Sex determination in plants gives rise to unisexual flowers. A better understanding of the regulatory mechanism underlying the production of unisexual flowers will illuminate sex determination in plants and allow researchers and farmers to harness heterosis. Androecious cucumber (Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus decreasing the cost of seed production. The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding. Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate (EMS)–mutagenized library of the cucumber inbred line ‘406’. Genetic analysis, whole-genome resequencing, and molecular marker–assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11 (ACS11) conferred androecy. The mutation caused an amino acid change from serine (Ser) to phenylalanine (Phe) at position 301 (S301F). In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzyme activity. This study thus provides a new germplasm for use in cucumber breeding as the androecious male parent and offers new insights into the catalytic mechanism of ACS enzymes.

ASFVs have been divided into at least 24 genotypes based on the C-terminus of the B646L gene with 478 nt (Bastos et al. 2003). B646L gene is one of the most used target genes for ASF diagnosis, which is also the target gene for the WOAH recommended PCR and fluorescent quantitative PCR assays (Agüero et al. 2003; King et al. 2003). Sanger sequencing of targeted amplification of the B646L genes is the main genotyping approach for ASFVs. Recently, Li et al. developed the duplex real-time PCR assay based on the ASFV E296R gene, and Cao et al. established the TaqMAN-MGB probe assay based on the N-terminal sequences of the B646L gene (Li et al. 2022; Cao et al. 2022), which could distinguish genotype I and genotype II ASFVs with detection limits of 10 copies. However, the target genes or regions in their methods were out of ASFV genotyping regions.

Single Nucleotide Polymorphism (SNP) is a single base change at a specific position in the genome of different individuals and can be used as a genotyping marker for the detection of different individual genotypes (Gut. 2001). The amplification refractory mutation system (ARMS), also named Allele-Specific PCR (AS-PCR), relies on the extension of primer only when its 3’end has a perfect complement to the template (Wang et al. 2019). ARMS-qPCR technology has been developed and widely used in SNP detection and genotyping (Ochsenreither et al. 2010; Shi et al. 2013; Wang et al. 2019). Compared with other assays for SNP detection and genotyping, ARMS-qPCR has the advantage of low-cost, simple operation, high sensitivity, and rapid and real-time detection.

Here, 126 complete or partial B646L genes of ASFVs, including 78 genotype I and 48 genotype II viruses, were obtained from the GenBank database, and their information is shown in Appendix A. After analyzing these genes by the MegAlign software (DNAStar), there were 4 SNPs in the C-terminus of the B646L gene, differentiating genotype I viruses from genotype II viruses (Fig. 1-A). Two SNPs at sites 1656 and 1710 were used to design primers and probes for differential detection of genotype I and II ASFVs (Fig. 1-A). As previously described (Huang et al. 1992; Liu et al. 2012), primers (F, I R, and II R) and probes (Probe 1 and Probe 2) were designed with the targeted gene sequences using Primer 5 Software (Fig. 1-B; Appendix B). For the positive sample of genotype I ASFV, FAM and Cy5 fluorophores could be detected; however, for the positive sample of genotype II ASFV, only FAM fluorophore could be detected (Fig. 1-B).

The standard curve test revealed that for the standard plasmids of genotype I ASFV, the slopes were -3.3825 for Cy5 and -3.1906 for FAM; the correlation coefficient R² was 0.999 for Cy5 and 0.998 for FAM; the amplification efficiency was 97.53% for Cy5 and 100.06 for FAM, respectively (Fig. 1-C); for the standard plasmids of genotype II ASFV, the slope was -3.2983 for FAM, the correlation coefficient R² was 0.992 for FAM, the amplification efficiency was 100.01% for FAM, whereas Cy5 fluorophore could not be detected (Fig. 1-C). In addition, the sensitivity of the duplex ARMS-qPCR was 10 copies per reaction for both genotype I and genotype II ASFVs (Fig. 1-D). Thus, these results indicated that the duplex ARMS-qPCR assay has high efficiency and sensitivity.

We then evaluated the specificity of the duplex ARMS-qPCR. The nucleic acids of 7 other swine viruses, including PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, were used as templates. 3 amplification curves were obtained for genotype I ASFV (FAM and Cy5 signals) and II ASFV (FAM signal), whereas no amplification curve was recorded for the nucleic acids of PRRSV, CSFV, PRV, PCV2, PEDV, TGEV, and PoRV, as well as genotype II ASFV (Cy5 signal) and ddH₂O (Fig. 1-E). The results demonstrated that the duplex ARMS-qPCR has a good specificity without cross-reactivity with other swine viruses.

The results of the stable detection limit test showed that for the standard plasmids of genotype I ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 7/12 replicates were tested positive at the dilution of 5 copies (Fig.  1-F); for the standard plasmids of genotype II ASFV, all 12 replicates were tested positive at the dilution of 10 copies, while 6/12 replicates were tested positive at the dilution of 1 copy (Fig. 1-F). Thus, the stable detection limit of the duplex ARMS-qPCR was 10 copies per reaction for both genotypes I and II ASFVs (Fig. 1-F).

We further assessed the repeatability and reproducibility of the duplex ARMS-qPCR. The assay tested the standard plasmids of 3 concentrations (10⁶, 10⁴, and 10² copies). For the standard plasmids of genotype I ASFV, the intra- and inter-assay variation (CV) of Ct value for the duplex ARMS-qPCR ranged from 0.07 to 0.93% and 1.2 to 2.17% in FAM fluorescence channel and from 0.38 to 1.02 and 0.85% to 1.27% in Cy5 fluorescence channel, respectively (Table 1). For the standard plasmids of genotype II ASFV, the intra- and inter-assay variation (CV) of Ct value for the duplex ARMS-qPCR ranged from 0.27 to 0.61% and 0.77 to 1.07% in FAM fluorescence channel (Table 1). These findings suggested that the duplex ARMS-qPCR assay has satisfactory repeatability and reproducibility.

Finally, we evaluated the duplex ARMS-qPCR compared with WOAH-qPCR. 40 samples were detected using both assays, including blood, oral and rectal swabs, tissues, and cell cultures from pigs or PAMs infected by genotypes I and II ASFVs. Animal studies have evaluated the virulence and transmissibility of genotype I ASFV SD/DY-I/21 and genotype II virus HLJ/18 (Zhao et al. 2019; Sun et al. 2021), respectively. The results showed that 36 samples, including 18 of genotype I ASFV and 18 of genotype II ASFV were detected to be positive and differentiated by the duplex ARMS-qPCR, which were consistent with the results of the WOAH-qPCR (Appendix C).

In summary, we developed a duplex ARMS-qPCR assay based on ASFV genotyping region of B646L gene, which can effectively differentiate genotypes I and II ASFVs. The assay had high sensitivity and specificity and exhibited good results in detecting samples, including blood, oral and rectal swabs, tissues, and cell culture. Whether our method could be used for differentiating other genotypes of ASFVs is needed for further evalution. However, just genotypes I and II ASFVs are spreading outside Africa. Thus, our method will provide an additional epidemiological investigation tool to implement effective ASFV control and prevention.

The reference genome for faba bean (Vicia faba L.) is lacking because of its large genome size (~13 Gb), and the genetic and gene mapping studies on faba bean are far behind compared with those for other legumes.  In this study, we selected three purified faba bean lines (Yundou 8137, H0003712, and H000572) as parents and constructed two F₂ populations.  And two F₂ populations, namely 167 F₂ plants in Pop1 (Yundou 8137×H0003712) and 204 F₂ plants in Pop2 (H000572×Yundou 8137), were genotyped using a targeted next-generation sequencing (TNGS) genotyping platform, and two high-density single nucleotide polymorphisms (SNP) genetic linkage maps of faba bean were constructed.  The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.  The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM. In these two F₂ populations, Quantitative trait locus (QTL) mapping identified 98 QTLs for 14 agronomic traits related to flowers, pods, plant types and grains.  Then, the two maps were merged into an integrated genetic linkage map containing 6895 SNPs, with a length of 3324.48 cM.  These results not only lay the foundation for fine mapping and map-based cloning of related genes, but also can accelerate molecular marker-assisted breeding of faba bean.

 Plant architecture and leaf color are important factors influencing cotton fiber yield.  In this study, based on genetic analysis, stem paraffin sectioning, and phytohormone treatments, we showed that the Dwarf and Red (DR) cotton mutant is a gibberellin-sensitive mutant and caused by mutation in a single dominant locus, designated GhDR.  Using Bulked Segregant Analysis (BSA) and genotyping by target sequencing (GBTS) approaches, we located the causative mutation to a ~197-kb genetic interval on chromosome A09 containing 25 annotated genes.  Based on gene annotation and expression changes between the mutant and normal plant, GH_A09G2280 was considered as the best candidate gene responsible for the dwarf and red mutant phenotypes.  A 2-nucleotide deletion was found in the coding region of GhDR/GH_A09G2280 in the DR mutant, which caused frameshift and truncation of GhDR.  GhDR is a homolog of Arabidopsis AtBBX24, encoding a B-box zinc finger protein.  The frameshift deletion eliminated the C-terminal nuclear localization domain and VP domain of GhDR and altered its subcellular localization.  Comparative transcriptome analysis demonstrated downregulation of the key genes involved in gibberellin biosynthesis and signaling transduction network and upregulation of the genes related to gibberellin degradation and anthocyanin biosynthetic pathway in the DR mutant.  The results of this study uncovered the potential molecular basis regulating plant architecture and anthocyanin accumulation in cotton. 

Genetic improvement has promoted grain yield and nitrogen use efficiency (NUE) of wheat during the past decades, therefore, grain yield and NUE of wheat cultivars were high as compared to previous cultivars in the Yangtze River Basin since the 2000s.  However, the critical traits and mechanisms of higher grain yield and NUE are not known.  To explore the higher grain yield and NUE mechanisms of these new cultivars, 21 local cultivars were cultivated for three growing seasons from 2016 to 2019.  Significantly positive correlations were observed between grain yield and NUE in the three years.  The cultivars were grouped into high, medium, and low grain yield and NUE groups (abbreviated to HH, MM, and LL, respectively).  Significantly high grain yield and NUE were observed in the HH group.  High grain yield was attributed to more effective ears by high tiller fertility and greater single-spike yield by increasing post-anthesis single-stem biomass.  Compared to other groups, longer leaf stay-green ability and greater flag leaf photosynthetic rate after anthesis were detected in HH group.  It also showed higher N accumulation at pre-anthesis which contributed to increasing N accumulation per stem, including stem and leaf sheath, leaf blade, and unit leaf area at pre-anthesis, and promoting N uptake efficiency, the main contribution of high NUE.  Moreover, Tiller fertility was positively related to N accumulation per stem, N accumulation per unit leaf area, leaf stay-green ability, and flag leaf photosynthetic rate, indicating that improving tiller fertility promoted N uptake, leaf N accumulation, and photosynthetic ability, thereby achieving synchronous improvements in grain yield and NUE.  Therefore, tiller fertility is proposed as an important kernel indicator that can be used in the breeding and management of cultivars to improve agricultural efficiency and sustainability. 

Lignin metabolism plays a pivotal role in plant defense against pathogens and is always positively correlated as a response to pathogen infection.  Thus, understanding resistance genes against pathogens in plants depends on a genetic analysis of lignin response.  In the study, eight upland cotton lines were used to construct a multi-parent advanced generation intercross (MAGIC) population (n=280), which exhibited peculiar characteristics from the convergence of various alleles coding for advantageous traits.  To measure the lignin response to Verticillium wilt (LRVW), artificial disease nursery (ADN) and rotation nursery (RN) were prepared for MAGIC population planting in four environments.  The stem lignin contents were collected, and the LRVW was measured with the lignin value of ADN/RN in each environment, which showed great variation.  A total of 9323 high-quality single-nucleotide polymorphism (SNP) markers obtained from the Cotton-SNP63K array were employed for genotyping the MAGIC population.  The SNPs were distributed through the whole genome with 4.78 SNP/Mb density, ranging from 1.14 (ChrA06) to 10.08 (ChrD08).  A genome-wide association study was performed using a mixed linear model (MLM) for LRVW, and three stable quantitative trait loci (QTLs), qLRVW-A04, qLRVW-A10 and qLRVW-D05, were identified in more than two environments.  Two key candidate genes, Ghi_D05G01046 and Ghi_D05G01221, were selected within the QTLs through the combination of variations in the coding sequence, induced expression patterns, and function annotations, both of which presented nonsynonymous mutations in coding regions and were strongly induced by Verticilliumdahliae. Ghi_D05G01046 encodes a leucine-rich extensin (LRx) protein, which is involved in Arabidopsis cell wall biosynthesis and organization.  Ghi_D05G01221 encodes a transcriptional co-repressor novel interactor of jaz (NINJA), which functions in the jasmonic acid (JA) signaling pathway.  In summary, the study creates valuable genetic resources for breeding and QTL mapping and opens up a new perspective to uncover the genetic basis of VW resistance in upland cotton.