A 43 Bp-Deletion in the F3'H Gene Reducing Anthocyanins Is Responsible for Keeping Buds Green at Low Temperatures in Broccoli.

Most broccoli cultivars or accessions exhibit green buds under appropriate growth conditions, which turn purple at cold temperatures. However, certain cultivars consistently maintain green buds both during normal growth and at cold temperatures. In this study, we used BSA-seq (bulked segregation analysis-sequencing), along with fine mapping and transcriptome analysis to identify a candidate gene (flavonoid 3'-hydroxylase, F3'H) responsible for reducing anthocyanin accumulation in the mutant GS and HX-16 broccoli (Brassica oleracea L. var. italica), which could retain green buds even at low temperatures. A 43-bp deletion was detected in the coding sequence (CDS) of the F3'H gene in HX-16 and the mutant GS, which significantly decreased F3'H expression and the accumulation of cyanidin and delphinidin in the mutant GS. Furthermore, the expression of F3'H was upregulated at low temperatures in the wild line PS. Our results demonstrated the efficacy of utilizing the 43-bp InDel (Insertion-Deletion) in predicting whether buds in B. oleracea L. will turn purple or remain green at cold temperatures across forty-two germplasm materials. This study provides critical genetic and molecular insights for the molecular breeding of B. oleracea and sheds light on the molecular mechanisms underlying the effect of low temperatures on bud color in broccoli.

Multi-Omics Approaches to Improve Clubroot Resistance in Brassica with a Special Focus on Brassica oleracea L.

Brassica oleracea is an agronomically important species of the Brassicaceae family, including several nutrient-rich vegetables grown and consumed across the continents. But its sustainability is heavily constrained by a range of destructive pathogens, among which, clubroot disease, caused by a biotrophic protist Plasmodiophora brassicae, has caused significant yield and economic losses worldwide, thereby threatening global food security. To counter the pathogen attack, it demands a better understanding of the complex phenomenon of Brassica-P. brassicae pathosystem at the physiological, biochemical, molecular, and cellular levels. In recent years, multiple omics technologies with high-throughput techniques have emerged as successful in elucidating the responses to biotic and abiotic stresses. In Brassica spp., omics technologies such as genomics, transcriptomics, ncRNAomics, proteomics, and metabolomics are well documented, allowing us to gain insights into the dynamic changes that transpired during host-pathogen interactions at a deeper level. So, it is critical that we must review the recent advances in omics approaches and discuss how the current knowledge in multi-omics technologies has been able to breed high-quality clubroot-resistant B. oleracea. This review highlights the recent advances made in utilizing various omics approaches to understand the host resistance mechanisms adopted by Brassica crops in response to the P. brassicae attack. Finally, we have discussed the bottlenecks and the way forward to overcome the persisting knowledge gaps in delivering solutions to breed clubroot-resistant Brassica crops in a holistic, targeted, and precise way.

Identification of QTLs associated with curd architecture in cauliflower.

BACKGROUND: Curd architecture is one of the most important characters determining the curd morphology of cauliflower. However, the genetic mechanism dissection of this complex trait at molecular level is lacking. Genes/QTLs responsible for the morphological differences between present-day loose-curd and compact-curd cauliflower haven't been well revealed. RESULTS: Herein, by using a common compact-curd parent and two loose-curd parents, we developed two double haploid (DH) populations including 122 and 79 lines, respectively. For each population, we decomposed the curd architecture concept into four parameters (basal diameter, stalk length, stalk angle and curd solidity), and collected corresponding phenotypic data for each parameter across two environments. The Kosambi function and composite interval mapping algorithm were conducted to construct the linkage map and analyze the QTLs associated with curd architecture parameters. A total of 20 QTLs were detected with the minimum likelihood of odd (LOD) values ranging from 2.61 to 8.38 and the percentage of the phenotypic variance explained by each QTL (PVE) varying between 7.69 and 25.10%. Of these, two QTLs controlling stalk length (qSL.C6-1, qSL.C6-2) and two QTLs controlling curd solidity (qCS.C6-1 and qCS.C6-2) were steadily expressed in both environments. Further, qSL.C6-1, qSL.C6-2, qCS.C6-1 and qCS.C6-4 fell into the same chromosomal region of the reference genome, indicating that these loci are involved in pleiotropic effects or are tightly linked. CONCLUSION: The current study identified a series of QTLs associated with curd architecture parameters, which might contribute essentially to the formation of present-day loose-curd cauliflower that is widely cultivated in China. These results may pave the way for intensive deciphering the molecular mechanisms of curd development and for marker-assisted selection of curd morphology in cauliflower breeding.

Identification of Candidate Genes Involved in Curd Riceyness in Cauliflower.

"Riceyness" refers to the precocious development of flower bud initials over the curd surface of cauliflower, and it is regarded as undesirable for the market. The present study aimed to identify the candidate loci and genes responsible for the morphological difference in riceyness between a pair of cauliflower sister lines. Genetic analysis revealed that riceyness is controlled by a single dominant locus. An F2 population derived from the cross between these sister lines was used to construct "riceyness" and "non-riceyness" bulks, and then it was subjected to BSA-seq. On the basis of the results of Δ(SNP-index) analysis, a 4.0 Mb candidate region including 22 putative SNPs was mapped on chromosome C04. Combining the RNA-seq, gene function annotation, and target sequence analysis among two parents and other breeding lines, an orthologous gene of the Arabidopsis gene SOC1, Bo4g024850 was presumed as the candidate gene, and an upstream SNP likely resulted in riceyness phenotype via influencing the expression levels of Bo4g024850. These results are helpful to understand the genetic mechanism regulating riceyness, and to facilitate the molecular improvement on cauliflower curds.

Construction of a high-density genetic map and identification of loci controlling purple sepal trait of flower head in Brassica oleracea L. italica.

BACKGROUND: Some broccoli (Brassica oleracea L. italic) accessions have purple sepals and cold weather would deepen the purple color, while the sepals of other broccoli lines are always green even in cold winter. The related locus or gene is still unknown. In this study, a high-density genetic map was constructed based on specific locus amplified fragment (SLAF) sequencing in a doubled-haploid segregation population with 127 individuals. And mapping of the purple sepal trait in flower heads based on phenotypic data collected during three seasons was performed. RESULTS: A genetic map was constructed, which contained 6694 SLAF markers with an average sequencing depth of 81.37-fold in the maternal line, 84-fold in the paternal line, and 15.76-fold in each individual population studied. In all of the annual data recorded, three quantitative trait loci (QTLs) were identified that were all distributed within the linkage group (LG) 1. Among them, a major locus, qPH.C01-2, located at 36.393 cM LG1, was consistently detected in all analysis. Besides this locus, another two minor loci, qPH.C01-4 and qPH.C01-5, were identified near qPH.C01-2, based on the phenotypic data from spring of 2018. CONCLUSION: The purple sepal trait could be controlled by a major single locus and two minor loci. The genetic map and location of the purple sepal trait of flower heads provide an important foundation for mapping other compound traits and the identification of the genes related to purple sepal trait in broccoli.

Genome wide analysis of MADS-box gene family in Brassica oleracea reveals conservation and variation in flower development.

BACKGROUND: MADS-box genes play important roles in vegetative growth and reproductive development and are essential for the correct development of plants (particularly inflorescences, flowers, and fruits). However, this gene family has not been identified nor their functions analyzed in Brassica oleracea. RESULTS: In this study, we performed a whole-genome survey of the complete set of MADS-box genes in B. oleracea. In total, 91 MADS-box transcription factors (TFs) were identified and categorized as type I (Mα, Mß, Mγ) and type II (MIKCC, MIKC*) groups according to the phylogeny and gene structure analysis. Among these genes, 59 were randomly distributed on 9 chromosomes, while the other 23 were assigned to 19 scaffolds and 9 genes from NCBI had no location information. Both RNA-sequencing and quantitative real-time-PCR analysis suggested that MIKC genes had more active and complex expression patterns than M type genes and most type II genes showed high flowering-related expression profiles. Additional quantitative real-time-PCR analysis of pedicel and four flower whorls revealed that the structure of the B.oleracea MIKC genes was conserved, but their homologues showed variable expression patterns compared to those in Arabidopsis thaliana. CONCLUSION: This paper gives a detailed overview of the BolMADS genes and their expression patterns. The results obtained in this study provide useful information for understanding the molecular regulation of flower development and further functional characterization of MADS-box genes in B. oleracea.

Novel and major QTL for branch angle detected by using DH population from an exotic introgression in rapeseed (Brassica napus L.).

KEY MESSAGE: A high-density SNP map was constructed and several novel QTL for branch angle across six environments in Brassica napus were identified. Branch angle is a major determinant for the ideotype of a plant, while the mechanisms underlying this trait in Brassica napus remain elusive. Herein, we developed one doubled haploid population from a cross involving one Capsella bursa-pastoris derived B. napus intertribal introgression line with the compressed branches and wooden stems, and constructed a high-density SNP map covering the genetic distance of 2242.14 cM, with an average marker interval of 0.73 cM. After phenotypic measurements across six environments, the inclusive composite interval mapping algorithm was conducted to analyze the QTL associated with branch angle. In single-environment analysis, a total of 17 QTL were detected and mainly distributed on chromosomes A01, A03, A09 and C03. Of these, three major QTL, qBA.A03-2, qBA.C03-3 and qBA.C03-4 were steadily expressed, each explaining more than 10% of the phenotypic variation in at least two environments. Compared with other results on rapeseed branch angle, these major QTL were newly detected. In QTL by environment interactions (QEI) mapping, 10 QTL were identified, and the QTL average effect and QEI effect were estimated. Of these, 7 QTL were detected in both single-environment analysis and QEI mapping. Based on the physical positions of SNPs and the functional annotation of the Arabidopsis thaliana genome, 27 genes within the QTL regions were selected as candidate genes, including early auxin-responsive genes, small auxin-up RNA, auxin/indoleacetic acid and gretchenhagen-3. These results may pave the way for deciphering the genetic control of branch angle in B. napus.

Unravelling Glucoraphanin and Glucoerucin Metabolism across Broccoli Sprout Development: Insights from Metabolite and Transcriptome Analysis.

Variations in the concentration of glucoraphanin (GRA) and glucoerucin (GER), as well as the corresponding breakdown products, isothiocyanates (ITCs) and nitriles, were investigated during the growth of broccoli sprouts. The concentrations of GRA and GER decreased sharply from 33.66 µmol/g to 11.48 µmol/g and 12.98 µmol/g to 8.23 µmol/g, respectively, after seed germination. From the third to the seventh day, both GRA and GER were maintained as relatively stable. The highest concentrations of sulforaphane (17.16 µmol/g) and erucin (12.26 µmol/g) were observed on the first day. Hereafter, the concentrations of nitrile hydrolyzed from GRA or GER were higher than those of the corresponding ITCs. Moreover, the ratio of sulforaphane to sulforaphane nitrile decreased from 1.35 to 0.164 from 1 d to 5 d, with a similar trend exhibited for erucin/erucin nitrile after 2 d. RNA-seq analysis showed that BolMYB28 and BolCYP83A1, involved in aliphatic glucosinolate (GSL) biosynthesis, remained largely unexpressed until the third day. In contrast, the genes operating within the GSL-myrosinase hydrolysis pathway were highly expressed right from the beginning, with their expression levels increasing significantly after the third day. Additionally, we identified two BolESPs and six BolNSPs that might play important roles in promoting the production of nitriles during the development of broccoli sprouts.

Comparative Transcriptome and Targeted Metabolome Profiling Unravel the Key Role of Phenylpropanoid and Glucosinolate Pathways in Defense against Alternaria brassicicola in Broccoli.

Alternaria brassicicola (Ab) can cause a major yield and quality-limiting disease of Brassica oleracea called black spot, and the genetic resources conferring complete resistance against Ab have not been identified to date. Here, comparative transcriptome and targeted metabolome analysis were performed utilizing a newly identified resistant (R) line and a broccoli susceptible (S) line at 6, 24, and 72 h post-inoculation (hpi). Kyoto encyclopedia of genes and genomes pathway enrichment and the weighted gene co-expression network analyses showed that the phenylpropanoid pathway regulates the resistance to Ab in broccoli. One metabolite, cinnamic acid, was significantly upregulated in the Ab_inoculated R line compared with the mock treatment but no significant difference in the S line, indicating that the cinnamic acid may cause the resistance difference between R and S lines. Our results also revealed that three indolic glucosinolates of I3G, 4MI3G, and 1MI3G were significantly increased in the Ab_inoculated R line compared with the mock treatment, and some related genes were differentially expressed between the R and S lines. These results provided new insights into the mechanism of Ab defense in B. oleracea and have laid a theoretical foundation for effectively utilizing resistant germplasm resources in broccoli breeding.

QTL Identification for Stem Fiber, Strength and Rot Resistance in a DH Population from an Alien Introgression of Brassica napus.

Stem fiber, stem strength and stem-rot resistance are important agronomic traits in Brassica napus. To understand the molecular mechanism that controls the stem-related traits, we investigated the stem lignin (ADL), cellulose (Cel), hemicellulose (Hem) content, S/G monolignol ratio (SG), stem breaking force (BF), breaking strength (F) and Sclerotinia sclerotiorum resistance (SSR). Each trait was significantly positively or negatively correlated with more than three of the other six traits. QTL mapping for ADL, Cel, Hem, SG, BF, F and SSR were performed using a doubled haploid population derived from an intertribal B. napus introgression line 'Y689' crossed with B. napus cv. 'Westar'. A total of 67 additive QTL were identified and integrated into 55 consensus QTL by meta-analysis. Among the 55 consensus QTL, 23 (41.8%) QTL were co-located and were integrated into 11 unique QTL. The QTL by environment (Q × E) interactions were analyzed and 22 combined QTL were identified. In addition, candidate genes within the QTL intervals were proposed based on the known function of Arabidopsis orthologs. These results provided valuable information for improving lodging resistance, S. sclerotiorum resistance and mechanized harvesting of B. napus.

Establishment of a stable, effective and universal genetic transformation technique in the diverse species of Brassica oleracea.

Brassica oleracea is an economically important species, including seven cultivated variants. Agrobacterium-mediated transformation of B. oleracea crops, mainly via hypocotyl and cotyledon, has been achieved in the past. However, previously established transformation methods showed low efficiency, severe genotype limitation and a prolonged period for transformants acquisition, greatly restricting its application in functional genomic studies and crop improvement. In this study, we have compared the shoot regeneration and genetic transformation efficiency of hypocotyl, cotyledon petiole and curd peduncle explants from twelve genotypes of cauliflower and broccoli. Finally, an Agrobacterium-mediated transformation method using curd peduncle as explant was established, which is rapid, efficient, and amenable to high-throughput transformation and genome editing. The average genetic transformation efficiency of this method is stable up to 11.87% and was successfully implemented in twelve different genotypes of cauliflower and broccoli and other B. oleracea crops with low genotype dependence. Peduncle explants were found to contain abundant cambial cells with a strong cell division and shoot regeneration ability, which might be why this method achieved stable and high genetic transformation efficiency with almost no genotype dependence.

Advances in Multi-Omics Approaches for Molecular Breeding of Black Rot Resistance in Brassica oleracea L.

Brassica oleracea is one of the most important species of the Brassicaceae family encompassing several economically important vegetables produced and consumed worldwide. But its sustainability is challenged by a range of pathogens, among which black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is the most serious and destructive seed borne bacterial disease, causing huge yield losses. Host-plant resistance could act as the most effective and efficient solution to curb black rot disease for sustainable production of B. oleracea. Recently, 'omics' technologies have emerged as promising tools to understand the host-pathogen interactions, thereby gaining a deeper insight into the resistance mechanisms. In this review, we have summarized the recent achievements made in the emerging omics technologies to tackle the black rot challenge in B. oleracea. With an integrated approach of the omics technologies such as genomics, proteomics, transcriptomics, and metabolomics, it would allow better understanding of the complex molecular mechanisms underlying black rot resistance. Due to the availability of sequencing data, genomics and transcriptomics have progressed as expected for black rot resistance, however, other omics approaches like proteomics and metabolomics are lagging behind, necessitating a holistic and targeted approach to address the complex questions of Xcc-Brassica interactions. Genomic studies revealed that the black rot resistance is a complex trait and is mostly controlled by quantitative trait locus (QTL) with minor effects. Transcriptomic analysis divulged the genes related to photosynthesis, glucosinolate biosynthesis and catabolism, phenylpropanoid biosynthesis pathway, ROS scavenging, calcium signalling, hormonal synthesis and signalling pathway are being differentially expressed upon Xcc infection. Comparative proteomic analysis in relation to susceptible and/or resistance interactions with Xcc identified the involvement of proteins related to photosynthesis, protein biosynthesis, processing and degradation, energy metabolism, innate immunity, redox homeostasis, and defence response and signalling pathways in Xcc-Brassica interaction. Specifically, most of the studies focused on the regulation of the photosynthesis-related proteins as a resistance response in both early and later stages of infection. Metabolomic studies suggested that glucosinolates (GSLs), especially aliphatic and indolic GSLs, its subsequent hydrolysis products, and defensive metabolites synthesized by jasmonic acid (JA)-mediated phenylpropanoid biosynthesis pathway are involved in disease resistance mechanisms against Xcc in Brassica species. Multi-omics analysis showed that JA signalling pathway is regulating resistance against hemibiotrophic pathogen like Xcc. So, the bonhomie between omics technologies and plant breeding is going to trigger major breakthroughs in the field of crop improvement by developing superior cultivars with broad-spectrum resistance. If multi-omics tools are implemented at the right scale, we may be able to achieve the maximum benefits from the minimum. In this review, we have also discussed the challenges, future prospects, and the way forward in the application of omics technologies to accelerate the breeding of B. oleracea for disease resistance. A deeper insight about the current knowledge on omics can offer promising results in the breeding of high-quality disease-resistant crops.

Molecular Breeding Strategy and Challenges Towards Improvement of Downy Mildew Resistance in Cauliflower (Brassica oleracea var. botrytis L.).

Cauliflower (Brassica oleracea var. botrytis L.) is one of the important, nutritious and healthy vegetable crops grown and consumed worldwide. But its production is constrained by several destructive fungal diseases and most importantly, downy mildew leading to severe yield and quality losses. For sustainable cauliflower production, developing resistant varieties/hybrids with durable resistance against broad-spectrum of pathogens is the best strategy for a long term and reliable solution. Identification of novel resistant resources, knowledge of the genetics of resistance, mapping and cloning of resistance QTLs and identification of candidate genes would facilitate molecular breeding for disease resistance in cauliflower. Advent of next-generation sequencing technologies (NGS) and publishing of draft genome sequence of cauliflower has opened the flood gate for new possibilities to develop enormous amount of genomic resources leading to mapping and cloning of resistance QTLs. In cauliflower, several molecular breeding approaches such as QTL mapping, marker-assisted backcrossing, gene pyramiding have been carried out to develop new resistant cultivars. Marker-assisted selection (MAS) would be beneficial in improving the precision in the selection of improved cultivars against multiple pathogens. This comprehensive review emphasizes the fascinating recent advances made in the application of molecular breeding approach for resistance against an important pathogen; Downy Mildew (Hyaloperonospora parasitica) affecting cauliflower and Brassica oleracea crops and highlights the QTLs identified imparting resistance against this pathogen. We have also emphasized the critical research areas as future perspectives to bridge the gap between availability of genomic resources and its utility in identifying resistance genes/QTLs to breed downy mildew resistant cultivars. Additionally, we have also discussed the challenges and the way forward to realize the full potential of molecular breeding for downy mildew resistance by integrating marker technology with conventional breeding in the post-genomics era. All this information will undoubtedly provide new insights to the researchers in formulating future breeding strategies in cauliflower to develop durable resistant cultivars against the major pathogens in general and downy mildew in particular.

Development of GBTS and KASP Panels for Genetic Diversity, Population Structure, and Fingerprinting of a Large Collection of Broccoli (Brassica oleracea L. var. italica) in China.

Broccoli (Brassica oleracea var. italica) is one of the most important and nutritious vegetables widely cultivated in China. In the recent four decades, several improved varieties were bred and developed by Chinese breeders. However, the efforts for improvement of broccoli are hindered by limited information of genetic diversity and genetic relatedness contained within the available germplasms. This study evaluated the genetic diversity, genetic relationship, population structure, and fingerprinting of 372 accessions of broccoli representing most of the variability of broccoli in China. Millions of SNPs were identified by whole-genome sequencing of 23 representative broccoli genotypes. Through several stringent selection criteria, a total of 1,167 SNPs were selected to characterize genetic diversity and population structure. Of these markers, 1,067 SNPs were genotyped by target sequencing (GBTS), and 100 SNPs were genotyped by kompetitive allele specific PCR (KASP) assay. The average polymorphism information content (PIC) and expected heterozygosity (gene diversity) values were 0.33 and 0.42, respectively. Diversity analysis revealed the prevalence of low to moderate genetic diversity in the broccoli accessions indicating a narrow genetic base. Phylogenetic and principal component analyses revealed that the 372 accessions could be clustered into two main groups but with weak groupings. STRUCTURE analysis also suggested the presence of two subpopulations with weak genetic structure. Analysis of molecular variance (AMOVA) identified 13% variance among populations and 87% within populations revealing very low population differentiation, which could be attributed to massive gene flow and the reproductive biology of the crop. Based on high resolving power, a set of 28 KASP markers was chosen for DNA fingerprinting of the broccoli accessions for seed authentication and varietal identification. To the best of our knowledge, this is the first comprehensive study to measure diversity and population structure of a large collection of broccoli in China and also the first application of GBTS and KASP techniques in genetic characterization of broccoli. This work broadens the understanding of diversity, phylogeny, and population structure of a large collection of broccoli, which may enhance future breeding efforts to achieve higher productivity.

Construction of a High-Density Genetic Map and Identification of Loci Related to Hollow Stem Trait in Broccoli (Brassic oleracea L. italica).

A high-quality genetic map is important for mapping of compound traits. In this study, a genetic map was constructed based on the reference genome TO1000 after specific locus amplified fragment (SLAF) sequencing in a double-haploid segregation population of broccoli, and loci controlling hollow stem trait were identified in the genetic map. The genetic map contains 4,787 SLAF markers, with a mean marker distance of 0.22 cM and the mean sequencing depths of 91.14-fold in the maternal line, 88.97-fold in the paternal line and 17.11-fold in each DH progeny. A locus controlling the hollow stem trait, QHS.C09-2, which could explain 14.1% of the phenotypic variation, was steadily detected on the linkage group nine in the indicated data of 3 years' trials and BLUE analysis. The genetic map could lay an important foundation for mapping of compound traits, and mapping of hollow stem trait would be basis to clone the genes related to hollow stems in broccoli.

Natural Variation of Glucosinolates and Their Breakdown Products in Broccoli (Brassica oleracea var. italica) Seeds.

Seeds of 32 pure lines and 6 commercial broccoli cultivars were used to investigate variation in glucosinolates and their breakdown products. The aliphatic glucosinolate content was 54.5-218.7 µmol/g fresh weight, accounting for >90% of the total glucosinolates. The major glucosinolates found were glucoraphanin and glucoerucin in 27 samples and progoitrin in 7 samples. A gas chromatography-flame ionization detector (GC-FID) method was used to identify glucosinolate breakdown products; nine products were directly determined using standards. Using Arabidopsis thaliana lines myb28myb29 and Landsberg erecta to hydrolyze each reference glucosinolate, seven products were tentatively identified. 4-(Methylsulfinyl)butyl isothiocyanate and 5-(methylsulfinyl)pentanenitrile contents were 2.6-91.1 µmol/g fresh weight and 0-35.4 µmol/g fresh weight, respectively, with epithionitriles being more common than nitriles in accessions rich in alkenyl glucosinolate. Additionally, (S)-5-vinyl-1,3-oxazolidine-2-thione was detected in accessions rich in progoitrin. Specific lines with altered glucosinolate profiles and breakdown products were obtained and discussed according to the putative glucosinolate metabolism pathway.

Major Co-localized QTL for Plant Height, Branch Initiation Height, Stem Diameter, and Flowering Time in an Alien Introgression Derived Brassica napus DH Population.

Plant height (PH), branch initiation height (BIH), and stem diameter (SD) are three stem-related traits that play crucial roles in plant architecture and lodging resistance. Herein, we show one doubled haploid (DH) population obtained from a cross between Y689 (one Capsella bursa-pastoris derived Brassica napus intertribal introgression) and Westar (B. napus cultivar) that these traits were significantly positively correlated with one another and with flowering time (FT). Based on a high-density SNP map, a total of 102 additive quantitative trait loci (QTL) were identified across six environments. Seventy-two consensus QTL and 49 unique QTL were identified using a two-round strategy of QTL meta-analysis. Notably, a total of 19 major QTL, including 11 novel ones, were detected for these traits, which comprised two QTL clusters on chromosomes A02 and A07. Conditional QTL mapping was performed to preliminarily evaluate the genetic basis (pleiotropy or tight linkage) of the co-localized QTL. In addition, QTL by environment interactions (QEI) mapping was performed to verify the additive QTL and estimate the QEI effect. In the genomic regions of all major QTL, orthologs of the genes involved in phytohormone biosynthesis, phytohormone signaling, flower development, and cell differentiation in Arabidopsis were proposed as candidate genes. Of these, BnaA02g02560, an ortholog of Arabidopsis GASA4, was suggested as a candidate gene for PH, SD, and FT; and BnaA02g08490, an ortholog of Arabidopsis GNL, was associated with PH, BIH and FT. These results provide useful information for further genetic studies on stem-related traits and plant growth adaptation.

High Density Linkage Map Construction and QTL Detection for Three Silique-Related Traits in Orychophragmus violaceus Derived Brassica napus Population.

Seeds per silique (SS), seed weight (SW), and silique length (SL) are important determinant traits of seed yield potential in rapeseed (Brassica napus L.), and are controlled by naturally occurring quantitative trait loci (QTLs). Mapping QTLs to narrow chromosomal regions provides an effective means of characterizing the genetic basis of these complex traits. Orychophragmus violaceus is a crucifer with long siliques, many SS, and heavy seeds. A novel B. napus introgression line with many SS was previously selected from multiple crosses (B. rapa ssp. chinesis × O. violaceus) × B. napus. In present study, a doubled haploid (DH) population with 167 lines was established from a cross between the introgression line and a line with far fewer SS, in order to detect QTLs for silique-related traits. By screening with a Brassica 60K single nucleotide polymorphism (SNP) array, a high-density linkage map consisting of 1,153 bins and spanning a cumulative length of 2,209.1 cM was constructed, using 12,602 high-quality polymorphic SNPs in the DH population. The average recombination bin densities of the A and C subgenomes were 1.7 and 2.4 cM, respectively. 45 QTLs were identified for the three traits in all, which explained 4.0-34.4% of the total phenotypic variation; 20 of them were integrated into three unique QTLs by meta-analysis. These unique QTLs revealed a significant positive correlation between SS and SL and a significant negative correlation between SW and SS, and were mapped onto the linkage groups A05, C08, and C09. A trait-by-trait meta-analysis revealed eight, four, and seven consensus QTLs for SS, SW, and SL, respectively, and five major QTLs (cqSS.A09b, cqSS.C09, cqSW.A05, cqSW.C09, and cqSL.C09) were identified. Five, three, and four QTLs for SS, SW, and SL, respectively, might be novel QTLs because of the existence of alien genetic loci for these traits in the alien introgression. Thirty-eight candidate genes underlying nine QTLs for silique-related traits were identified.