Genome-Wide Variation Analysis of Four Vegetable Soybean Cultivars Based on Re-Sequencing

Vegetable soybean is a type of value-added specialty soybean, served as a fresh vegetable or snack in China. Due to the difference from other types, it is important to understand the genetic structure and diversity of vegetable soybean for further utilization in breeding programs. The four vegetable cultivars, Taiwan-75, Zhexiandou No. 8, Zhexian No. 9 and Zhexian No. 10 are popular soybean varieties planted in Zhejiang province, and have large pods and intermediate maturity. The clustering showed a close relationship of these four cultivars in simple sequence repeat analysis. To reveal the genome variation of vegetable soybean, these four improved lines were analyzed by whole-genome re-sequencing. The average sequencing depth was 7X and the coverage ratio of each cultivar was at least more than 94%. Compared with the reference genome, a large number of single-nucleotide polymorphisms, insertion/deletions and structure variations were identified with different chromosome distributions. The average heterozygosity rate of the single-nucleotide polymorphisms was 11.99% of these four cultivars. According to the enrichment analysis, there were 23,371 genes identified with putative modifications, and a total of 282 genes were related to carbohydrate metabolic processes. These results provide useful information for genetic research and future breeding, which can facilitate the selection procedures in vegetable soybean breeding.

Applying Biotechnology in the Propagation and Further Selection of Vaccinium uliginosum × (V. corymbosum × V. angustifolium) Hybrids

The most serious problem of intergeneric and interspecific hybridization is related to overcoming the reproductive isolation of different species. We assessed the efficiency of reproduction under in vitro conditions and the ex vitro growth capacity of interspecific hybrids of Vaccinium uliginosum × (V. corymbosum × V. angustifolium). The percentage of seed germination in in vitro culture was 88% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), SC5-8, while it was 42% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), ‘Northcountry’. The analysis of mean value showed that the multiplication rate increased and the shoot height decreased as the 2-isopentenyl adenine (2iP) concentration was increased in the nutrient medium of the studied hybrids. The maximum rate was achieved using 15 μM 2iP. A detailed analysis of the hybrids indicated that the hybrid variant reliably affected growth and development indicators. Inter simple sequence repeat analysis demonstrated that all analyzed hybrids inherited DNA fragments of the parent plants in various combinations, confirming their hybrid nature. Thus, the use of in vitro methods for the propagation and further selection of genotypes is demonstrated as being an effective approach for developing interspecific hybrids of V. uliginosum × (V. corymbosum × V. angustifolium).

Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome

Background: The number of studies using third-generation sequencing using Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas. Among them, plant full-length single-molecule transcriptome studies have mostly used PacBio sequencing, whereas ONT is rarely used. Therefore, in this study, we examined ONT RNA sequencing methods in plants. We performed a detailed evaluation of reads from PacBio, Nanopore direct cDNA (ONT Dc), and Nanopore PCR cDNA (ONT Pc) sequencing including characteristics of raw data and identification of transcripts. In addition, matched Illumina data were generated for comparison. Results: ONT Pc showed overall better raw data quality, whereas PacBio generated longer read lengths. In the transcriptome analysis, PacBio and ONT Pc performed similarly in transcript identification, simple sequence repeat analysis, and long non-coding RNA prediction. PacBio was superior in identifying alternative splicing events, whereas ONT Pc could estimate transcript expression levels. Conclusions: This paper made a comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome, the results indicate that ONT Pc is more cost-effective for generating extremely long reads and can characterise the transcriptome as well as quantify transcript expression. Therefore, ONT Pc is a new cost-effective and worthwhile method for full-length single-molecule transcriptome analysis in plants.

Assessing Salinity Tolerance in Rice Mutants by Phenotypic Evaluation Alongside Simple Sequence Repeat Analysis

Salinity stress is one of the most severe constraints limiting rice production worldwide. Thus, the development of salt-tolerant rice promises to deal with increasing food demand due to climate change effects. This study investigated the salinity tolerance of mutant rice by evaluating phenotype and genotype, using forty-two simple sequence repeat (SSR) markers linked to the salinity tolerance Saltol quantitative trait locus (QTL) in ten cultivars and mutant lines. Results of phenotypic screening showed that the mutant line SKLo/BC15TB and cultivar BC15TB performed salt tolerance, while the mutant line Bao Thai/DT 84 and cultivar DT84DB were sensitive to salt stress. The markers RM 493, RM 562, RM 10748, RM 518, RM 237, and RM 20224 were the most polymorphic in salinity tolerance. Among them, RM 237, RM 10748, and RM 224 showed the highest polymorphism information (PIC = 0.58). This study reveals that the three markers are profitable for classification of salinity tolerance in both cultivar and mutant rice. The mutant line SKLo/BC15TB and cultivar BC15TB were found to be promising candidates for diversity analysis of salt-tolerant rice. Findings of this study are useful for developing new salinity-tolerant rice cultivars towards climate change.

Population Structure and Genetic Diversity of Fungi Causing Rice Seedling Blight in Northeast China Based on Microsatellite Markers

Rice seedling blight, which is caused by diverse pathogenic microorganisms, occurs worldwide and is the most important seedling disease affecting rice production in Northeast China. To further characterize the population structure and genetic diversity of the fungi responsible for rice seedling blight in Northeast China, 225 fungal strains were isolated from diseased rice seedlings collected from various rice-producing areas. The isolated strains included Fusarium oxysporum (48.0%), F. verticillioides (11.6%), F. tricinctum (8.0%), F. redolens (6.7%), F. equiseti (6.2%), F. solani (6.2%), Rhizoctonia solani (6.7%), Alternaria alternata (4.0%), and Curvularia coatesiae (2.7%). F. oxysporum was the dominant fungal species causing rice seedling blight, with most isolates exhibiting moderate pathogenicity. Moreover, to our knowledge, this is the first study to identify A. alternata and C. coatesiae as causal agents of rice seedling blight in Northeast China. None of the F. oxysporum isolates were sensitive to 10 μg/ml of carbendazim, implying that carbendazim is ineffective for controlling rice seedling blight in Northeast China. The F. oxysporum isolates were divided into nine groups based on a simple sequence repeat analysis involving 14 primer pairs. In addition, an analysis of molecular variance revealed a significant correlation between the F. oxysporum population and geographical location, which had a significant effect on the differentiation of the dominant isolate population. The results of this study provide insights into the genetic diversity of F. oxysporum strains causing rice seedling blight and may be useful for selecting isolates to screen for disease-resistant rice varieties, evaluating fungicide efficacy, and developing effective disease management strategies.

Analysis and comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome

Background The number of studies using third-generation sequencing using Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) is rapidly increasing in many different research areas. Among them, plant full-length single-molecule transcriptome studies have mostly used PacBio sequencing, whereas ONT is rarely used. Therefore, in this study, we examined ONT RNA sequencing methods in plants. We performed a detailed evaluation of reads from PacBio, Nanopore direct cDNA (ONT Dc), and Nanopore PCR cDNA (ONT Pc) sequencing including characteristics of raw data and identification of transcripts. In addition, matched Illumina data were generated for comparison.Results ONT Pc showed overall better raw data quality, whereas PacBio generated longer read lengths. In the transcriptome analysis, PacBio and ONT Pc performed similarly in transcript identification, simple sequence repeat analysis, and long non-coding RNA prediction. PacBio was superior in identifying alternative splicing events, whereas ONT Pc could estimate transcript expression levels.Conclusions This paper made a comprehensive comparison of PacBio and nanopore-based RNA sequencing of the Arabidopsis transcriptome, the results indicate that ONT Pc is more cost-effective for generating extremely long reads and can characterise the transcriptome as well as quantify transcript expression. Therefore, ONT Pc is a new cost-effective and worthwhile method for full-length single-molecule transcriptome analysis in plants.

Comparative analysis of the complete chloroplast genome sequences of six species of Pulsatilla Miller, Ranunculaceae

Background Baitouweng is a traditional Chinese medicine with a long history of different applications. Although referred to as a single medicine, Baitouweng is actually comprised of many closely related species. It is therefore critically important to identify the different species that are utilized in these medicinal applications. Knowledge about their phylogenetic relationships can be derived from their chloroplast genomes and may provide additional insights into development of molecular markers. Methods Genomic DNA was extracted from six species of Pulsatilla and then sequenced on an Illumina HiSeq 4000. Sequences were assembled into contigs by SOAPdenovo 2.04, aligned to the reference genome using BLAST, and then manually corrected. Genome annotation was performed by the online DOGMA tool. General characteristics of the cp genomes of the six species were analyzed and compared with closely related species. Additionally, phylogenetic trees were constructed, based on single nucleotide polymorphisms (SNPs) and 51 shared protein-coding gene sequences in the cp genome among all 31 species via maximum likelihood. Results The size of cp genomes of P. chinensis (Bge.) Regel, P. chinensis (Bge.) Regel var. kissii (Mandl) S. H. Li et Y. H. Huang, P. cernua (Thunb.) Bercht. et Opiz f. plumbea J. X. Ji et Y. T. zhao, P. dahurica (Fisch.) Spreng, P. turczaninovii Kryl. et Serg, and P. cernua (Thunb.) Bercht. et Opiz. were 163,851 bp, 163,756 bp, 162,481 bp, 162,450 bp, 162,795 bp, and 162,924 bp, respectively. Each species included two inverted repeat regions, a small single-copy region, and a large single-copy region. A total of 134 genes were annotated, including 90 protein-coding genes, 36 tRNAs, and eight rRNAs across all species. In simple sequence repeat analysis, only P. dahurica was found to contain hexanucleotide repeats. A total of 26, 39, 32, 37, 32 and 43 large repeat sequences were identified in the genic regions of the six Pulsatilla species. Nucleotide diversity analysis revealed that the rpl36 gene and ccsA-ndhD region have the highest Pi value. In addition, two phylogenetic trees of the cp genomes were constructed, which laced all Pulsatilla species into one branch within Ranunculaceae. Conclusions We identified and analyzed the cp genome features of six species of P. Miller, with implications for species identification and phylogenetic analysis.