Maize-legume intercropping achieves yield advantages by improving leaf functions and dry matter partition.

Intercropping can obtain yield advantages, but the mechanism of yield advantages of maize-legume intercropping is still unclear. Then, we explored the effects of cropping systems and N input on yield advantages in a two-year experiment. Cropping systems included monoculture maize (Zea mays L.) (MM), monoculture soybean (Glycine max L. Merr.) (MS), monoculture peanut (Arachis hypogaea L.) (MP), maize-soybean substitutive relay intercropping (IMS), and maize-peanut substitutive strip intercropping (IMP). N input included without N (N0) and N addition (N1). Results showed that maize's leaf area index was 31.0% and 34.6% higher in IMS and IMP than in MM. The specific leaf weight and chlorophyll a (chl a) of maize were notably higher by 8.0% and 18.8% in IMS, 3.1%, and 18.6% in IMP compared with MM. Finally, N addition resulted in a higher thousand kernels weight of maize in IMS and IMP than that in MM. More dry matter accumulated and partitioned to the grain, maize's averaged partial land equivalent ratio and the net effect were 0.76 and 2.75 t ha-1 in IMS, 0.78 and 2.83 t ha-1 in IMP. The leaf area index and specific leaf weight of intercropped soybean were 16.8% and 26% higher than MS. Although soybean suffers from shade during coexistence, recovered growth strengthens leaf functional traits and increases dry matter accumulation. The averaged partial land equivalent ratio and the net effect of intercropped soybean were 0.76 and 0.47 t ha-1. The leaf area index and specific leaf weight of peanuts in IMP were 69.1% and 14.4% lower than in the MP. The chlorophyll a and chlorophyll b of peanut in MP were 17.0% and 24.4% higher than in IMP. A less dry matter was partitioned to the grain for intercropped peanut. The averaged pLER and NE of intercropped peanuts were 0.26 and -0.55 t ha-1. In conclusion, the strengthened leaf functional traits promote dry matter accumulation, maize-soybean relay intercropping obtained a win-win yield advantage, and maize-peanut strip intercropping achieved a trade-off yield advantage.

Analysis of Altered Flowering Related Genes in a Multi-Silique Rapeseed (Brassica napus L.) Line zws-ms Based on Combination of Genome, Transcriptome and Proteome Data.

Based on previous researches, we further investigated the multi-silique trait in rapeseed (Brassica napus L.) line zws-ms. In this study, we used a relatively comprehensive list of flowering related genes in rapeseed and compared them between zws-ms and its near-isogenic line (NIL) zws-217. Genes were studied on genome, transcriptome and proteome levels and then we focused on genes with non-synonymous single nucleotide polymorphism (SNP) or frame-shift insertion-deletion (InDel), finding some genes on the list which changes their sequences. Then, combined with their annotation and the information of their orthologs, certain genes such as BnaA09g05900D, ortholog of AGAMOUS-LIKE 42 (AGL42), which encodes an MADS-box protein, were assumed as probably responsible for the multi-silique trait. Also, we analyzed the Differentially Accumulated Proteins (DAPs) between zws-ms and zws-217, revealing some genes involved in homologous recombination and mismatch repair pathways. Since the development of flowers/siliques is crucial to crops and it influences the yield of rapeseed, this study paved a way to deeply understand the mechanism of the multi-pistil flower formation, which may facilitate researches on rapeseed production in future.

Biochar and biofertilizer reduced nitrogen input and increased soybean yield in the maize soybean relay strip intercropping system.

Applying Biochar (BC) or biofertilizers (BF) are potential approaches to reduce the nitrogen input and mitigate soil degradation in the maize soybean relay strip intercropping system (IS). In 2019 and 2020, a two-factor experiment was carried out to examine the effects of BC and BF on soil productivity and yield production in IS. 4 N input levels (8.4, 22.5, 45 kg, and 67.5 kg ha - 1) referred to as N0, N1, N2, and N3 were paired with various organic treatments, including BC (150 kg ha - 1), BF (300 kg ha - 1), and without organic amendments (CK). The results demonstrated that, despite BF decreasing the biomass and N distribution into grains, BF performed better on improved soybean yield (5.2-8.5%) by increasing the accumulation of soybean biomass (7.2 ~ 11.6%) and N (7.7%). Even though BC and BF have a detrimental effect on soybean nitrogen fixation by reducing nodule number and weight, the values of soybean nitrogenase activity and nitrogen fixation potential in BF were higher than those in BC. Additionally, BF performs better at boosting the soil's nitrogen content and nitrate reductase and urease activity. BF increased the concentration of total N, soil organic matter, Olsen-phosphorus, and alkaline hydrolyzable N in the soil by 13.0, 17.1, 22.0, and 7.4%, respectively, compared to CK. Above all, applying BF combination with N2 (45 kg ha - 1 N) is a feasible strategy to raise crop grain output and keep soil productivity over the long term in IS.

Straw incorporation and nitrogen reduction effect on the uptake and use efficiency of nitrogen as well as soil CO2 emission of relay strip intercropped soybean.

Intercropping can increase crop N uptake and reduce carbon emissions. However, the effects of straw incorporation and N reduction on N use and carbon emissions in intercropping are still unclear. We explored the mechanism of N uptake, N use efficiency, and CO2 emissions in the wheat-maize-soybean relay strip intercropping system. A two-year field experiment was conducted with two straw managements, i.e., wheat straw incorporation (SI) and straw removal (SR), and four N application levels of soybean, i.e., 60 (N60), 30 (N30), 15 (N15), and 0 kg N ha-1 (N0). We assessed soil properties, CO2 emissions, and characteristics of roots, nodules, and aboveground N uptake of intercropped soybean. Results showed that geometry mean diameter of aggregate, soil porosity, soil total N, and soil urease activity were notably greater in SI than in SR. N input reduced from N60 to N30 did not significantly affect the soil total N content and urease activity in SI. The root length, root surface area, root volume, root biomass, root bleeding intensity, and inorganic N content of bleeding sap were greater in SI than in SR. In the SI, although the root length and surface area peaked at N60, the root biomass and inorganic N content of bleeding sap were insignificant between N60 and N30. The nodule number, nodule dry weight, nodule nitrogenase activity, and nodule nitrogen fixation potential in SI were notably increased compared with SR. The nodule nitrogen fixation potential in SI notably increased with the decrease of N input at the R3 stage, but it peaked in N30 at the R5 stage. On average, the aboveground N uptake and nitrogen recovery efficiency (RE) was notably higher by 43.7% and 76.8% in SI than in SR. SI+N30 achieved the greatest aboveground N uptake and RE. The CO2 emission and accumulated CO2 emission were notably greater in SI than in SR, and the accumulated CO2 emission of SI was the lowest with N30 input. In conclusion, SI+N30 promoted N uptake and utilization efficiency with reduced CO2 emissions during the soybean cropping season. It provides a potential strategy for sustainable agricultural development in intercropping systems.

Investigation of Thermomorphogenesis-Related Genes for a Multi-Silique Trait in Brassica napus by Comparative Transcriptome Analysis.

In higher plants, the structure of a flower is precisely controlled by a series of genes. An aberrance flower results in abnormal fruit morphology. Previously, we reported multi-silique rapeseed (Brassica napus) line zws-ms. We identified two associated regions and investigated differentially expressed genes (DEGs); thus, some candidate genes underlying the multi-silique phenotype in warm area Xindu were selected. However, this phenotype was switched off by lower temperature, and the responsive genes, known as thermomorphogenesis-related genes, remained elusive. So, based on that, in this study, we further investigated the transcriptome data from buds of zws-ms and its near-isogenic line zws-217 grown in colder area Ma'erkang, where both lines showed normal siliques only, and the DEGs between them analyzed. We compared the 129 DEGs from Xindu to the 117 ones from Ma'erkang and found that 33 of them represented the same or similar expression trends, whereas the other 96 DEGs showed different expression trends, which were defined as environment-specific. Furthermore, we combined this with the gene annotations and ortholog information and then selected BnaA09g45320D (chaperonin gene CPN10-homologous) and BnaC08g41780D [Seryl-tRNA synthetase gene OVULE ABORTION 7 (OVA7)-homologous] the possible thermomorphogenesis-related genes, which probably switched off the multi-silique under lower temperature. This study paves a way to a new perspective into flower/fruit development in Brassica plants.

Improving maize's N uptake and N use efficiency by strengthening roots' absorption capacity when intercropped with legumes.

Maize's nitrogen (N) uptake can be improved through maize-legume intercropping. N uptake mechanisms require further study to better understand how legumes affect root growth and to determine maize's absorptive capacity in maize-legume intercropping. We conducted a two-year field experiment with two N treatments (zero N (N0) and conventional N (N1)) and three planting patterns (monoculture maize (Zea mays L.) (MM), maize-soybean (Glycine max L. Merr.) strip intercropping (IMS), and maize-peanut (Arachis hypogaea L.) strip intercropping (IMP)). We sought to understand maize's N uptake mechanisms by investigating root growth and distribution, root uptake capacity, antioxidant enzyme activity, and the antioxidant content in different maize-legume strip intercropping systems. Our results showed that on average, the N uptake of maize was significantly greater by 52.5% in IMS and by 62.4% in IMP than that in MM. The average agronomic efficiency (AE) of maize was increased by 110.5 % in IMS and by 163.4 % in IMP, compared to MM. The apparent recovery efficiency (RE) of maize was increased by 22.3% in IMS. The roots of intercropped maize were extended into soybean and peanut stands underneath the space and even between the inter-rows of legume, resulting in significantly increased root surface area density (RSAD) and total root biomass. The root-bleeding sap intensity of maize was significantly increased by 22.7-49.3% in IMS and 37.9-66.7% in IMP, compared with the MM. The nitrate-N content of maize bleeding sap was significantly greater in IMS and IMP than in MM during the 2018 crop season. The glutathione (GSH) content, superoxide dismutase (SOD), and catalase (CAT) activities in the root significantly increased in IMS and IMP compared to MM. Strip intercropping using legumes increases maize's aboveground N uptake by promoting root growth and spatial distribution, delaying root senescence, and strengthening root uptake capacity.

Detection of Blackleg Resistance Gene Rlm1 in Double-Low Rapeseed Accessions from Sichuan Province, by Kompetitive Allele-Specific PCR.

Blackleg is a serious disease in Brassica plants, causing moderate to severe yield losses in rapeseed worldwide. Although China has not suffered from this disease yet (more aggressive Leptosphaeria maculans is not present yet), it is crucial to take provisions in breeding for disease resistance to have excellent blackleg-resistant cultivars already in the fields or in the breeding pipeline. The most efficient strategy for controlling this disease is breeding plants with identified resistance genes. We selected 135 rapeseed accessions in Sichuan, including 30 parental materials and 105 hybrids, and we determined their glucosinolate and erucic acid content and confirmed 17 double-low materials. A recently developed single-nucleotide polymorphism (SNP) marker, SNP_208, was used to genotype allelic Rlm1/rlm1 on chromosome A07, and 87 AvrLm1-resistant materials. Combined with the above-mentioned seed quality data, we identified 11 AvrLm1-resistant double-low rapeseed accessions, including nine parental materials and two hybrids. This study lays the foundation of specific R gene-oriented breeding, in the case that the aggressive Leptosphaeria maculans invades and establishes in China in the future and a robust and less labor consuming method to identify resistance in canola germplasm.

Diethyl Aminoethyl Hexanoate Increase Relay Strip Intercropping Soybean Grain by Optimizing Photosynthesis Aera and Delaying Leaf Senescence.

Insufficient and unbalanced biomass supply inhibited soybean [Glycine max (L.) Merr.] yield formation in the maize-soybean relay strip intercropping (IS) and monoculture soybean (SS). A field experiment was conducted to explore the soybean yield increase mechanism of DA-6 in IS and SS treatments. In this 2-year experiment, compact maize "Denghai 605" and shade-tolerant soybean "Nandou 25" were selected as cultivated materials. DA-6 with four concentrations, i.e., 0 mg/L (CK), 40 mg/L (D40), 60 mg/L (D60), and 80 mg/L (D80), were sprayed on soybean leaves at the beginning of flowering stage of soybean. Results showed that DA-6 treatments significantly (p < 0.05) increased soybean grain yield, and the yield increase ratio was higher in IS than SS. The leaf area index values and net photosynthesis rate of IS peaked at D60 and were increased by 32.2-49.3% and 24.1-27.2% compared with the corresponding CK. Similarly, DA-6 treatments increased the aboveground dry matter and the amount of soybean dry matter accumulation from the R1 stage to the R8 stage (VDMT) and highest at D60 both in IS and SS. D60 increased the VDMT by 29.0-47.1% in IS and 20.7-29.2% in SS. The TR G at D60 ranged 72.4-77.6% in IS and 61.4-62.5% in SS. The MDA content at D60 treatment was decreased by 38.3% in IS and 25.8% in SS. The active grain-filling day in IS was about 7 days longer than in SS. In D60 treatment, the Vmean and Vmax increased by 6.5% and 6.5% in IS and 5.7% and 4.3% in SS compared with the corresponding CK. Although the pod number and hundred-grain weight were significantly (p < 0.05) increased by DA-6 treatments, the grains per pod were maintained stable. The pod number and hundred-grain weight were increased by 30.1-36.8% and 4.5-6.7% in IS and 6.3-13% and 3.6-5.6% in SS. Thus, the grain yield at D60 was increased by 36.7-38.4% in IS and 21.7-26.6% in SS. DA-6 treatments significantly (p < 0.05) increased soybean grain yield and peaked D60 treatments both in IS and SS.

Investigation for a multi-silique trait in Brassica napus by alternative splicing analysis.

BACKGROUND: Flower and fruit development are vital stages of the angiosperm lifecycle. We previously investigated the multi-silique trait in the rapeseed (Brassica napus) line zws-ms on a genomic and transcriptomic level, leading to the identification of two genomic regions and several candidate genes associated with this trait. However, some events on the transcriptome level, like alternative splicing, were poorly understood. METHODS: Plants from zws-ms and its near-isogenic line (NIL) zws-217 were both grown in Xindu with normal conditions and a colder area Ma'erkang. Buds from the two lines were sampled and RNA was isolated to perform the transcriptomic sequencing. The numbers and types of alternative splicing (AS) events from the two lines were counted and classified. Genes with AS events and expressed differentially between the two lines, as well as genes with AS events which occurred in only one line were emphasized. Their annotations were further studied. RESULTS: From the plants in Xindu District, an average of 205,496 AS events, which could be sorted into five AS types, were identified. zws-ms and zws-217 shared highly similar ratios of each AS type: The alternative 5' and 3' splice site types were the most common, while the exon skipping type was observed least often. Eleven differentially expressed AS genes were identified, of which four were upregulated and seven were downregulated in zws-ms. Their annotations implied that five of these genes were directly associated with the multi-silique trait. While samples from colder area Ma'erkang generated generally reduced number of each type of AS events except for Intron Retention; but the number of differentially expressed AS genes increased significantly. Further analysis found that among the 11 differentially expressed AS genes from Xindu, three of them maintained the same expression models, while the other eight genes did not show significant difference between the two lines in expression level. Additionally, the 205 line-specific expressed AS genes were analyzed, of which 187 could be annotated, and two were considered to be important. DISCUSSION: This study provides new insights into the molecular mechanism of the agronomically important multi-silique trait in rapeseed on the transcriptome level and screens out some environment-responding candidate genes.

Fine Mapping of a Locus Underlying the Ectopic Blade-Like Outgrowths on Leaf and Screening Its Candidate Genes in Rapeseed (Brassica napus L.).

Leaf is an important organ for higher plants, and the shape of it is one of the crucial traits of crops. In this study, we investigated a unique aberrant leaf morphology trait in a mutational rapeseed material, which displayed ectopic blade-like outgrowths on the adaxial side of leaf. The abnormal line 132000B-3 was crossed with the normal line 827-3. Based on the F2 : 3 family, we constructed two DNA pools (normal pool and abnormal pool) by the bulked segregant analysis (BSA) method and performed whole genome re-sequencing (WGR), obtaining the single-nucleotide polymorphism (SNP) and insertion/deletion (InDel) data. The SNP-index method was used to calculate the Δ(SNP/InDel-index), and then an association region was identified on chromosome A10 with a length of 5.5 Mbp, harboring 1048 genes totally. Subsequently, the fine mapping was conducted by using the penta-primer amplification refractory mutation system (PARMS), and the associated region was narrowed down to a 35.1-kbp segment, containing only seven genes. These seven genes were then analyzed according to their annotations and finally, BnA10g0422620 and BnA10g0422610, orthologs of LATE MERISTEM IDENTITY1 (LMI1) gene from Arabidopsis and REDUCED COMPLEXITY (RCO) gene from its relative Cardamine hirsuta, respectively, were identified as the candidate genes responding to this blade-like outgrowth trait in rapeseed. This study provides a novel perspective into the leaf formation in Brassica plants.

Identification of genomic regions associated with multi-silique trait in Brassica napus.

BACKGROUND: Although rapeseed (Brassica napus L.) mutant forming multiple siliques was morphologically described and considered to increase the silique number per plant, an important agronomic trait in this crop, the molecular mechanism underlying this beneficial trait remains unclear. Here, we combined bulked-segregant analysis (BSA) and whole genome re-sequencing (WGR) to map the genomic regions responsible for the multi-silique trait using two pools of DNA from the near-isogenic lines (NILs) zws-ms (multi-silique) and zws-217 (single-silique). We used the Euclidean Distance (ED) to identify genomic regions associated with this trait based on both SNPs and InDels. We also conducted transcriptome sequencing to identify differentially expressed genes (DEGs) between zws-ms and zws-217. RESULTS: Genetic analysis using the ED algorithm identified three SNP- and two InDel-associated regions for the multi-silique trait. Two highly overlapped parts of the SNP- and InDel-associated regions were identified as important intersecting regions, which are located on chromosomes A09 and C08, respectively, including 2044 genes in 10.20-MB length totally. Transcriptome sequencing revealed 129 DEGs between zws-ms and zws-217 in buds, including 39 DEGs located in the two abovementioned associated regions. We identified candidate genes involved in multi-silique formation in rapeseed based on the results of functional annotation. CONCLUSIONS: This study identified the genomic regions and candidate genes related to the multi-silique trait in rapeseed.

Genome-Wide Identification of Flowering-Time Genes in Brassica Species and Reveals a Correlation between Selective Pressure and Expression Patterns of Vernalization-Pathway Genes in Brassica napus.

Flowering time is a key agronomic trait, directly influencing crop yield and quality. Many flowering-time genes have been identified and characterized in the model plant Arabidopsis thaliana; however, these genes remain uncharacterized in many agronomically important Brassica crops. In this study, we identified 1064, 510, and 524 putative orthologs of A. thaliana flowering-time genes from Brassica napus, Brassica rapa, and Brassica oleracea, respectively, and found that genes involved in the aging and ambient temperature pathways were fewer than those in other flowering pathways. Flowering-time genes were distributed mostly on chromosome C03 in B. napus and B. oleracea, and on chromosome A09 in B. rapa. Calculation of non-synonymous (Ka)/synonymous substitution (Ks) ratios suggested that flowering-time genes in vernalization pathways experienced higher selection pressure than those in other pathways. Expression analysis showed that most vernalization-pathway genes were expressed in flowering organs. Approximately 40% of these genes were highly expressed in the anther, whereas flowering-time integrator genes were expressed in a highly organ-specific manner. Evolutionary selection pressures were negatively correlated with the breadth and expression levels of vernalization-pathway genes. These findings provide an integrated framework of flowering-time genes in these three Brassica crops and provide a foundation for deciphering the relationship between gene expression patterns and their evolutionary selection pressures in Brassica napus.

Genome-wide identification, putative functionality and interactions between lncRNAs and miRNAs in Brassica species.

Non-coding RNA (ncRNA) is abundant in plant genomes, but is poorly described with unknown functionality in most species. Using whole genome RNA sequencing, we identified 1885, 1910 and 1299 lncRNAs and 186, 157 and 161 miRNAs at the whole genome level in the three Brassica species B. napus, B. oleracea and B. rapa, respectively. The lncRNA sequences were divergent between the three Brassica species. One quarter of lncRNAs were located in tandem repeat (TR) region. The expression of both lncRNAs and miRNAs was strongly biased towards the A rather than the C subgenome in B. napus, unlike mRNA expression. miRNAs in genic regions had higher average expression than miRNAs in non-genic regions in B. napus and B. oleracea. We provide a comprehensive reference for the distribution, functionality and interactions of lncRNAs and miRNAs in Brassica.

Transcriptome Analysis of Interspecific Hybrid between Brassica napus and B. rapa Reveals Heterosis for Oil Rape Improvement.

The hybrid between Brassica napus and B. rapa displays obvious heterosis in both growth performance and stress tolerances. A comparative transcriptome analysis for B. napus (A(n)A(n)CC genome), B. rapa (A(r)A(r) genome), and its hybrid F1 (A(n)A(r)C genome) was carried out to reveal the possible molecular mechanisms of heterosis at the gene expression level. A total of 40,320 nonredundant unigenes were identified using B. rapa (AA genome) and B. oleracea (CC genome) as reference genomes. A total of 6,816 differentially expressed genes (DEGs) were mapped in the A and C genomes with 4,946 DEGs displayed nonadditively by comparing the gene expression patterns among the three samples. The coexistence of nonadditive DEGs including high-parent dominance, low-parent dominance, overdominance, and underdominance was observed in the gene action modes of F1 hybrid, which were potentially related to the heterosis. The coexistence of multiple gene actions in the hybrid was observed and provided a list of candidate genes and pathways for heterosis. The expression bias of transposable element-associated genes was also observed in the hybrid compared to their parents. The present study could be helpful for the better understanding of the determination and regulation of mechanisms of heterosis to aid Brassica improvement.