the other groups. The present study provides SNP markers for lotus genomic diversity analysis, which will be useful for guiding lotus breeding.

Investigation of an Antioxidative System for Salinity Tolerance in Oenanthe javanica.

Abiotic stress, such as drought and salinity, severely affect the growth and yield of many plants. Oenanthe javanica (commonly known as water dropwort) is an important vegetable that is grown in the saline-alkali soils of East Asia, where salinity is the limiting environmental factor. To study the defense mechanism of salt stress responses in water dropwort, we studied two water dropwort cultivars, V11E0022 and V11E0135, based on phenotypic and physiological indexes. We found that V11E0022 were tolerant to salt stress, as a result of good antioxidant defense system in the form of osmolyte (proline), antioxidants (polyphenols and flavonoids), and antioxidant enzymes (APX and CAT), which provided novel insights for salt-tolerant mechanisms. Then, a comparative transcriptomic analysis was conducted, and Gene Ontology (GO) analysis revealed that differentially expressed genes (DEGs) involved in the carbohydrate metabolic process could reduce oxidative stress and enhance energy production that can help in adaptation against salt stress. Similarly, lipid metabolic processes can also enhance tolerance against salt stress by reducing the transpiration rate, H2O2, and oxidative stress. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs involved in hormone signals transduction pathway promoted the activities of antioxidant enzymes and reduced oxidative stress; likewise, arginine and proline metabolism, and flavonoid pathways also stimulated the biosynthesis of proline and flavonoids, respectively, in response to salt stress. Moreover, transcription factors (TFs) were also identified, which play an important role in salt stress tolerance of water dropwort. The finding of this study will be helpful for crop improvement under salt stress.

Construction of a high-density, high-quality genetic map of cultivated lotus (Nelumbo nucifera) using next-generation sequencing.

BACKGROUND: The sacred lotus (Nelumbo nucifera) is widely cultivated in China for its edible rhizomes and seeds. Traditional plant breeding methods have been used to breed cultivars with increased yields and quality of rhizomes and seeds with limited success. Currently, the available genetic maps and molecular markers in lotus are too limited to be useful for molecular genetics based breeding programs. However, the development of next-generation sequencing (NGS) technologies has enabled large-scale identification of single-nucleotide polymorphisms (SNPs) for genetic map construction. In this study, we constructed an SNP-based high-density genetic map for cultivated lotus using double digest restriction site-associated DNA sequencing (ddRADseq). RESULTS: An F2 population of 96 individuals was derived from a cross between the rhizome lotus cultivar 'Juwuba' (male parent) and the seed lotus cultivar 'Mantianxing' (female parent). Genomic DNAs from this population were digested with the restriction enzymes EcoRI and MspI and then sequenced. In total, 133.65 Gb of raw data containing 1,088,935,610 pair-end reads were obtained. The coverage of reads on a reference genome was 7.2 % for the female parent, 6.56 % for the male parent, and 1.46 % for F2 individuals. From these reads, 10,753 valid SNP markers were used for genetic map construction. Finally, 791 bin markers (so-segregated adjacent SNPs treated as a bin marker), consisting of 8,971 SNP markers, were sorted into 8 linkage groups (LGs) that spanned 581.3 cM, with an average marker interval of 0.74 cM. A total of 809 genome sequence scaffolds, covering about 565.9 cM of the wild sacred lotus genome, were anchored on the genetic map, accounting for 70.6 % of the genome assembly. CONCLUSIONS: This study reports the large-scale discovery of SNPs between cultivars of rhizome and seed lotus using a ddRADseq library combined with NGS. These SNPs have been used to construct the first high-density genetic map for cultivated lotus that can serve as a genomic reference and will facilitate genetic mapping of important traits in the parental cultivars.

Genome-Wide Identification of SSR and SNP Markers Based on Whole-Genome Re-Sequencing of a Thailand Wild Sacred Lotus (Nelumbo nucifera).

Genomic resources such as single nucleotide polymorphism (SNPs), insertions and deletions (InDels) and SSRs (simple sequence repeats) are essential for crop improvement and better utilization in genetic breeding. However, the resources for the sacred lotus (Nelumbo nucifera Gaertn.) are still limited. In the present study, to dissect large-scale genomic molecular marker resources for sacred lotus, we re-sequenced a Thailand sacred lotus cultivar 'Chiang Mai wild lotus' and compared with the reported lotus genome 'Middle lake wild lotus'. A total of 3,180,059 SNPs, 328, 251 InDels and 14,191 SVs were found between the two genomes. The functional impact analyses of these SNPs indicated that they may be involved in metabolic processes, binding, catalytic activity, etc. Mining the genome sequences for SSRs showed that 191,657 SSRs were identified with a frequency of one SSR per 4.23 kb and 103,656 SSR primer pairs were designed. Furthermore, 14, 502 EST-SSRs were also indentified using the available RNA-seq data in the NCBI. A subset of 150 SSRs (genomic and EST-SSRs) was randomly selected for validation and genetic diversity analysis. The genotypes could be easily distinguished using these SSR markers and the 'Chiang Mai wild lotus' was obviously differentiated from the other Chinese accessions. This study provides considerable amounts of genomic resources and markers for the quantitative trait locus (QTL) identification and molecular selection of the species, which could have a potential role in various applications in sacred lotus breeding.

Transcriptomic Analysis of the Regulation of Rhizome Formation in Temperate and Tropical Lotus (Nelumbo nucifera).

Rhizome is the storage organ of lotus derived from modified stems. The development of rhizome is a complex process and depends on the balanced expression of the genes that is controlled by environmental and endogenous factors. However, little is known about the mechanism that regulates rhizome girth enlargement. In this study, using RNA-seq, transcriptomic analyses were performed at three rhizome developmental stages-the stolon, middle swelling and later swelling stage -in the cultivars 'ZO' (temperate lotus with enlarged rhizome) and 'RL' (tropical lotus with stolon). About 348 million high-quality reads were generated, and 88.5% of the data were mapped to the reference genome. Of 26783 genes identified, 24069 genes were previously predicted in the reference, and 2714 genes were novel transcripts. Moreover, 8821 genes were differentially expressed between the cultivars at the three stages. Functional analysis identified that these genes were significantly enriched in pathways carbohydrate metabolism and plant hormone signal transduction. Twenty-two genes involved in photoperiod pathway, starch metabolism and hormone signal transduction were candidate genes inducing rhizome girth enlargement. Comparative transcriptomic analysis detected several differentially expressed genes and potential candidate genes required for rhizome girth enlargement, which lay a foundation for future studies on molecular mechanisms underlying rhizome formation.

A precise chloroplast genome of Nelumbo nucifera (Nelumbonaceae) evaluated with Sanger, Illumina MiSeq, and PacBio RS II sequencing platforms: insight into the plastid evolution of basal eudicots.

BACKGROUND: The chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as 'SMRT (Single Molecule, Real-Time) sequencing', has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots. RESULTS: The sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently. CONCLUSIONS: Because of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate 'finished' genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.

The sacred lotus genome provides insights into the evolution of flowering plants.

Sacred lotus (Nelumbo nucifera) is an ornamental plant that is also used for food and medicine. This basal eudicot species is especially important from an evolutionary perspective, as it occupies a critical phylogenetic position in flowering plants. Here we report the draft genome of a wild strain of sacred lotus. The assembled genome is 792 Mb, which is approximately 85-90% of genome size estimates. We annotated 392 Mb of repeat sequences and 36,385 protein-coding genes within the genome. Using these sequence data, we constructed a phylogenetic tree and confirmed the basal location of sacred lotus within eudicots. Importantly, we found evidence for a relatively recent whole-genome duplication event; any indication of the ancient paleo-hexaploid event was, however, absent. Genomic analysis revealed evidence of positive selection within 28 embryo-defective genes and one annexin gene that may be related to the long-term viability of sacred lotus seed. We also identified a significant expansion of starch synthase genes, which probably elevated starch levels within the rhizome of sacred lotus. Sequencing this strain of sacred lotus thus provided important insights into the evolution of flowering plant and revealed genetic mechanisms that influence seed dormancy and starch synthesis.

Comparative analysis of genetic diversity in sacred lotus (Nelumbo nucifera Gaertn.) using AFLP and SSR markers.

The sacred lotus (Nelumbo nucifera Gaertn.) is an aquatic plant of economic and ornamental importance in China. In this study, we developed twenty novel sacred lotus SSR markers, and used AFLP and SSR markers to investigate the genetic diversity and genetic relationships among 58 accessions of N. nucifera including 15 seed lotus, 12 rhizome lotus, 24 flower lotus and 7 wild lotus. Our results showed that sacred lotus exhibited a low level of genetic diversity, which may attribute to asexual reproduction and long-term artificial selection. A dendrogram based on both AFLP and SSR clustering data showed that: (1) the seed lotus accessions and rhizome lotus accessions were distinctly clustered into different groups, which indicated the significant genetic differentiation between them. This may be attributed to the two modes of reproduction and lack of genetic exchange; (2) the accessions of Thailand wild lotus were separated from other wild lotus accessions. This implied that the Thailand lotus might be genetically differentiated from other wild lotuses. In addition, Mantel test conducted gave highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with the values of r = 0.941 and r = 0.879, respectively, indicating the higher efficiency of the combination of these techniques (AFLP and SSR) in estimation and validation of the genetic diversity among the accession of sacred lotus. This knowledge of the genetic diversity and genetic relatedness of N. nucifera is potentially useful to improve the current strategies in breeding and germplasm conservation to enhance the ornamental and economic value of sacred lotus.

Development and characterization of microsatellite markers for Sagittaria trifolia var. sinensis (Alismataceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the aquatic plant Sagittaria trifolia var. sinensis to assess its genetic diversity and population structure. Cross-species transferability was assayed in eight congeneric species. METHODS AND RESULTS: Seventeen microsatellite markers were isolated and characterized in Sagittaria trifolia var. sinensis using Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) protocol. Across the evaluated populations, 14 of the markers showed polymorphisms with 3 to 11 alleles per locus; the observed and expected heterozygosity (H(o) and H(E)) ranged from 0.0000 to 0.6364 and from 0.0000 to 0.8386, respectively. Nine of the loci were successfully amplified in the congeneric species. CONCLUSIONS: These markers will be useful for further investigation of population genetics in Sagittaria trifolia var. sinensis and related research in Sagittaria species.

Development of novel EST-SSRs from sacred lotus (Nelumbo nucifera Gaertn) and their utilization for the genetic diversity analysis of N. nucifera.

Expressed sequence tags (ESTs) provide a valuable resource for the development of simple sequence repeat (SSR) or microsatellite markers. This study identified SSRs within ESTs from Nelumbo nucifera (lotus or sacred lotus), developed markers from them, and assessed the potential of those markers for diversity analysis. Within 2207 ESTs from N. nucifera downloaded from GenBank, 1483 unigenes (303 contigs and 1180 singletons) were identified. After eliminating for redundancy, 125 SSR-containing ESTs were derived, and 71 unique SSRs were detected with an average density of one SSR per 13.04 kb. Dinucleotide repeats were the dominant motif in N. nucifera, whereas the sequences AG/TC/GA/CT, AAG/TTC/GAT/AGA, and AAAGCC were the most frequent of di-, tri-, and hexanucleotide motifs, respectively. The AG/TC (40.85%) and AAG (5.63%) motifs were predominant for the di- and trinucleotide repeats, respectively. Sixty-two SSR-containing ESTs were suitable for primer design. From these sequences, 23 EST-SSR markers were developed and were applied to 39 cultivated varieties of N. nucifera, 10 accessions of wild N. nucifera, and 1 accession of Nelumbo lutea (American lotus). Genetic diversity and genetic relationships were examined by constructing unweighted pair-group method with arithmetic average dendrograms and principal coordinates analysis plots based on SSR polymorphisms. Results indicated genetic differentiation between cultivated and wild lotus and between seed lotus cultivars and rhizome lotus cultivars. These EST-SSR markers will be useful for further studies of the evolution and diversity of Nelumbo.

Polymorphic microsatellite markers in Euryale ferox Salisb. (Nymphaeaceae).

Eleven polymorphic microsatellite markers were isolated and identified in the aquatic plant Euryale ferox Salisb. (Nymphaeaceae). This species, which belongs to basal Magnoliophyta, reproduces sexually. All of these 11 microsatellite markers yielded 25 alleles in a survey of a wild population of 34 individuals. Two or three alleles per locus were detected, with expected heterozygosity ranging from 0.056 to 0.634 and observed heterozygosity from 0.000 to 0.088. These simple sequence repeat markers will be useful for evaluating the genetic structure of the E. ferox population in the future.

Sixteen polymorphic microsatellite markers from Zizania latifolia Turcz. (Poaceae).

Sixteen polymorphic microsatellite markers were isolated and identified in Zizania latifolia Turcz. (Poaceae), a perennial aquatic plant widespread in Eastern Asia. The microsatellite-enriched library was constructed using the fast isolation by AFLP of sequences containing repeats method. These markers revealed two to 14 alleles, with an average of 5.6 alleles per locus. The observed and expected heterozygosities varied from 0.071 to 0.690 and from 0.174 to 0.812, respectively. These markers will be useful for studying of gene flow and evaluating the genetic diversity of the Zizania latifolia population.