Genetic Diversity and Association Mapping of Grain-Size Traits in Rice Landraces from the Honghe Hani Rice Terraces System in Yunnan Province.

The Honghe Hani Rice Terraces System (HHRTS) of Yunnan Province is an important agricultural and cultural heritage landscape. Until now, a large number of local rice landraces have been planted. Mining excellent genes contained in these landraces provides a reference for variety improvement and new variety breeding. In this study, 96 rice landraces collected from the Hani terraces were planted in Honghe Mengzi, Yunnan Province, in 2013, 2014, 2015, and 2021, and five major grain traits were measured and analyzed. The genomic variation of 96 rice landraces was scanned by 201 simple sequence repeat (SSR) markers. The genetic diversity, population structure, and genetic relationships of the natural population were analyzed. The mixed linear model (MLM) method of the TASSEL software was used to analyze the associations between markers and traits. A total of 936 alleles were amplified by 201 pairs of SSR primers. The average number of observed alleles (Na), the effective number of alleles (Ne), Shannon's information index (I), heterozygosity (H), and the polymorphism information content (PIC) per marker were 4.66, 2.71, 1.08, 0.15, and 0.55, respectively. Ninety-six landraces were divided into two groups by population structure, clustering, and principal component analysis, and indica rice was the main group. The coefficients of variation of the five traits ranged from 6.80 to 15.24%, and their broad heritabilities were more than 70%. In addition, there were positive correlations among the same grain traits between different years. Through MLM analysis, 2, 36, 7, 7, and 4 SSR markers were significantly associated with grain length (GL), grain width (GW), grain thickness (GT), grain length-width ratio (LWR), and thousand-grain weight (TGW), respectively. The explanation rates of phenotypic variation were 16.31 (RM449, Chr. 1)-23.51% (RM316, Chr. 9), 10.84 (RM523, Chr. 3; RM161/RM305, Chr. 5)-43.01% (RM5496, Chr. 1), 11.98 (RM161/RM305, Chr. 5)-24.72% (RM275, Chr. 6), 12.68 (RM126, Chr. 8)-36.96% (RM5496, Chr. 1), and 17.65 (RM4499, Chr. 2)-26.32% (RM25, Chr. 8), respectively. The associated markers were distributed on 12 chromosomes of the genome.

De novo transcriptome assembly, gene annotation, and EST-SSR marker development of an important medicinal and edible crop, Amomum tsaoko (Zingiberaceae).

BACKGROUND: Amomum tsaoko is a medicinal and food dual-use crop that belongs to the Zingiberaceae family. However, the lack of transcriptomic and genomic information has limited the understanding of the genetic basis of this species. Here, we performed transcriptome sequencing of samples from different A. tsaoko tissues, and identified and characterized the expressed sequence tag-simple sequence repeat (EST-SSR) markers. RESULTS: A total of 58,278,226 high-quality clean reads were obtained and de novo assembled to generate 146,911 unigenes with an N50 length of 2002 bp. A total of 128,174 unigenes were successfully annotated by searching seven protein databases, and 496 unigenes were identified as annotated as putative terpenoid biosynthesis-related genes. Furthermore, a total of 55,590 EST-SSR loci were detected, and 42,333 primer pairs were successfully designed. We randomly selected 80 primer pairs to validate their polymorphism in A. tsaoko; 18 of these primer pairs produced distinct, clear, and reproducible polymorphisms. A total of 98 bands and 96 polymorphic bands were amplified by 18 pairs of EST-SSR primers for the 72 A. tsaoko accessions. The Shannon's information index (I) ranged from 0.477 (AM208) to 1.701 (AM242) with an average of 1.183, and the polymorphism information content (PIC) ranged from 0.223 (AM208) to 0.779 (AM247) with an average of 0.580, indicating that these markers had a high level of polymorphism. Analysis of molecular variance (AMOVA) indicated relatively low genetic differentiation among the six A. tsaoko populations. Cross-species amplification showed that 14 of the 18 EST-SSR primer pairs have transferability between 11 Zingiberaceae species. CONCLUSIONS: Our study is the first to provide transcriptome data of this important medicinal and edible crop, and these newly developed EST-SSR markers are a very efficient tool for germplasm evaluation, genetic diversity, and molecular marker-assisted selection in A. tsaoko.

Antiviral activity of Mulberroside C against enterovirus A71 in vitro and in vivo.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease which seriously threatens young children's health and lives. However, there is no effective therapy currently available for treating these infections. Therefore, effective drugs to prevent and treat EV-A71 infections are urgently needed. Here, we identified Mulberroside C potently against the proliferation of EV-A71. The in-vitro anti-EV-A71 activity of Mulberroside C was assessed by cytopathic effect inhibition and viral plaque reduction assays, and the results showed that Mulberroside C significantly inhibited EV-A71 infection. The downstream assays affirmed that Mulberroside C inhibited viral protein and RNA synthesis. Furthermore, Mulberroside C effectively reduced clinical symptoms in EV-A71 infected mice and reduced mortality at higher concentrations. The mechanism study indicated that Mulberroside C bound to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and genome release. Taken together, our study indicated that Mulberroside C could be a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Licochalcone A inhibits enterovirus A71 replication in vitro and in vivo.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand-foot-mouth disease (HFMD) and causes serious neurological complications. However, no effective therapy is currently available for treating these infections. Therefore, effective drugs to prevent and treat EV-A71 infections are urgently needed. Here, we demonstrated that treatment with Licochalcone A (LCA) significantly inhibited EV-A71 replication in a dose-dependent manner, with an EC50 of 9.30 µM in RD cells and 5.73 µM in Vero cells. The preliminary results on the inhibition mechanism showed that LCA exerted antiviral effects by interfering with the early step of viral replication. We further demonstrated that LCA showed potent antiviral activity against many enteroviruses, including EV-A71 (strain C4), EV-A71 (strain H), and coxsackievirus A16 (CV-A16). Furthermore, LCA could effectively prevent the clinical symptoms and death of virus infected mice and decreased viral load in EV-A71-infected mice. Taken together, our studies showed for the first time, that LCA is a promising EV-A71 inhibitor and provide important information for the clinical development of LCA as a potential new anti-EV-A71 agent.

Development of 23 novel microsatellite markers of Amomum tsao-ko (Zingiberaceae) based on restriction-site-associated DNA sequencing.

Amomum tsao-ko (Zingiberaceae) is a traditional Chinese medicine and condiment, and an important economic crop in the tropical forest of southwest China. However, few simple sequence repeat (SSR) markers are available in A. tsao-ko, which is hindering genetic research in this species. The aim of this study was to develop and characterize microsatellite markers for A. tsao-ko using restriction-site-associated DNA sequencing. A total of 115,482 microsatellites were identified using MISA software, and 13,411 SSR primer pairs were designed. 100 pairs of SSR primers were selected at random and used to evaluate polymorphisms among 4 A. tsao-ko samples. Finally, 23 pairs of SSR primers with clear bands and obvious polymorphism were selected for genetic diversity analysis of 72 A. tsao-ko accessions. The number of alleles and effective number of alleles per locus ranged from 2 to 6 and from 1.315 to 3.776, respectively. The observed heterozygosity ranged from 0.208 to 0.779, and the expected heterozygosity was from 0.239 to 0.735. The average values of the polymorphic information content were 0.454. Hardy-Weinberg equilibrium (HWE) analysis showed that 10 loci significantly deviated from HWE (P < 0.05). The pairwise FST and genetic distance values revealed low levels of genetic differentiation and high genetic similarity among six A. tsao-ko populations. These microsatellite markers developed will provide a valuable tool for further germplasm characterization, genetic diversity, and breeding studies in A. tsao-ko.

Remediation of Arsenic contaminated soil using malposed intercropping of Pteris vittata L. and maize.

Intercropping of arsenic (As) hyperaccumulator and cash crops during remediation of contaminated soil has been applied in farmland remediation project. However, little is known about the fate of As fractions in the soil profile and As uptake within the intercropping plants under field condition. In this study, As removal, uptake, and translocation were investigated within an intercropping system of Pteris vittata L. (P. vittata) and maize (Zea mays). Results indicated that the concentration of As associated with amorphous Fe (hydr)oxides in the 10-20 cm soil layer was significantly lower under malposed intercropping of P. vittata and maize, and As accumulation in P. vittata and biomass of P. vittata were simultaneously higher under malposed intercropping than under coordinate intercropping, leading to a 2.4 times higher rate of As removal. Although maize roots absorbed over 13.4 mg kg-1 As and maize leaves and flowers accumulated over 21.5 mg kg-1 As (translocation factor higher than 1), grains produced in all intercropping modes accumulated lower levels of As, satisfying the standard for human consumption. Our results suggested that malposed intercropping of a hyperaccumulator and a low-accumulation cash crop was an ideal planting pattern for As remediation in soil. Furthermore, timely harvest of P. vittata, agronomic strategies during remediation, and appropriate management of the above ground parts of P. vittata and high-As tissues of cash crops may further improve remediation efficiency.