Paternal chromosome elimination of inducer triggers induction of double haploids in Brassica napus

A synthetic octoploid rapeseed, Y3380, induces maternal doubled haploids when used as a pollen donor to pollinate plant. However, the mechanism underlying doubled haploid formation remains elusive. We speculated that double haploid induction occurs as the inducer line’s chromosomes pass to the maternal egg cell, and the zygote is formed through fertilization. In the process of zygotic mitosis, the paternal chromosome is specifically eliminated. Part of the paternal gene might have infiltrated the maternal genome through homologous exchange during the elimination process. Then, the zygote haploid genome doubles (early haploid doubling, EH phenomenon), and the doubled zygote continues to develop into a complete embryo, finally forming doubled haploid offspring. To test our hypothesis, in the current study, the octoploid Y3380 line was back bred with the 4122-cp4-EPSPS exogenous gene used as a marker into hexaploid Y3380-cp4-EPSPS as paternal material to pollinate three different maternal materials. The fertilization process of crossing between the inducer line and the maternal parent was observed 48 h after pollination, and the fertilization rate reached 97.92% and 98.72%. After 12 d of pollination, the presence of cp4-EPSPS in the embryo was detected by in situ PCR, and at 13–23 d after pollination, the probability of F1 embryos containing cp4-EPSPS gene was up to 97.27%, but then declined gradually to 0% at 23–33 d. At the same time, the expression of cp4-EPSPS was observed by immunofluorescence in the 3rd to 29th day embryo. As the embryos developed, cp4-EPSPS marker genes were constantly lost, accompanied by embryonic death. After 30 d, the presence of cp4-EPSPS was not detected in surviving embryos. Meanwhile, SNP detection of induced offspring confirmed the existence of double haploids, further indicating that the induction process was caused by the loss of specificity of the paternal chromosome. The tetraploid-induced offspring showed infiltration of the induced line gene loci, with heterozygosity and homozygosity. Results indicated that the induced line chromosomes were eliminated during embryonic development, and the maternal haploid chromosomes were synchronously doubled in the embryo. These findings support our hypothesis and lay a theoretical foundation for further localization or cloning of functional genes involved in double haploid induction in rapeseed

Responses to shade and subsequent recovery of soya bean in maize-soya bean relay strip intercropping

In relay intercropping systems, late-planted crops often grow under the shade of the canopy of early-planted tall crops and then transfer to full sunlight after the harvest of the early-planted crops. In order to know the effects of recovery growth of the late-planted soya bean in maize–soya bean relay intercropping, a field experiment was carried out to observe architectural, morphological, physiological and anatomical traits of soya bean plants related to shade and subsequent removal in intercropping before and after maize harvest, respectively. During shade period, soya bean biomass was severely reduced, and stem elongation was stimulated. Typical features of shade grown leaves were found, such as lower LMA (leaf mass per unit area), thinner thickness, higher chlorophyll content, lower chlorophyll a:b ratio. Whole-plant leaf area analysis found that soya bean increased leaf area ratio by adjusting leaf morphology rather than by dry mass allocation. After maize harvest, leaf area and leaf mass increased rapidly, contributing to compensation growth in intercropped soya bean. Meanwhile, physiological and anatomical traits of leaf went back to similar levels as grown in sole cropping. However, stem morphological traits were irreversible after removal of shade. Finally, no difference on seed weight per plant of soya bean was observed between relay intercropping and sole cropping. Based on these findings, we speculated the recovery growth might be the direct determining factor on pod formation in soya bean, and improvement on the capacity of recovery growth could increase yield of relay intercropped soya bean

Seed Treatment with Uniconazole Powder Improves Soybean Seedling Growth under Shading by Corn in Relay Strip Intercropping System

The relay strip intercropping system of wheat-corn-soybean is widely used in southwest China. However, it is hard to obtain a stable production of soybean with this system, since soybean plants grow under shading by corn; the stems are thinner and susceptible to lodging. We examined the effects of seed treatment with uniconazole powder (0, 2, 4 and 8 mg kg-1 seed) on the growth of soybean seedlings under relay strip intercropping, some morphological characteristics and yield. The seedling height, first internode length, cotyledonary node height and leaf area per plant were decreased, while the stem diameter, root dry weight, shoot dry weight, root volume, leaf greenness and root to shoot dry weight ratio were increased by uniconazole treatment. The root vigor and root active absorption area were also increased significantly by uniconazole treatment. Moreover, 2 and 4 mg kg-1 uniconazole powder treatment increased shoot dry weight, number of pods per plant, number of seeds per pod and seed yield significantly. Thus, the results suggested that seed treatment with uniconazole powder at a suitable concentration can improve soybean seedling growth, resist the lodging and also increase the seed yield under shading by corn in relay strip intercropping system

Photosynthetic Acclimation of Shade-Grown Soybean Seedlings to a High-Light Environment

Soybean in relay intercropping is initially exposed to a shade environment, followed by exposure to full sunlight after the harvesting of primary crops, e.g., maize. Therefore, soybean’s ability to acclimate to this changing light environment determines its growth and yield formation. However, the changes in soybean photosynthesis under such light alternations in relay intercropping are poorly understood. This study compared the photosynthetic acclimation of two soybean varieties with contrasting shade tolerance, i.e., Gongxuan1 (shade-tolerant) and C103 (shade-intolerant). The two soybean genotypes were grown in a greenhouse under full sunlight (HL) and 40% full sunlight (LL) conditions. Subsequently, after the fifth compound leaf expanded, half of the LL plants were transferred to a high-sunlight environment (LL-HL). Morphological traits were measured at 0 and 10 days, while chlorophyll content, gas exchange characteristics and chlorophyll fluorescence were assayed at 0, 2, 4, 7 and 10 days after transfer to an HL environment (LL-HL). Shade-intolerant C103 showed photoinhibition 10 days after transfer, and the net photosynthetic rate (Pn) did not completely recover to that under a high light level. On the day of transfer, the shade-intolerant variety, C103, exhibited a decrease in net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (E) in the low-light (LL) and low-light-to-high-light (LL-HL) treatments. Additionally, intercellular CO2 concentration (Ci) increased in low light, suggesting that non-stomatal factors were the primary limitations to photosynthesis in C103 following the transfer. In contrast, the shade-tolerant variety, Gongxuan1, displayed a greater increase in Pn 7 days after transfer, with no difference observed between the HL and LL-HL treatments. Ten days after transfer, the shade-tolerant Gongxuan1 exhibited 24.1%, 10.9% and 20.9% higher biomass, leaf area and stem diameter than the intolerant C103. These findings suggest that Gongxuan1 possesses a higher capacity to adapt to variations in light conditions, making it a potential candidate for variety selection in intercropping systems

Rapid Creation of Interspecific Hybrid Progeny to Broaden Genetic Distance through Double Haploid (DH) Inducer in Brassica napus

Interspecific hybridization of rapeseed is an important way to innovate breeding resources. This research used Brassica napus and Brassica rapa for artificial synthesis interspecific hybridization of F1. The F1 self-fruiting rate was particularly low. By comparing the fertilization rate and seed setting rate of nine crosses and selfing combinations of interspecific hybrid progeny F1 and control B. napus, the results proved that the genetic stability of egg cells was greater than that of sperm cells, so the F1 could get seed by artificial pollination with other normal pollen. Based on these results, interspecific maternal inbred offspring (induced F1) from egg cells was obtained by emasculation and pollination with the pollen of DH inducer Y3380. It was found through morphological analysis, flow cytometry identification, and meiotic observation of induced F1, the plants had most normal fertile tetraploid and the meiosis was normal. The FISH results showed that the induced F1 were B. napus (2n = 4x = 38, AACC), 20 A and 19 C chromosomes. The results of SNP chip detection and genetic cluster analysis found that the genetic variation between interspecies could be preserved or broadened in the induced F1. The use of DH inducer created special breeding resources for interspecific hybridization and distant hybridization of rapeseed while shortening time, improving efficiency, and providing a new insight into innovate breeding resources

Data_Sheet_1_Morphological and physiological variation of soybean seedlings in response to shade.docx

Soybean (Glycine max) is a legume species that is widely used in intercropping. Quantitative analyses of plasticity and genetic differences in soybean would improve the selection and breeding of soybean in intercropping. Here, we used data of 20 varieties from one year artificial shading experiment and one year intercropping experiment to characterize the morphological and physiological traits of soybean seedlings grown under shade and full sun light conditions. Our results showed that shade significantly decreased biomass, leaf area, stem diameter, fraction of dry mass in petiole, leaf mass per unit area, chlorophyll a/b ratio, net photosynthetic rate per unit area at PAR of 500 μmol m–2 s–1 and 1,200 μmol m–2 s–1 of soybean seedling, but significantly increased plant height, fraction of dry mass in stem and chlorophyll content. Light × variety interaction was significant for all measured traits, light effect contributed more than variety effect. The biomass of soybean seedlings was positively correlated with leaf area and stem diameter under both shade and full sunlight conditions, but not correlated with plant height and net photosynthetic rate. The top five (62.75% variation explained) most important explanatory variables of plasticity of biomass were that the plasticity of leaf area, leaf area ratio, leaflet area, plant height and chlorophyll content, whose total weight were 1, 0.9, 0.3, 0.2, 0.19, respectively. The plasticity of biomass was positively correlated with plasticity of leaf area and leaflet area but significant negative correlated with plasticity of plant height. The principal component one account for 42.45% variation explain. A cluster analysis further indicated that soybean cultivars were classified into three groups and cultivars; Jiandebaimaodou, Gongdou 2, and Guixia 3 with the maximum plasticity of biomass. These results suggest that for soybean seedlings grown under shade increasing the capacity for light interception by larger leaf area is more vital than light searching (plant height) and light conversion (photosynthetic rate).</p

Modelling soybean and maize growth and grain yield in strip intercropping systems with different row configurations

Intercropping of two or more crop species increases the efficiency of resource use and often produces a greater yield per unit land area. The relative efficiency of intercropping depends on row configuration, but there is a shortage of modelling-based evaluation of alternative intercropping options due to the inadequacy of standard process-based crop models to simulate resource capture, growth and yield formation when the canopy is spatially structured in strips. We implemented a light interception model for strip crops into the APSIM Classic model and combined it with a quasi-Bayesian approach to derive the model parameters to simulate crop growth and grain yield in maize-soybean strip intercropping. We used 4 years of field data for 5 different row configurations to derive key model parameters for simulation of light interception, LAI dynamics, biomass growth and grain yield of maize and soybean intercrops. Key model parameters (e.g. RUE, k etc.) were found to change with row-spacing and configuration, posing challenges to simulate different configurations with a single parameter set. The potential ranges of these key parameters were derived by constraining the model to observed data. The model can be potentially used to evaluate impact of planting configurations on productivity of strip intercropping systems, but the variability of key model parameters among configuration treatments calls for further in-depth research to improve modelling physiology of strip intercrops.</p

Transcriptome Analysis of Shade-Induced Inhibition on Leaf Size in Relay Intercropped Soybean

<div><p>Multi-species intercropping is a sustainable agricultural practice worldwide used to utilize resources more efficiently. In intercropping systems, short crops often grow under vegetative shade of tall crops. Soybean, one important legume, is often planted in intercropping. However, little is known about the mechanisms of shade inhibition effect on leaf size in soybean leaves at the transcriptome level. We analyzed the transcriptome of shaded soybean leaves via RNA-Seq technology. We found that transcription 1085 genes in mature leaves and 1847 genes in young leaves were significantly affected by shade. Gene ontology analyses showed that expression of genes enriched in polysaccharide metabolism was down-regulated, but genes enriched in auxin stimulus were up-regulated in mature leaves; and genes enriched in cell cycling, DNA-replication were down-regulated in young leaves. These results suggest that the inhibition of higher auxin content and shortage of sugar supply on cell division and cell expansion contribute to smaller and thinner leaf morphology, which highlights potential research targets such as auxin and sugar regulation on leaves for crop adaptation to shade in intercropping.</p></div

Functional categorization of genes showing differential transcription patterns in response to shade in mature and young leaves.

<p>Functional categorization was performed using AgriGO. (A) Functional categorization of differentially transcribed genes in mature leaves; (B) Functional categorization of differentially transcribed genes in young leaves. Blue and green columns represent query (input) and reference lists, respectively.</p

Correlation between the RNA-Seq and qRT-PCR data on identified differentially transcribed genes.

<p>Data are log₂FC values from RNA-Seq and qPCR analyses. 24 genes were randomly selected for comparison between RNA-Seq and qRT-PCR. Filled circles represent data in mature leaves, open circles represent data in young leaves. The overall Spearman correlation of mature and young leaves is 0.910 (<i>p</i><0.001).</p