The story of a decade: Genomics, functional genomics, and molecular breeding in Brassica napus.

Rapeseed (Brassica napus L.) is one of the major global sources of edible vegetable oil and is also used as a feed and pioneer crop and for sightseeing and industrial purposes. Improvements in genome sequencing and molecular marker technology have fueled a boom in functional genomic studies of major agronomic characters such as yield, quality, flowering time, and stress resistance. Moreover, introgression and pyramiding of key functional genes have greatly accelerated the genetic improvement of important traits. Here we summarize recent progress in rapeseed genomics and genetics, and we discuss effective molecular breeding strategies by exploring these findings in rapeseed. These insights will extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process, ultimately contributing to more sustainable agriculture throughout the world.

Fine mapping of a major QTL, qKl-1BL controlling kernel length in common wheat.

KEY MESSAGE: A major stable QTL, qKl-1BL, for kernel length of wheat was narrowed down to a 2.04-Mb interval on chromosome 1BL; the candidate genes were predicated and the genetic effects on yield-related traits were characterized. As a key factor influencing kernel weight, wheat kernel shape is closely related to yield formation, and in turn affects both wheat processing quality and market value. Fine mapping of the major quantitative trait loci (QTL) for kernel shape could provide genetic resources and a theoretical basis for the genetic improvement of wheat yield-related traits. In this study, a major QTL for kernel length (KL) on 1BL, named qKl-1BL, was identified from the recombinant inbred lines (RIL) in multiple environments based on the genetic map and physical map, with 4.76-21.15% of the phenotypic variation explained. To fine map qKl-1BL, the map-based cloning strategy was used. By using developed InDel markers, the near-isogenic line (NIL) pairs and eight key recombinants were identified from a segregating population containing 3621 individuals derived from residual heterozygous lines (RHLs) self-crossing. In combination with phenotype identification, qKl-1BL was finely positioned into a 2.04-Mb interval, KN1B:698.15-700.19 Mb, with eight differentially expressed genes enriched at the key period of kernel elongation. Based on transcriptome analysis and functional annotation information, two candidate genes for qKl-1BL controlling kernel elongation were identified. Additionally, genetic effect analysis showed that the superior allele of qKl-1BL from Jing411 could increase KL, thousand kernel weight (TKW), and yield per plant (YPP) significantly, as well as kernel bulk density and stability time. Taken together, this study identified a QTL interval for controlling kernel length with two possible candidate genes, which provides an important basis for qKl-1BL cloning, functional analysis, and application in molecular breeding programs.

Enhanced ε­poly­L­lysine production in Streptomyces species by combining interspecific hybridization with multiple antibiotic resistance.

To improve the ε-PL production in wild-type strains of Streptomyces. albulus, Streptomyces. noursei, Streptomyces. rochei and Streptomyces. yunnanensis, the interspecific hybridization based on protoplast fusion was first performed. Two-species hybridizations failed to obtain hybrids with significant increase in ε-PL production, but four-species hybridizations succeed in acquiring many high-yield hybrids. 16S rDNA homology alignment and RAPD confirmed that the hybrid HX17 was restructured by integrating gene fragments from S. albulus and S. rochei with S. noursei as the carrier. S. noursei HX17 was subsequently suffered from mutagenesis and genome shuffling combining with multiple antibiotic resistance, and a mutant S. noursei GX6 was obtained with ε-PL yield of 2.23 g/L in shake-flask fermentation. In fed-batch fermentation, the ε-PL production of GX6 reached 47.2 g/L, which was increased by 95.6% to 136.8% over the wild parents. Ribosomal genes associated with antibiotics were sequenced and majority of mutant strains had mutations at different sites, indicating that the increase of antibiotic resistance was strongly associated with them. This research proved that combining interspecific hybridization with multiple antibiotic resistance was as an effective approach to rapidly improve the ε-PL production in Streptomyces species.

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences.

Pleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding. KEY POINTS: • Various genetic techniques are available in Pleurotus ostreatus. • P. ostreatus can be used as an alternative model mushroom in genetic analyses. • New frontiers in mushroom science are being developed using the fungus.

Shifting the pH profiles of Staphylococcus epidermidis lipase (SEL) and Staphylococcus hyicus lipase (SHL) through generating chimeric lipases by DNA shuffling strategy.

Enzymes are often required to function in a particular reaction condition by the industrial procedure. In order to identify critical residues affecting the optimum pH of Staphylococcal lipases, chimeric lipases from homologous lipases were generated via a DNA shuffling strategy. Chimeric 1 included mutations of G166S, K212E, T243A, H271Y. Chimeric 2 consisted of substitutions of K212E, T243A, H271Y. Chimeric 3 contained substitutions of K212E, R359L. From the screening results, the pH profiles for chimeric 1 and 2 lipases were shifted from pH 7 to 6. While the pH of chimeric 3 was shifted to 8. It seems the mutation of K212E in chimeric 1 and 2 decreased the pH to 6 by changing the electrostatic potential surface. Furthermore, chimeric 3 showed 10 ˚C improvement in the optimum temperature due to the rigidification of the catalytic loop through the hydrophobic interaction network. Moreover, the substrate specificity of chimeric 1 and 2 was increased towards the longer carbon length chains due to the mutation of T243A adjacent to the lid region through increasing the flexibility of the lid. Current study illustrated that directed evolution successfully modified lipase properties including optimum pH, temperature and substrate specificity through mutations, especially near catalytic and lid regions.

A Comprehensive Analysis of Auxin Response Factor Gene Family in Melastoma dodecandrum Genome.

Auxin Response Factors (ARFs) mediate auxin signaling and govern diverse biological processes. However, a comprehensive analysis of the ARF gene family and identification of their key regulatory functions have not been conducted in Melastoma dodecandrum, leading to a weak understanding of further use and development for this functional shrub. In this study, we successfully identified a total of 27 members of the ARF gene family in M. dodecandrum and classified them into Class I-III. Class II-III showed more significant gene duplication than Class I, especially for MedARF16s. According to the prediction of cis-regulatory elements, the AP2/ERF, BHLH, and bZIP transcription factor families may serve as regulatory factors controlling the transcriptional pre-initiation expression of MedARF. Analysis of miRNA editing sites reveals that miR160 may play a regulatory role in the post-transcriptional expression of MeARF. Expression profiles revealed that more than half of the MedARFs exhibited high expression levels in the stem compared to other organs. While there are some specific genes expressed only in flowers, it is noteworthy that MedARF16s, MedARF7A, and MedARF9B, which are highly expressed in stems, also demonstrate high expressions in other organs of M. dodecandrum. Further hormone treatment experiments revealed that these MedARFs were sensitive to auxin changes, with MedARF6C and MedARF7A showing significant and rapid changes in expression upon increasing exogenous auxin. In brief, our findings suggest a crucial role in regulating plant growth and development in M. dodecandrum by responding to changes in auxin. These results can provide a theoretical basis for future molecular breeding in Myrtaceae.

[Exploration of cross-cultivar group characteristics of a new cultivar of Prunus mume 'Zhizhang Guhong Chongcui'].

'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.

CRISPR-Cas9 based molecular breeding in crop plants: a review.

Traditional crop breeding techniques are not quickly boosting yields to fulfill the expanding population needs. Long crop lifespans hinder the ability of plant breeding to develop superior crop varieties. Due to the arduous crossing, selecting, and challenging processes, it can take decades to establish new varieties with desired agronomic traits. Develop new plant varieties instantly to reduce hunger and improve food security. As a result of the adoption of conventional agricultural techniques, crop genetic diversity has decreased over time. Several traditional and molecular techniques, such as genetic selection, mutant breeding, somaclonal variation, genome-wide association studies, and others, have improved agronomic traits associated with agricultural plant productivity, quality, and resistance to biotic and abiotic stresses. In addition, modern genome editing approaches based on programmable nucleases, CRISPR, and Cas9 proteins have escorted an exciting new era of plant breeding. Plant breeders and scientists worldwide rely on cutting-edge techniques like quick breeding, genome editing tools, and high-throughput phenotyping to boost crop breeding output. This review compiles discoveries in numerous areas of crop breeding, such as using genome editing tools to accelerate the breeding process and create yearly crop generations with the desired features, to describe the shift from conventional to modern plant breeding techniques.

De Novo Pterostilbene Production from Glucose Using Modular Coculture Engineering in Escherichia coli.

Pterostilbene, a derivative of resveratrol, is of increasing interest due to its increased bioavailability and potential health benefits. Sustainable production of pterostilbene is important, especially given the challenges of traditional plant extraction and chemical synthesis methods. While engineered microbial cell factories provide a potential alternative for pterostilbene production, most approaches necessitate feeding intermediate compounds. To address these limitations, we adopted a modular coculture engineering strategy, dividing the pterostilbene biosynthetic pathway between two engineered E. coli strains. Using a combination of gene knockout, atmospheric and room-temperature plasma mutagenesis, and error-prone PCR-based whole genome shuffling to engineer strains for the coculture system, we achieved a pterostilbene production titer of 134.84 ± 9.28 mg/L from glucose using a 1:3 inoculation ratio and 0.1% dimethyl sulfoxide supplementation. This represents the highest reported de novo production titer. Our results underscore the potential of coculture systems and metabolic balance in microbial biosynthesis.

Identification of hub genes that variate the qCSS12-mediated cold tolerance between indica and japonica rice using WGCNA.

KEY MESSAGE: Cold-tolerant QTL qCSS12-regulated 14 hub genes are involved in the chloroplastic biological processes and in the protein synthesis and degradation processes in japonica rice. Low temperature is a main constraint factor for rice growth and production. To better understand the regulatory mechanisms underlying the cold tolerance phenotype in rice, here, we selected a cold-sensitive nearly isogenic line (NIL) NIL(qcss12) as materials to identify hub genes that are mediated by the cold-tolerant locus qCSS12 through weighted gene co-expression network analysis (WGCNA). Fourteen cold-responsive genes were identified, of which, 6 are involved in regulating biological processes in chloroplasts, including the reported EF-Tu, Prk, and ChlD, and 8 are involved in the protein synthesis and degradation processes. Differential expression of these genes between NIL(qcss12) and its controls under cold stress may be responsible for qCSS12-mediated cold tolerance in japonica rice. Moreover, natural variations in 12 of these hub genes are highly correlated with the cold tolerance divergence in two rice subspecies. The results provide deep insights into a better understanding of the molecular basis of cold adaptation in rice and provide a theoretical basis for molecular breeding.

Dissection and validation of a promising QTL controlling spikelet number on 5B in bread wheat.

KEY MESSAGE: Five environmentally stable QTLs for spikelet number per spike and days to heading were identified using a high-genetic map containing 95,444 SNPs, among which QSns.ucas-5B was validated using residual heterozygous line at multiple environments. Spikelet number per spike (SNS) and days to heading (DTH) play pivotal roles in the improvement of wheat yield. In this study, a high-density genetic map for a recombinant inbred lines (RILs) population derived from Zhengnong 17 (ZN17) and Yangbaimai (YBM) was constructed using 95,444 single-nucleotide polymorphism (SNP) markers from the Wheat660K SNP array. Our study identified a total of five environmentally stable QTLs for SNS and DTH, one of which was named QSns.ucas-5B, with a physical interval of approximately 545.4-552.1 Mb on the 5BL chromosome arm. Importantly, the elite haplotype within QSns.ucas-5B showed a consistent and positive effect on SNS, grain number and weight per spike, without extending the days to heading. These findings provide a foundation for future efforts to map and clone the gene(s) responsible for QSns.ucas-5B and further indicate the potential application of the developed and validated InDel marker of QSns.ucas-5B for molecular breeding purposes, aimed at improving wheat grain yield.

Improving the Herbicide Resistance of Rice 4-Hydroxyphenylpyruvate Dioxygenase by DNA Shuffling Basis-Directed Evolution.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an ideal target for herbicide resistance genetic engineering. In this study, a mutant MFRR-2 with mesotrione resistance was screened from an Oryza sativa HPPD and mutant-Zea mays HPPD DNA shuffling library. The enzyme properties showed that although the stability of the mutant decreased in vitro, the enzyme activity of MFRR-2 at the optimum temperature of 25 °C was still equivalent to that of OsHPPD. Under 50 µM mesotrione treatment, MFRR-2 enzyme activity remained at approximately 90%, while the enzyme activity of OsHPPD decreased by approximately 50%. Surprisingly, Fe2+ was found to have an inhibitory effect on the enzyme activity. Then, the transgenic rice of the MFRR-2 gene showed approximately 1.5 times mesotrione resistance compared to OsHPPD transgenic rice. In conclusion, this study has conducted a beneficial exploration on the use of DNA shuffling for HPPD-directed evolution, and the mutant has potential application value for herbicide resistance genetic engineering.

Advances in Mechanism and Application of Molecular Breeding of Medicinal Mushrooms: A Review.

With the development of molecular biology and genomics technology, mushroom breeding methods have changed from single traditional breeding to molecular breeding. Compared with traditional breeding methods, molecular breeding has the advantages of short time and high efficiency. It breaks through the restrictive factors of conventional breeding and improves the accuracy of breeding. Molecular breeding technology is gradually applied to mushroom breeding. This paper summarizes the concept of molecular breeding and the application progress of various molecular breeding technologies in mushroom breeding, in order to provide reference for future research on mushroom breeding.

Improved pearl millet genomes representing the global heterotic pool offer a framework for molecular breeding applications.

High-quality reference genome assemblies, representative of global heterotic patterns, offer an ideal platform to accurately characterize and utilize genetic variation in the primary gene pool of hybrid crops. Here we report three platinum grade de-novo, near gap-free, chromosome-level reference genome assemblies from the active breeding germplasm in pearl millet with a high degree of contiguity, completeness, and accuracy. An improved Tift genome (Tift23D2B1-P1-P5) assembly has a contig N50 ~ 7,000-fold (126 Mb) compared to the previous version and better alignment in centromeric regions. Comparative genome analyses of these three lines clearly demonstrate a high level of collinearity and multiple structural variations, including inversions greater than 1 Mb. Differential genes in improved Tift genome are enriched for serine O-acetyltransferase and glycerol-3-phosphate metabolic process which play an important role in improving the nutritional quality of seed protein and disease resistance in plants, respectively. Multiple marker-trait associations are identified for a range of agronomic traits, including grain yield through genome-wide association study. Improved genome assemblies and marker resources developed in this study provide a comprehensive framework/platform for future applications such as marker-assisted selection of mono/oligogenic traits as well as whole-genome prediction and haplotype-based breeding of complex traits.

A major QTL simultaneously increases the number of spikelets per spike and thousand-kernel weight in a wheat line.

KEY MESSAGE: A novel and stably expressed QTL QSNS.sicau-SSY-7A for spikelet number per spike in wheat without negative effects on thousand-kernel weight was identified and validated in different genetic backgrounds. Spikelet number per spike (SNS) is an important determinant of yield in wheat. In the present study, we combined bulked segregant analysis (BSA) and the wheat 660 K single-nucleotide polymorphism (SNP) array to rapidly identify genomic regions associated with SNS from a recombinant inbred line (RIL) population derived from a cross between the wheat lines S849-8 and SY95-71. A genetic map was constructed using Kompetitive Allele Specific PCR markers in the SNP-enriched region on the long arm of chromosome 7A. A major and stably expressed QTL, QSNS.sicau-SSY-7A, was detected in multiple environments. It was located in a 1.6 cM interval on chromosome arm 7AL flanked by the markers AX-109983514 and AX-109820548. This QTL explained 6.86-15.72% of the phenotypic variance, with LOD values ranging from 3.66 to 8.66. Several genes associated with plant growth and development were identified in the interval where QSNS.sicau-SSY-7A was located on the 'Chinese Spring' wheat and wild emmer reference genomes. Furthermore, the effects of QSNS.sicau-SSY-7A and WHEAT ORTHOLOG OFAPO1(WAPO1) on SNS were analyzed. Interestingly, QSNS.sicau-SSY-7A significantly increased SNS without negative effects on thousand-kernel weight, anthesis date and plant height, demonstrating its great potential for breeding aimed at improving grain yield. Taken together, these results indicate that QSNS.sicau-SSY-7A is a promising locus for yield improvement, and its linkage markers are helpful for fine mapping and molecular breeding.

Characterization and fine mapping analysis of a major stable QTL qKnps-4A for kernel number per spike in wheat.

KEY MESSAGE: A major stable QTL for kernel number per spike was narrowed down to a 2.19-Mb region containing two potential candidate genes, and its effects on yield-related traits were characterized. Kernel number per spike (KNPS) in wheat is a key yield component. Dissection and characterization of major stable quantitative trait loci (QTLs) for KNPS would be of considerable value for the genetic improvement of yield potential using molecular breeding technology. We had previously reported a major stable QTL controlling KNPS, qKnps-4A. In the current study, primary fine-mapping analysis, based on the primary mapping population, located qKnps-4A to an interval of approximately 6.8-Mb from 649.0 to 655.8 Mb on chromosome 4A refering to 'Kenong 9204' genome. Further fine-mapping analysis based on a secondary mapping population narrowed qKnps-4A to an approximately 2.19-Mb interval from 653.72 to 655.91 Mb. Transcriptome sequencing, gene function annotation analysis and homologous gene related reports showed that TraesKN4A01HG38570 and TraesKN4A01HG38590 were most likely to be candidate genes of qKnps-4A. Phenotypic analysis based on paired near-isogenic lines in the target region showed that qKnps-4A increased KNPS mainly by increasing the number of central florets per spike. We also evaluated the effects of qKnps-4A on other yield-related traits. Moreover, we dissected the QTL cluster of qKnps-4A and qTkw-4A and proved that the phenotypic effects were probably due to close linkage of two or more genes rather than pleiotropic effects of a single gene. This study provides molecular marker resource for wheat molecular breeding designed to improve yield potential, and lay the foundation for gene functional analysis of qKnps-4A.

Cloning of PmMYB6 in Pinus massoniana and an Analysis of Its Function.

Phenylpropanoids are crucial for the growth and development of plants and their interaction with the environment. As key transcriptional regulators of plant growth and development, MYB-like transcription factors play a vital role in the biosynthesis of phenylpropanoid metabolites. In this study, we functionally characterized PmMYB6, a Pinus massoniana gene that encodes an R2R3-MYB transcription factor. It was confirmed by qPCR that PmMYB6 was highly expressed in the flowers, xylem, and phloem of P. massoniana. By overexpressing PmMYB6 in tobacco and poplar, we found that transgenic plants had enlarged xylem, increased content of lignin and flavonoids, and up-regulated expression of several enzyme genes of the phenylpropane metabolism pathway to different degrees. The above research results indicate that PmMYB6 is involved in the metabolic flux distribution of different branches of the phenylpropane metabolic pathway, and the results may provide clues for the regulation of metabolic fluxes between flavonoids and the lignin biosynthesis pathways of P. massoniana, as well as provide a basis for the molecular breeding of P. massoniana.

Association Mapping and Expression Analysis of the Genes Involved in the Wood Formation of Poplar.

Xylogenesis is a complex and sequential biosynthetic process controlled by polygenes. Deciphering the genetic architecture of this complex quantitative trait could provide valuable information for increasing wood biomass and improving its properties. Here, we performed genomic resequencing of 64 24-year-old trees (64 hybrids of section Aigeiros and their parents) grown in the same field and conducted full-sib family-based association analyses of two growth and six woody traits using GEMMA as a choice of association model selection. We identified 1342 significantly associated single nucleotide polymorphisms (SNPs), 673 located in the region upstream and downstream of 565 protein-encoding genes. The transcriptional regulation network of secondary cell wall (SCW) biosynthesis was further constructed based on the published data of poplar miRNA, transcriptome, and degradome. These provided a certain scientific basis for the in-depth understanding of the mechanism of poplar timber formation and the molecular-assisted breeding in the future.

Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

OBJECTIVE: In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). RESULTS: The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20 g·l-1 cellulase and 15 g·l-1 macerozyme for 6 h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. CONCLUSIONS: We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.