Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.).

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Transcriptome Analyses Revealed the Key Metabolic Genes and Transcription Factors Involved in Terpenoid Biosynthesis in Sacred Lotus.

As the largest group of structurally diverse metabolites, terpenoids are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. However, few terpenoid compounds have been identified in plant parts of sacred lotus (Nelumbo nucifera Gaertn.). To elucidate the molecular genetic basis of the terpene biosynthetic pathway, terpenes from different parts of the plant, including seeds (S), young leaves (YL), mature leaves (ML), white flowers (WF), yellow flowers (YF), and red flowers (RF), were identified by LC-MS/MS and the relative contents of the same terpenes in different parts were compared. The results indicate that all plant parts primarily consist of triterpenes, with only minor quantities of sesquiterpenes and diterpenes, and there were differences in the terpene content detected in different plant parts. To illustrate the biosynthesis of various terpenoids, RNA sequencing was performed to profile the transcriptomes of various plant parts, which generated a total of 126.95 GB clean data and assembled into 29,630 unigenes. Among these unigenes, 105 candidate unigenes are involved in the mevalonate (MVA) pathway, methyl-erythritol phosphate (MEP) pathway, terpenoid backbone biosynthesis pathway, and terpenoid synthases pathway. Moreover, the co-expression network between terpene synthase (TPS) and WRKY transcription factors provides new information for the terpene biosynthesis pathway.

Genomic variation reveals demographic history and biological adaptation of the ancient relictual, lotus (Nelumbo Adans).

Lotus (Nelumbo Adans.), a relict plant, is the testimony of long-term sustained ecological success, but the underlying genetic changes related to its survival strategy remains unclear. Here, we assembled the high-quality lotus genome, investigated genome variation of lotus mutation accumulation (MA) lines and reconstructed the demographic history of wild Asian lotus, respectively. We identified and validated 43 base substitutions fixed in MA lines, implying a spontaneous mutation rate of 1.4 × 10-9 base/generation in lotus shoot stem cells. The past history of lotus revealed that the ancestors of lotus in eastern and southern Asia could be traced back ~20 million years ago (Mya) and experienced twice significant bottlenecks and population splits. We further identified the selected genes among three lotus groups in different habitats, suggesting that 453 genes between tropical and temperate group and 410 genes between two subgroups from Northeastern China and the Yangtze River - Yellow River Basin might play important roles in natural selection in lotus's adaptation and resilience. Our findings not only improve an understanding of the lotus evolutionary history and the genetic basis of its survival advantages, but also provide valuable data for addressing various questions in evolution and protection for the relict plants.

First Report of Lotus Seedpod Withering Caused by Fusarium proliferatum on Nelumbo nucifera Plants in China.

Nelumbo nucifera (Nymphaeaceae family) is a well-known plant in China and with the increasing value of this crop, the planting area of lotus is expanding. In May 2019, an unknown withering lotus seedpod was obtained in Guangchang County of Jiangxi Province (26.79°N, 116.31°E). The disease arose between May and July of each year, resulted in the withering and consequent death of ~10% of lotus seedpods, with the disease being most serious during the rainy season. The initial symptoms of this disease include the shrinking of young lotus seedpods with concomitant yellowing of the epidermal tissue layer. These pods failed to grow normally and could to wither and die within one week, with the withering symptoms gradually spreading to associated stem tissues. To characterize the pathogens responsible for this disease, ten diseases seedpods were collected and cut into pieces of ~5×5 mm, then sterilized with 75% ethanol for 30 s, and treated with 0.1% mercuric chloride for 5 min. After being washed four times under sterilized water, samples were then transferred onto potato dextrose agar (PDA) and incubated for 7 d at 28℃ in the dark. Eight purified isolates yielded large numbers of aerial mycelium that were initially white in color, but then changed to a purple-red color over the course of this incubation period. The average mycelial growth rate was 6.3 mm per day (n=5). On PDA, macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The microconidia were hyaline, ovoid or ellipse and 4.6-13.5×2.2-4.3 µm in size (average: 8.7×3.1 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). To confirm the identity of this putative pathogen at the molecular level, the universal ITS4/ITS5 primers (White et al., 1990), the Fusarium specific pair PRO1/PRO2 (Mulè et al., 2004), EF1T/EF2T (O'Donnell et a., 1998) and RPB2F/R (O'Donnell et al., 2010) primers were utilized to amplify the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-internal transcribed spacer 2 (ITS2), calmodulin, alpha elongation factor genes, and RNA-dependent DNA polymerase II subunit from these isolates. Following alignment of the resultant sequences with GenBank via a BLAST analysis, the sequences (GenBank accession numbers: MW862499, MW762531, MW767988, MW831311, respectively.) showed 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank accession numbers: MW817705, LS423443, MH153750, and MW091308, respectively.). Based upon these morphological and molecular findings, this pathogen was identified as F. proliferatum. Pathogenicity testing was then performed using five plump healthy lotus seedpods. Sterile needles were used to generate wounds (2 mm deep, 1 mm in diameter) a 10 µL suspension of prepared spores (1.0×106 spores/mL) derived from a 7-day-old culture grown on PDA was injected into the wound sites of the lotus seedpod. As a control, give seedpods were additionally wounded and injected as the same as treated with 10 µL of sterile water. The experiments were repeated three times with five biological replicates. All seedpods were then incubated at 28℃ in a growth chamber (12 h light/dark) with 80% relative humidity. After a 3-day incubation period, wounded sites injected with spore suspensions exhibited browning. Following a 5-day incubation period, a mean lesion diameter of 9.8 mm was observed, with white mycelia growing on the wound surface and with evident withering of the internal and external tissues near the wounded site. In contrast, blank control wound sites remained healthy. We were again able to isolate F. proliferatum from the infected lotus seedpods. Finally, eight isolates were obtained were identified as the pathogen based on these morphological and molecular analyses, thus fulfilling Koch's postulates. This is the first report to our knowledge to have described a case of F. proliferatum causing lotus seedpod withering in China, providing a foundation for future research efforts aimed at presenting diseases caused by this pathogen.

Genetic Diversity and DNA Fingerprints of Three Important Aquatic Vegetables by EST-SSR Markers.

Twenty-two sacred lotus (Nelumbo nucifera), 46 taros (Colocasia esculenta) and 10 arrowheads (Sagittaria trifolia) were used as materials and combined with EST-SSR (expressed sequence tag-simple sequence repeats) primers developed by our laboratory. Core primers were screened from a large number of primers that were able to distinguish all materials with a high frequency of polymorphisms. Six pairs, twenty pairs and three pairs of core primers were screened from sacred lotus, taro, and arrowhead, respectively. The SSR fingerprints of these three important aquatic vegetables, producing 17-, 87- and 14-bit binary molecular identity cards, respectively, were separately determined by using the core primers. Since there were few core primers of sacred lotus and arrowhead, 3 and 9 primer pairs with higher polymorphic information content (PIC), respectively, were selected as candidate primers. These core and candidate primers were used to identify the purities of No.36 space lotus, Shandong 8502 taro and Wuhan arrowhead, which were 93.3% (84/90), 98.9% (89/90) and 100.0% (90/90), respectively. The fingerprints, displayed as binary molecular identification cards of three important aquatic vegetables, were obtained, and their purity was successfully determined with EST-SSR labeling technology. Phylogenetic trees were also constructed to analyze the genetic diversity of 22 sacred lotus, 46 taros and 10 arrowheads. This study classifies and identifies germplasm resources and is an important reference to test the authenticity and variety purity of other aquatic vegetables in the future.