Summary
Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major ...challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T0 plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T1 and T2 progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.
Seed weight (SW), which is one of the three major factors influencing grain yield, has been widely accepted as a complex trait that is controlled by polygenes, particularly in polyploid crops. ...Brassica napus L., which is the second leading crop source for vegetable oil around the world, is a tetraploid (4×) species. In the present study, we identified a major quantitative trait locus (QTL) on chromosome A9 of rapeseed in which the genes for SW and silique length (SL) were colocated. By fine mapping and association analysis, we uncovered a 165-bp deletion in the auxin-response factor 18 (ARF18) gene associated with increased SW and SL. ARF18 encodes an auxin-response factor and shows inhibitory activity on downstream auxin genes. This 55-aa deletion prevents ARF18 from forming homodimers, in turn resulting in the loss of binding activity. Furthermore, reciprocal crossing has shown that this QTL affects SW by maternal effects. Transcription analysis has shown that ARF18 regulates cell growth in the silique wall by acting via an auxin-response pathway. Together, our results suggest that ARF18 regulates silique wall development and determines SW via maternal regulation. In addition, our study reveals the first (to our knowledge) QTL in rapeseed and may provide insights into gene cloning involving polyploid crops.
Rapeseed (Brassica napus L.) is the largest oilseed crop in China and accounts for about 20% of world production. For the last 10years, the production, planting area, and yield of rapeseed have been ...stable, with improvement of seed quality and especially seed oil content. China is among the leading countries in rapeseed genomic research internationally, having jointly with other countries accomplished the whole genome sequencing of rapeseed and its two parental species, Brassica oleracea and Brassica rapa. Progress on functional genomics including the identification of QTL governing important agronomic traits such as yield, seed oil content, fertility regulation, disease and insect resistance, abiotic stress, nutrition use efficiency, and pod shattering resistance has been achieved. As a consequence, molecular markers have been developed and used in breeding programs. During 2005–2014, 215 rapeseed varieties were registered nationally, including 210 winter- and 5 spring-type varieties. Mechanization across the whole process of rapeseed production was investigated and operating instructions for all relevant techniques were published. Modern techniques for rapeseed field management such as high-density planting, controlled-release fertilizer, and biocontrol of disease and pests combined with precision tools such as drones have been developed and are being adopted in China. With the application of advanced breeding and production technologies, in the near future, the oil yield and quality of rapeseed varieties will be greatly increased, and more varieties with desirable traits, especially early maturation, high yield, high resistance to biotic and abiotic stress, and suitability for mechanized harvesting will be developed. Application of modern technologies on the mechanized management of rapeseed will greatly increase grower profit.
Seed weight (SW) and silique length (SL) are important determinants of the yield potential in rapeseed (Brassica napus L.). However, the genetic basis of both traits is poorly understood. The main ...objectives of this study were to dissect the genetic basis of SW and SL in rapeseed through the preliminary mapping of quantitative trait locus (QTL) by linkage analysis and fine mapping of the target major QTL by regional association analysis.
Preliminary linkage mapping identified thirteen and nine consensus QTLs for SW and SL, respectively. These QTLs explained 0.7-67.1% and 2.1-54.4% of the phenotypic variance for SW and SL, respectively. Of these QTLs, three pairs of SW and SL QTLs were co-localized and integrated into three unique QTLs. In addition, the significance level and genetic effect of the three co-localized QTLs for both SW and SL showed great variation before and after the conditional analysis. Moreover, the allelic effects of the three QTLs for SW were highly consistent with those for SL. Two of the three co-localized QTLs, uq.A09-1 (mean R(2) = 20.1% and 19.0% for SW and SL, respectively) and uq.A09-3 (mean R(2) = 13.5% and 13.2% for SW and SL, respectively), were detected in all four environments and showed the opposite additive-effect direction. These QTLs were validated and fine mapped (their confidence intervals were narrowed down from 5.3 cM to 1 cM for uq.A09-1 and 13.2 cM to 2.5 cM for uq.A09-3) by regional association analysis with a panel of 576 inbred lines, which has a relatively rapid linkage disequilibrium decay (0.3 Mb) in the target QTL region.
A few QTLs with major effects and several QTLs with moderate effects might contribute to the natural variation of SW and SL in rapeseed. The meta-, conditional and allelic effect analyses suggested that pleiotropy, rather than tight linkage, was the genetic basis of the three pairs of co-localized of SW and SL QTLs. Regional association analysis was an effective and highly efficient strategy for the direct fine mapping of target major QTL identified by preliminary linkage mapping.
BACKGROUND: Although Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by ...Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3’end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge. RESULTS: In this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3ʳᵈ nucleotide from the 3’ end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species. CONCLUSION: The method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.
Selenium is an essential micronutrient that plays a crucial role in maintaining human health. Selenium deficiency is seriously associated with various diseases such as Keshan disease, Kashin–Beck ...disease, cataracts, and others. Conversely, selenium supplementation has been found to have multiple effects, including antioxidant, anti-inflammatory, and anticancer functions. Compared with inorganic selenium, organic selenium exhibits higher bioactivities and a wider range of safe concentrations. Consequently, there has been a significant development of selenium-enriched foods which contain large amounts of organic selenium in order to improve human health. This review summarizes the physiological role and metabolism of selenium, the development of selenium-enriched foods, the physiological functions of selenium-enriched foods, and provides an analysis of total selenium and its species in selenium-enriched foods, with a view to laying the foundation for selenium-enriched food development.
As plant-specific transcription factors, the TIFY family genes are involved in the responses to a series of biotic and abiotic stresses and the regulation of the development of multiple organs. To ...explore the potential roles of the TIFY gene family in shoot branching, which can shape plant architecture and finally determine seed yield, we conducted comprehensive genome-wide analyses of the TIFY gene family in
. Here, HMMER search and BLASTp were used to identify the TIFY members. A total of 70 TIFY members were identified and divided into four subfamilies based on the conserved domains and motifs. These TIFY genes were distributed across 19 chromosomes. The predicted subcellular localizations revealed that most TIFY proteins were located in the nucleus. The tissue expression profile analyses indicated that TIFY genes were highly expressed in the stem, flower bud, and silique at the transcriptional level. High-proportioned activation of the dormant axillary buds on stems determined the branch numbers of rapeseed plants. Here, transcriptome analyses were conducted on axillary buds in four sequential developing stages, that is, dormant, temporarily dormant, being activated, and elongating (already activated). Surprisingly, the transcription of the majority of TIFY genes (65 of the 70) significantly decreased on the activation of buds. GO enrichment analysis and hormone treatments indicated that the transcription of TIFY family genes can be strongly induced by jasmonic acid, implying that the TIFY family genes may be involved in the regulation of jasmonic acid-mediated branch development. These results shed light on the roles of TIFY family genes in plant architecture.
Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available ...to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were
synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.
Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.
In May 2016, a highly pathogenic avian influenza A(H5N8) virus strain caused deaths among 3 species of wild migratory birds in Qinghai Lake, China. Genetic analysis showed that the novel reassortant ...virus belongs to group B H5N8 viruses and that the reassortment events likely occurred in early 2016.
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