Evaluating the fermentation quality and bacterial community of high-moisture whole-plant quinoa silage ensiled with different additives.

AIM: To explore the potential of whole-plant quinoa (WPQ) as a high-protein source for livestock feed, this study evaluated the effects of additives on the fermentation quality and bacterial community of high-moisture WPQ silage. METHODS AND RESULTS: High-moisture WPQ was ensiled with one of the following additives: untreated control (C), fibrolytic enzyme (E), molasses (M), LAB inoculant (L), a combination of fibrolytic enzyme and LAB inoculant (EL) and a combination of molasses and LAB inoculant (ML). The fermentation quality and bacterial community after 60 days of ensiling were analysed. Naturally fermented WPQ exhibited acetic acid-type fermentation dominated by enterobacteria, with low lactic acid content (37.0 g/kg DM), and high pH value (5.65), acetic acid (70.8 g/kg DM) and NH3 -N production (229 g/kg TN). Adding molasses alone or combined with LAB inoculant shifted the fermentation pattern towards increased intensity of lactic acid fermentation, lowering the pH value (<4.56), contents of acetic acid (<46.7 g/kg DM) and NH3 -N (<140 g/kg TN) and total abundance of enterobacteria (<16.0%), and increasing the lactic acid content (>60.5 g/kg DM), lactic/acetic acid ratio (>1.40) and the relative abundance of Lactobacillus (>83.0%). CONCLUSIONS: The results suggested that the lack of fermentable sugar could be the main factor of restricting extensive lactic acid fermentation in WPQ silage. Supplementing fermentable sugar or co-ensiling with materials with high WSC content and low moisture content are expected to be beneficial strategies for producing high-quality WPQ silage. SIGNIFICANCE AND IMPACT OF STUDY: High biomass production and high protein content make WPQ to be an ideal forage source for livestock feed. Results of this study revealed the restricting factor for extensive lactic acid fermentation in WPQ silage, which could be helpful in producing high-quality WPQ silage.

Transcriptome analysis and differential gene expression profiling of two contrasting quinoa genotypes in response to salt stress.

BACKGROUND: Soil salinity is one of the major abiotic stress factors that affect crop growth and yield, which seriously restricts the sustainable development of agriculture. Quinoa is considered as one of the most promising crops in the future for its high nutrition value and strong adaptability to extreme weather and soil conditions. However, the molecular mechanisms underlying the adaptive response to salinity stress of quinoa remain poorly understood. To identify candidate genes related to salt tolerance, we performed reference-guided assembly and compared the gene expression in roots treated with 300 mM NaCl for 0, 0.5, 2, and 24 h of two contrasting quinoa genotypes differing in salt tolerance. RESULTS: The salt-tolerant (ST) genotype displayed higher seed germination rate and plant survival rate, and stronger seedling growth potential as well than the salt-sensitive (SS) genotype under salt stress. An average of 38,510,203 high-quality clean reads were generated. Significant Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified to deeper understand the differential response. Transcriptome analysis indicated that salt-responsive genes in quinoa were mainly related to biosynthesis of secondary metabolites, alpha-Linolenic acid metabolism, plant hormone signal transduction, and metabolic pathways. Moreover, several pathways were significantly enriched amongst the differentially expressed genes (DEGs) in ST genotypes, such as phenylpropanoid biosynthesis, plant-pathogen interaction, isoquinoline alkaloid biosynthesis, and tyrosine metabolism. One hundred seventeen DEGs were common to various stages of both genotypes, identified as core salt-responsive genes, including some transcription factor members, like MYB, WRKY and NAC, and some plant hormone signal transduction related genes, like PYL, PP2C and TIFY10A, which play an important role in the adaptation to salt conditions of this species. The expression patterns of 21 DEGs were detected by quantitative real-time PCR (qRT-PCR) and confirmed the reliability of the RNA-Seq results. CONCLUSIONS: We identified candidate genes involved in salt tolerance in quinoa, as well as some DEGs exclusively expressed in ST genotype. The DEGs common to both genotypes under salt stress may be the key genes for quinoa to adapt to salinity environment. These candidate genes regulate salt tolerance primarily by participating in reactive oxygen species (ROS) scavenging system, protein kinases biosynthesis, plant hormone signal transduction and other important biological processes. These findings provide theoretical basis for further understanding the regulation mechanism underlying salt tolerance network of quinoa, as well establish foundation for improving its tolerance to salinity in future breeding programs.

Development of novel InDel markers and genetic diversity in Chenopodium quinoa through whole-genome re-sequencing.

BACKGROUND: Quinoa (Chenopodium quinoa Willd.) is a balanced nutritional crop, but its breeding improvement has been limited by the lack of information on its genetics and genomics. Therefore, it is necessary to obtain knowledge on genomic variation, population structure, and genetic diversity and to develop novel Insertion/Deletion (InDel) markers for quinoa by whole-genome re-sequencing. RESULTS: We re-sequenced 11 quinoa accessions and obtained a coverage depth between approximately 7× to 23× the quinoa genome. Based on the 1453-megabase (Mb) assembly from the reference accession Riobamba, 8,441,022 filtered bi-allelic single nucleotide polymorphisms (SNPs) and 842,783 filtered InDels were identified, with an estimated SNP and InDel density of 5.81 and 0.58 per kilobase (kb). From the genomic InDel variations, 85 dimorphic InDel markers were newly developed and validated. Together with the 62 simple sequence repeat (SSR) markers reported, a total of 147 markers were used for genotyping the 129 quinoa accessions. Molecular grouping analysis showed classification into two major groups, the Andean highland (composed of the northern and southern highland subgroups) and Chilean coastal, based on combined STRUCTURE, phylogenetic tree and PCA (Principle Component Analysis) analyses. Further analysis of the genetic diversity exhibited a decreasing tendency from the Chilean coast group to the Andean highland group, and the gene flow between subgroups was more frequent than that between the two subgroups and the Chilean coastal group. The majority of the variations (approximately 70%) were found through an analysis of molecular variation (AMOVA) due to the diversity between the groups. This was congruent with the observation of a highly significant FST value (0.705) between the groups, demonstrating significant genetic differentiation between the Andean highland type of quinoa and the Chilean coastal type. Moreover, a core set of 16 quinoa germplasms that capture all 362 alleles was selected using a simulated annealing method. CONCLUSIONS: The large number of SNPs and InDels identified in this study demonstrated that the quinoa genome is enriched with genomic variations. Genetic population structure, genetic core germplasms and dimorphic InDel markers are useful resources for genetic analysis and quinoa breeding.

Genome-Wide Analysis and Expression Profiles of the VOZ Gene Family in Quinoa (Chenopodium quinoa).

Vascular plant one zinc-finger (VOZ) proteins are a plant-specific transcription factor family and play important roles in plant development and stress responses. However, little is known about the VOZ genes in quinoa. In the present study, a genome-wide investigation of the VOZ gene family in quinoa was performed, including gene structures, conserved motifs, phylogeny, and expression profiles. A total of four quinoa VOZ genes distributed on three chromosomes were identified. Based on phylogenetic analysis, CqVOZ1 and CqVOZ3 belong to subfamily II, and CqVOZ2 and CqVOZ4 belong to subfamily III. Furthermore, the VOZ transcription factors of quinoa and sugarbeet were more closely related than other species. Except for CqVOZ3, all the other three CqVOZs have four exons and four introns. Analysis of conserved motifs indicated that each CqVOZ member contained seven common motifs. Multiple sequence alignment showed that the CqVOZ genes were highly conserved with consensus sequences, which might be plausibly significant for the preservation of structural integrity of the family proteins. Tissue expression analysis revealed that four CqVOZ genes were highly expressed in inflorescence and relatively low in leaves and stems, suggesting that these genes had obvious tissue expression specificity. The expression profiles of the quinoa CqVOZs under various abiotic stresses demonstrated that these genes were differentially induced by cold stress, salt stress, and drought stress. The transcript level of CqVOZ1 and CqVOZ4 were down-regulated by salt stress and drought stress, while CqVOZ2 and CqVOZ3 were up-regulated by cold, salt, and drought stress, which could be used as abiotic stress resistance candidate genes. This study systematically identifies the CqVOZ genes at the genome-wide level, contributing to a better understanding of the quinoa VOZ transcription factor family and laying a foundation for further exploring the molecular mechanism of development and stress resistance of quinoa.

Transcriptome analyses revealed molecular responses of Cynanchum auriculatum leaves to saline stress.

Cynanchum auriculatum is a traditional herbal medicine in China and can grow in saline soils. However, little is known in relation to the underlying molecular mechanisms. In the present study, C. auriculatum seedlings were exposed to 3.75‰ and 7.5‰ salinity. Next, transcriptome profiles of leaves were compared. Transcriptome sequencing showed 35,593 and 58,046 differentially expressed genes (DEGs) in treatments with 3.75‰ and 7.5‰, compared with the control, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of these DEGs enriched various defense-related biological pathways, including ROS scavenging, ion transportation, lipid metabolism and plant hormone signaling. Further analyses suggested that C. auriculatum up-regulated Na+/H+ exchanger and V-type proton ATPase to avoid accumulation of Na+. The flavonoid and phenylpropanoids biosynthesis pathways were activated, which might increase antioxidant capacity in response to saline stress. The auxin and ethylene signaling pathways were upregulated in response to saline treatments, both of which are important plant hormones. Overall, these results raised new insights to further investigate molecular mechanisms underlying resistance of C. auriculatum to saline stress.

Selection of solvent for extraction of antioxidant components from Cynanchum auriculatum, Cynanchum bungei, and Cynanchum wilfordii roots.

In east Asia, "Baishouwu" has been used as an herbal drug and functional dietary supplement for hundreds of years. Actually, "Baishouwu" is the common name of the roots of Cynanchum auriculatum, Cynanchum bungei, and Cynanchum wilfordii. In the present study, roots of these three specie were extracted and then fractionated using petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EA), and water. DPPH scavenging experiments revealed high antioxidant activity of DCM and EA fractions of C. bungei and the EA fraction of C. wilfordii. Treatments with these three fractions significantly reduced malondialdehyde content in heat-stressed Daphnia magna, validating in vivo antioxidant activity. Gas chromatography-mass spectrometer (GC-MS) analyses demonstrated that the chemical components of fractions extracted from C. bungei, C. bungei, and C. wilfordii were different. Further determination of total phenol and total flavonoids contents showed that DCM and EA fractions of C. bungei and EA fraction of C. wilfordii had much higher contents of total phenol and total flavonoids, which might be the reason to explain their strong antioxidant activity. Overall, the present study suggested that these three plants have different chemical components and biological activities. They could not be used as the same drug.

Comparative expression analysis of Calcineurin B-like family gene CBL10A between salt-tolerant and salt-sensitive cultivars in B. oleracea.

Calcineurin B-like proteins (CBLs) are plant calcium sensors that play a critical role in the regulation of plant growth and response to stress. Many CBLs have been identified in the calcium signaling pathway in both Arabidopsis and rice. However, information about BoCBLs genes from Brassica oleracea has not been reported. In the present study, we identified 13 candidate CBL genes in the B. oleracea genome based on the conserved domain of the Calcineurin B-like family, and we carried out a phylogenetic analysis of CBLs among Arabidopsis, rice, maize, cabbage and B. oleracea. For B. oleracea, the distribution of the predicted BoCBL genes was uneven among the five chromosomes. Sequence analysis showed that the nucleotide sequences and corresponding protein structure of BoCBLs were highly conserved, i.e., all of the putative BoCBLs contained 6-8 introns, and most of the exons of those genes contained the same number of nucleotides and had high sequence identities. All BoCBLs consisted of four EF-Hand functional domains, and the distance between the EF-hand motifs was conserved. Evolutionary analysis revealed that the CBLs were classified into two subgroups. Additionally, the CBL10A gene was cloned from salt-tolerant (CB6) and salt-sensitive (CB3) cultivars using RT-PCR. The results indicated that the cloned gene had a substantial difference in length (741bp in CB3 and 829bp in CB6) between these two cultivars. The deduced CBL10A protein in CB6 had four EF-hand structural domains, which have an irreplaceable role in calcium-binding and have calcineurin A subunit binding sites, while the BoCBL10A protein in CB3 had only two EF-hand structural domains and lacked calcineurin A subunit binding sites. The expression level of the BoCBL10A gene between salt tolerance (CB6)and sensitive varieties(CB3) under salt stress was significantly different (P<0.01 and P<0.05). The expression of BoCBL10A gene was relatively higher in salt-tolerant (CB6) cultivar under salt stress, with a longer period of up-regulation expression and a shorter time responding to salt, compared with the salt-sensitive (CB3) cultivar. We speculate that these differences in the coding region of BoCBL10A may lead to the different salt responses between these two cultivars.