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 auxinresponse 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.seed weight | silique length | ARF18 | cell growth | maternal effect T he rapid growth of the world population has increased the global requirement for food, which in turn warrants significant improvement in crop grain yield. As one of the three direct factors influencing crop grain yield, seed weight (SW) has been widely accepted as a complex trait that is controlled by polygenes. Therefore understanding the genetic and molecular basis of SW is extremely important for crop-improvement programs.The size of seeds is influenced by a variety of cellular processes (1). In Arabidopsis, some mutants such as ap2, arf2, da1, eod3, ttg2, and klu control seed size mainly by regulating cell elongation in the integument surrounding the seed (1-5). In mini3, iku1, iku2, and shb1 mutants, premature cellularization or proliferation of the endosperm in the early phase of seed development affects seed mass (6-10). The met1 gene has been determined to have parent-of-origin effects on seed size because of the loss of methylation in cytosine residues in CG islands (11). In rice, a total of 47 quantitative trait loci (QTLs) for grain length and 48 for grain width have been identified (12). Recent studies have shown that certain genes such as GW2, GIF1, qSW5, GS3, GS5, GW8, and qGL3 regulate grain size (13)(14)(15)(16)(17)(18)(19)(20). Among these, GW2 and qSW5 were determined to regulate grain weight by increasing cell number in the outer glume, whereas the others affected grain weight by directly regulating cell division and/or cell expansion of grain. Despite this progress, no genes responsible for SW have been identified in polyploid crops.Polyploidy is produced by the multiplication of a single genome (autopolyploid) or the c...

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 ( Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S- BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S- BnGRF2a transgenic line, Napin- BnGRF2a transgenic lines were further used to identify the function of BnGRF2 , and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

Two experiments were conducted to evaluate the effects ofBacillus subtilis natto, which was initially isolated from fermented soybeans on milk production, rumen fermentation and ruminal microbiome in dairy cows. In Experiment 1, 36 early lactation Chinese Holstein dairy cows (56 ± 23 days in milk) were randomly assigned to three groups: Control, cows were fed total mixed ration (TMR); BSNLOW, TMR plus 0.5 × 1011colony-forming units (cfu) ofB. subtilis natto/cow per day; and BSNHIGH, TMR plus 1.0 × 1011cfu ofB. subtilis natto/cow per day. During the 70-day treatment period, daily milk production and daily milk composition were determined in individual cows. The results showed that supplementing dairy cows with 0.5 × 1011and 1.0 × 1011cfu ofB. subtilis nattolinearly increased (P< 0.01) milk production (25.2 and 26.4 kg/dayv. 23.0 kg/day), 4% fat-corrected milk (27.3 and 28.1 kg/dayv. 24.2 kg/day), energy-corrected milk (27.3 and 28.2 kg/dayv. 24.2 kg/day), as well as milk fat (1.01 and 1.03 kg/dayv. 0.88 kg/day), protein (0.77 and 0.82 kg/dayv. 0.69 kg/day) and lactose yield (1.16 and 1.22 kg/dayv. 1.06 kg/day) but decreased milk somatic cell counts (SCC) by 3.4% to 5.5% (P< 0.01) in BSNLOW and BSNHIGH treatments compared with Control. In Experiment 2, four rumen-cannulated dairy cows were fed the basal diet from 1 to 7 days (pre-trial period) and rumen samples were collected on days 6 and 7; the same cows then were fed 1.0 × 1011cfu/dayB. subtilis nattofrom days 8 to 21 (trial period) and rumen samples were collected on days 20 and 21.B. subtilis nattowas discontinued from days 22 to 28 (post-trial period) and rumen samples were collected on days 27 and 28. Compared with the pre- and post-periods, ruminal pH decreased by 2.7% to 3.0% during the trial period (P< 0.01), whereas ammonia nitrogen (NH3-N), total volatile fatty acids and molar proportion of propionate (P< 0.01) and valerate (P< 0.05) increased. Molar proportion of acetate decreased and the acetate to propionate ratio was lower (P< 0.01) during the trial period. However, no differences for 24-hin saccodry matter digestibility were detected among different periods (treatments) though NDF digestibility was reduced in the trial and post-trial periods (P< 0.01). Compared with pre-trial period, total ruminal bacteria, proteolytic and amylolytic bacteria in rumen enumerated by culture methods increased by 15.0%, 16.2% and 11.7%, respectively (P< 0.01) but protozoa decreased to 5.35 log10cfu/ml (P< 0.01) during the trial period. These results demonstrate thatB. subtilis nattoimproves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate thatB. subtilis nattohas potential to be applied as a probiotic for dairy cows.

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BackgroundRapeseed (Brassica napus L.) is an important oil crop in the world, and increasing its oil content is a major breeding goal. The studies on seed structure and characteristics of different oil content rapeseed could help us to understand the biological mechanism of lipid accumulation, and be helpful for rapeseed breeding.Methodology/Principal FindingsHere we report on the seed ultrastructure of an ultrahigh oil content rapeseed line YN171, whose oil content is 64.8%, and compared with other high and low oil content rapeseed lines. The results indicated that the cytoplasms of cotyledon, radicle, and aleuronic cells were completely filled with oil and protein bodies, and YN171 had a high oil body organelle to cell area ratio for all cell types. In the cotyledon cells, oil body organelles comprised 81% of the total cell area in YN171, but only 53 to 58% in three high oil content lines and 33 to 38% in three low oil content lines. The high oil body organelle to cotyledon cell area ratio and the cotyledon ratio in seed were the main reasons for the ultrahigh oil content of YN171. The correlation analysis indicated that oil content is significantly negatively correlated with protein content, but is not correlated with fatty acid composition.Conclusions/SignificanceOur results indicate that the oil content of YN171 could be enhanced by increasing the oil body organelle to cell ratio for some cell types. The oil body organelle to seed ratio significantly highly positively correlates with oil content, and could be used to predict seed oil content. Based on the structural analysis of different oil content rapeseed lines, we estimate the maximum of rapeseed oil content could reach 75%. Our results will help us to screen and identify high oil content lines in rapeseed breeding.

BackgroundSingle nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.ResultsA total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.ConclusionsDespite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.

Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.

Cosuppression of an Arabidopsis Rubisco small subunit gene RBCS3B at Arabidopsis resulted in albino or pale green phenotypes which were caused by ROS accumulation As the most abundant protein on Earth, Rubisco has received much attention in the past decades. Even so, its function is still not understood thoroughly. In this paper, four Arabidopsis transgenic lines (RBCS3B-7, 18, 33, and 35) with albino or pale green phenotypes were obtained by transformation with a construct driving expression of sense RBCS3B, a Rubisco small subunit gene. The phenotypes produced in these transgenic lines were found to be caused by cosuppression. Among these lines, RBCS3B-7 displayed the most severe phenotypes including reduced height, developmental arrest and plant mortality before flowering when grown under normal light on soil. Chloroplast numbers in mesophyll cells were decreased compared to WT, and stacked thylakoids of chloroplasts were broken down gradually in RBCS3B-7 throughout development. In addition, the RBCS3B-7 line was light sensitive, and PSII activity measurement revealed that RBCS3B-7 suffered severe photoinhibition, even under normal light. We found that photoinhibition was due to accumulation of ROS, which accelerated photodamage of PSII and inhibited the repair of PSII in RBCS3B-7.