A systematic dissection in oilseed rape provides insights into the genetic architecture and molecular mechanism of yield heterosis.

Heterosis refers to the better performance of cross progeny compared with inbred parents, and its utilization contributes greatly to agricultural production. Several hypotheses have been proposed to explain heterosis mainly including dominance, over-dominance (or pseudo-overdominance) and epistasis. However, systematic dissection and verification of these hypotheses are rarely documented. Here, comparison of heterosis level across different traits showed that the strong heterosis of composite traits (such as yield) could be attributed to the multiplicative effects of moderate heterosis of component traits, whether at the genome or locus level. Yield heterosis was regulated by a complex trait-QTL network that was characterized by obvious centre-periphery structure, hub QTL, complex up/downstream and positive/negative feedback relationships. More importantly, we showed that better-parent heterosis on yield could be produced in a cross of two near-isogenic lines by the pyramiding and complementation of two major heterotic QTL showing partial-dominance on yield components. The causal gene (BnaA9.CYP78A9) of QC14 was identified, and its heterotic effect results from the heterozygous status of a CACTA-like transposable element in its upstream regulatory region, which led to partial dominance at expression and auxin levels, thus resulting in non-additive expression of downstream responsive genes involved in cell cycle and proliferation, eventually leading to the heterosis of cell number. Taken together, the results at the phenotypic, genetic and molecular levels were highly consistent, which demonstrated that the pyramiding effect of heterotic QTL and the multiplicative effect of individual component traits could well explain substantial parts of yield heterosis in oilseed rape. These results provide in-depth insights into the genetic architecture and molecular mechanism of yield heterosis.

A systematic dissection of the mechanisms underlying the natural variation of silique number in rapeseed (Brassica napus L.) germplasm.

Silique number is the most important component of yield in rapeseed (Brassica napus L.). To dissect the mechanism underlying the natural variation of silique number in rapeseed germplasm, a series of studies were performed. A panel of 331 core lines was employed to genome-wide association study (GWAS), and 27 loci (including 20 novel loci) were identified. The silique number difference between the more- and fewer-silique lines can be attributed to the accumulative differences in flower number and silique setting rate. Each of them accounted for 75.2% and 24.8%, respectively. The silique number was highly associated with the total photosynthesis and biomass. Microscopic analysis showed that the difference between extremely more- and fewer-silique lines normally occurred at the amount of flower bud but not morphology. Transcriptome analysis of shoot apical meristem (SAM) suggested that most of enriched groups were associated with the auxin biosynthesis/metabolism, vegetative growth and nutrition/energy accumulation. By integrating GWAS and RNA-seq results, six promising candidate genes were identified, and some of them were related to biomass accumulation. In conclusion, the natural variation of silique number is largely affected by the biomass and nutrition accumulation, which essentially reflects the positive regulatory relationship between the source and sink. Our study provides a comprehensive and systematic explanation for natural variation of silique number in rapeseed, which provides a foundation for its improvement.

Genetic and signalling pathways of dry fruit size: targets for genome editing-based crop improvement.

Fruit is seed-bearing structures specific to angiosperm that form from the gynoecium after flowering. Fruit size is an important fitness character for plant evolution and an agronomical trait for crop domestication/improvement. Despite the functional and economic importance of fruit size, the underlying genes and mechanisms are poorly understood, especially for dry fruit types. Improving our understanding of the genomic basis for fruit size opens the potential to apply gene-editing technology such as CRISPR/Cas to modulate fruit size in a range of species. This review examines the genes involved in the regulation of fruit size and identifies their genetic/signalling pathways, including the phytohormones, transcription and elongation factors, ubiquitin-proteasome and microRNA pathways, G-protein and receptor kinases signalling, arabinogalactan and RNA-binding proteins. Interestingly, different plant taxa have conserved functions for various fruit size regulators, suggesting that common genome edits across species may have similar outcomes. Many fruit size regulators identified to date are pleiotropic and affect other organs such as seeds, flowers and leaves, indicating a coordinated regulation. The relationships between fruit size and fruit number/seed number per fruit/seed size, as well as future research questions, are also discussed.

Maternal control of seed weight in rapeseed (Brassica napus L.): the causal link between the size of pod (mother, source) and seed (offspring, sink).

Seed size/weight is one of the key traits related to plant domestication and crop improvement. In rapeseed (Brassica napus L.) germplasm, seed weight shows extensive variation, but its regulatory mechanism is poorly understood. To identify the key mechanism of seed weight regulation, a systematic comparative study was performed. Genetic, morphological and cytological evidence showed that seed weight was controlled by maternal genotype, through the regulation of seed size mainly via cell number. The physiological evidence indicated that differences in the pod length might result in differences in pod wall photosynthetic area, carbohydrates and the final seed weight. We also identified two pleiotropic major quantitative trait loci that acted indirectly on seed weight via their effects on pod length. RNA-seq results showed that genes related to pod development and hormones were significantly differentially expressed in the pod wall; genes related to development, cell division, nutrient reservoir and ribosomal proteins were all up-regulated in the seeds of the large-seed pool. Finally, we proposed a potential seed weight regulatory mechanism that is specific to rapeseed and novel in plants. The results demonstrate a causal link between the size of the pod (mother, source) and the seed (offspring, sink) in rapeseed, which provides novel insight into the maternal control of seed weight and will open a new research field in plants.

An integrated analysis of QTL mapping and RNA sequencing provides further insights and promising candidates for pod number variation in rapeseed (Brassica napus L.).

BACKGROUND: As the most important yield component in rapeseed (Brassica napus L.), pod number is determined by a series of successive growth and development processes. Pod number shows extensive variation in rapeseed natural germplasm, which is valuable for genetic improvement. However, the genetic and especially the molecular mechanism for this kind of variation are poorly understood. In this study, we conducted QTL mapping and RNA sequencing, respectively, using the BnaZNRIL population and its two parental cultivars Zhongshuang11 and No.73290 which showed significant difference in pod number, primarily due to the difference in floral organ number. RESULT: A total of eight QTLs for pod number were identified using BnaZNRIL population with a high-density SNP linkage map, each was distributed on seven linkage groups and explained 5.8-11.9% of phenotypic variance. Then, they were integrated with those previously detected in BnaZNF2 population (deriving from same parents) and resulted in 15 consensus-QTLs. Of which, seven QTLs were identical to other studies, whereas the other eight should be novel. RNA sequencing of the shoot apical meristem (SAM) at the formation stage of floral bud primordia identified 9135 genes that were differentially expressed between the two parents. Gene ontology (GO) analysis showed that the top two enriched groups were S-assimilation, providing an essential nutrient for the synthesis of diverse metabolites, and polyamine metabolism, serving as second messengers that play an essential role in flowering genes initiation. KEGG analysis showed that the top three overrepresented pathways were carbohydrate (707 genes), amino acid (390 genes) and lipid metabolisms (322 genes). In silico mapping showed that 647 DEGs were located within the confidence intervals of 15 consensus QTLs. Based on annotations of Arabidopsis homologs corresponding to DEGs, nine genes related to meristem growth and development were considered as promising candidates for six QTLs. CONCLUSION: In this study, we discovered the first repeatable major QTL for pod number in rapeseed. In addition, RNA sequencing was performed for SAM in rapeseed, which provides new insights into the determination of floral organ number. Furthermore, the integration of DEGs and QTLs identified promising candidates for further gene cloning and mechanism study.

Precise Determination of Selenium Forms and Contents in Selenium-Enriched Rapeseed Seedlings and Flowering Stalks by HPLC-ICP-MS.

Rapeseed (Brassica napus L.) has the ability of selenium (Se) enrichment. Identification of selenides in Se-rich rapeseed products will promote the development and utilization of high value. By optimizing the Se species extraction process (protease type, extraction reagent, enzyme sample ratio, extraction time, etc.) and chromatographic column, an efficient, stable, and accurate method was established for the identification of Se species and content in rapeseed seedlings and flowering stalks, which were cultured by inorganic Se hydroponics. Five Se compounds, including selenocystine (SeCys2), methylselenocysteine (MeSeCys), selenomethionine (SeMet), selenite (SeIV), and selenate (SeVI) were qualitatively and quantitatively identified. Organoselenium was absolutely dominant in both seedlings and flowering stalks among the detected rapeseed varieties, with 64.18-90.20% and 94.38-98.47%, respectively. Further, MeSeCys, a highly active selenide, predominated in rapeseed flowering stalks with a proportion of 56.36-72.93% and a content of 1707.3-5030.3 µg/kg. This study provides a new source of MeSeCys supplementation for human Se fortification.

Genome-wide analysis of the JAZ subfamily of transcription factors and functional verification of BnC08.JAZ1-1 in Brassica napus.

BACKGROUND: JAZ subfamily plays crucial roles in growth and development, stress, and hormone responses in various plant species. Despite its importance, the structural and functional analyses of the JAZ subfamily in Brassica napus are still limited. RESULTS: Comparing to the existence of 12 JAZ genes (AtJAZ1-AtJAZ12) in Arabidopsis, there are 28, 31, and 56 JAZ orthologues in the reference genome of B. rapa, B. oleracea, and B. napus, respectively, in accordance with the proven triplication events during the evolution of Brassicaceae. The phylogenetic analysis showed that 127 JAZ proteins from A. thaliana, B. rapa, B. oleracea, and B. napus could fall into five groups. The structure analysis of all 127 JAZs showed that these proteins have the common motifs of TIFY and Jas, indicating their conservation in Brassicaceae species. In addition, the cis-element analysis showed that the main motif types are related to phytohormones, biotic and abiotic stresses. The qRT-PCR of the representative 11 JAZ genes in B. napus demonstrated that different groups of BnJAZ individuals have distinct patterns of expression under normal conditions or treatments with distinctive abiotic stresses and phytohormones. Especially, the expression of BnJAZ52 (BnC08.JAZ1-1) was significantly repressed by abscisic acid (ABA), gibberellin (GA), indoleacetic acid (IAA), polyethylene glycol (PEG), and NaCl treatments, while induced by methyl jasmonate (MeJA), cold and waterlogging. Expression pattern analysis showed that BnC08.JAZ1-1 was mainly expressed in the vascular bundle and young flower including petal, pistil, stamen, and developing ovule, but not in the stem, leaf, and mature silique and seed. Subcellular localization showed that the protein was localized in the nucleus, in line with its orthologues in Arabidopsis. Overexpression of BnC08.JAZ1-1 in Arabidopsis resulted in enhanced seed weight, likely through regulating the expression of the downstream response genes involved in the ubiquitin-proteasome pathway and phospholipid metabolism pathway. CONCLUSIONS: The systematic identification, phylogenetic, syntenic, and expression analyses of BnJAZs subfamily improve our understanding of their roles in responses to stress and phytohormone in B. napus. In addition, the preliminary functional validation of BnC08.JAZ1-1 in Arabidopsis demonstrated that this subfamily might also play a role in regulating seed weight.

Validation and Characterization of a Seed Number Per Silique Quantitative Trait Locus qSN.A7 in Rapeseed (Brassica napus L.).

Seed number is a key character/trait tightly related to the plant fitness/evolution and crop domestication/improvement. The seed number per silique (SNPS) shows a huge variation from several to more than 30, however the underlying regulatory mechanisms are poorly known, which has hindered its improvement. To answer this question, several representative lines with extreme SNPS were previously subjected to systematic genetic and cytological analyses. The results showed that the natural variation of seed number per silique is mainly controlled by maternal and embryonic genotype, which are co-determined by ovule number per ovary, fertile ovule ratio, ovule fertilization rate, and fertilized ovule development rate. More importantly, we also mapped two repeatable quantitative trait loci (QTLs) for SNPS using the F2:3 population derived from Zhongshuang11 and No. 73290, of which the major QTL qSN.A6 has been fine-mapped. In the current study, the near-isogenic lines (NILs) of qSN.A7 were successfully developed by the successive backcross of F1 with Zhongshuang11. First, the effect of qSN.A7 was validated by evaluating the SNPS of two types of homozygous NILs from BC3F2 population, which showed a significant difference of 2.23 on average. Then, qSN.A7 was successfully fine-mapped from the original 4.237 to 1.389 Mb, using a BC4F2 segregating population of 2,551 individuals. To further clarify the regulatory mechanism of qSN.A7, the two types of homologous NILs were subjected to genetic and cytological analyses. The results showed that the difference in SNPS between the two homologous NILs was determined by the embryonic genotypic effect. Highly accordant with this, no significant difference was observed in ovule number per ovary, ovule fertility, fertilization rate, and pollen fertility between the two homologous NILs. Therefore, the regulatory mechanism of qSN.A7 is completely different from the cloned qSS.C9 and qSN.A6. These results will advance the understanding of SNPS and facilitate gene cloning and molecular breeding in Brassica napus.

Genetic and Cytological Analyses of the Natural Variation of Seed Number per Pod in Rapeseed (Brassica napus L.).

Seed number is one of the key traits related to plant evolution/domestication and crop improvement/breeding. In rapeseed germplasm, the seed number per pod (SNPP) shows a very wide variation from several to nearly 30; however, the underlying causations/mechanisms for this variation are poorly known. In the current study, the genetic and cytological bases for the natural variation of SNPP in rapeseed was firstly and systematically investigated using the representative four high-SNPP and five low-SNPP lines. The results of self- or cross-pollination experiment between the high- and low-SNPP lines showed that the natural variation of SNPP was mainly controlled by maternal effect (mean = 0.79), followed by paternal effect (mean = 0.21). Analysis of the data using diploid seed embryo-cytoplasmic-maternal model further showed that the maternal genotype, embryo, and cytoplasm effects, respectively, explained 47.6, 35.2, and 7.5% of the genetic variance. In addition, the analysis of combining ability showed that for the SNPP of hybrid F1 was mainly determined by the general combining ability of parents (63.0%), followed by special combining ability of parental combination (37.0%). More importantly, the cytological observation showed that the SNPP difference between the high- and low-SNPP lines was attributable to the accumulative differences in its components. Of which, the number of ovules, the proportion of fertile ovules, the proportion of fertile ovules to be fertilized, and the proportion of fertilized ovules to develop into seeds accounted for 30.7, 18.2, 7.1, and 43.9%, respectively. The accordant results of both genetic and cytological analyses provide solid evidences and systematic insights to further understand the mechanisms underlying the natural variation of SNPP, which will facilitate the development of high-yield cultivars in rapeseed.

Linkage and regional association analysis reveal two new tightly-linked major-QTLs for pod number and seed number per pod in rapeseed (Brassica napus L.).

To facilitate the pseudochromosomes assembly and gene cloning in rapeseed, we developed a reference genetic population/map (named BnaZNF2) from two sequenced cultivars, Zhongshuang11 and No.73290, those exhibit significant differences in many traits, particularly yield components. The BnaZNF2 genetic map exhibited perfect collinearity with the physical map of B. napus, indicating its high quality. Comparative mapping revealed several genomic rearrangements between B. napus and B. rapa or B. oleracea. A total of eight and 16 QTLs were identified for pod number and seed number per pod, respectively, and of which three and five QTLs are identical to previously identified ones, whereas the other five and 11 are novel. Two new major QTL respectively for pod number and seed number per pod, qPN.A06-1 and qSN.A06-1 (R(2 )= 22.8% and 32.1%), were colocalised with opposite effects, and only qPN.A06-1 was confirmed and narrowed by regional association analysis to 180 kb including only 33 annotated genes. Conditional QTL analysis and subsequent NILs test indicated that tight linkage, rather than pleiotropy, was the genetic causation of their colocalisation. Our study demonstrates potential of this reference genetic population/map for precise QTL mapping and as a base for positional gene cloning in rapeseed.

Genome-wide microsatellite characterization and marker development in the sequenced Brassica crop species.

Although much research has been conducted, the pattern of microsatellite distribution has remained ambiguous, and the development/utilization of microsatellite markers has still been limited/inefficient in Brassica, due to the lack of genome sequences. In view of this, we conducted genome-wide microsatellite characterization and marker development in three recently sequenced Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. The analysed microsatellite characteristics of these Brassica species were highly similar or almost identical, which suggests that the pattern of microsatellite distribution is likely conservative in Brassica. The genomic distribution of microsatellites was highly non-uniform and positively or negatively correlated with genes or transposable elements, respectively. Of the total of 115 869, 185 662 and 356 522 simple sequence repeat (SSR) markers developed with high frequencies (408.2, 343.8 and 356.2 per Mb or one every 2.45, 2.91 and 2.81 kb, respectively), most represented new SSR markers, the majority had determined physical positions, and a large number were genic or putative single-locus SSR markers. We also constructed a comprehensive database for the newly developed SSR markers, which was integrated with public Brassica SSR markers and annotated genome components. The genome-wide SSR markers developed in this study provide a useful tool to extend the annotated genome resources of sequenced Brassica species to genetic study/breeding in different Brassica species.