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.

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.

Morphological and physiological variation of soybean seedlings in response to shade.

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).

Rapid and Synchronous Breeding of Cytoplasmic Male Sterile and Maintainer Line Through Mitochondrial DNA Rearrangement Using Doubled Haploid Inducer in Brassica napus.

When homozygously fertile plants were induced using doubled haploid (DH) induction lines Y3380 and Y3560, the morphology of the induced F1 generation was basically consistent with the female parent, but the fertility was separated, showing characteristics similar to cytoplasmic male sterile (CMS) and maintainer lines. In this study, the morphology, fertility, ploidy, and cytoplasm genotype of the induced progeny were identified, and the results showed that the sterile progeny was polima cytoplasm sterile (pol CMS) and the fertile progeny was nap cytoplasm. The molecular marker and test-cross experimental results showed that the fertile progeny did not carry the restorer gene of pol CMS and the genetic distance between the female parent and the offspring was 0.002. This suggested that those inductions which produced sterile and fertile progeny were coordinated to CMS and maintainer lines. Through the co-linearity analysis of the mitochondrial DNA (mtDNA), it was found that the rearrangement of mtDNA by DH induction was the key factor that caused the transformation of fertility (nap) into sterility (pol). Also, when heterozygous females were induced with DH induction lines, the induction F2 generation also showed the segregation of fertile and sterile lines, and the genetic distance between sterile and fertile lines was approximately 0.075. Therefore, the induction line can induce different types of female parents, and the breeding of the sterile line and the maintainer line can be achieved through the rapid synchronization of sister crosses and self-crosses. The induction of DH inducer in B. napus can provide a new model for the innovation of germplasm resources and open up a new way for its application.

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.

Genetic characteristics and ploidy trigger the high inducibility of double haploid (DH) inducer in Brassica napus.

BACKGROUND: Our recently reported doubled haploid (DH) induction lines e.g., Y3380 and Y3560 are allo-octoploid (AAAACCCC, 2n = 8× ≈ 76), which can induce the maternal parent to produce DH individuals. Whether this induction process is related to the production of aneuploid gametes form male parent and genetic characteristics of the male parent has not been reported yet. RESULTS: Somatic chromosome counts of DH inducer parents, female wax-less parent (W1A) and their F1 hybrid individuals revealed the reliability of flow cytometry analysis. Y3560 has normal chromosome behavior in metaphase I and anaphase I, but chromosome division was not synchronized in the tetrad period. Individual phenotypic identification and flow cytometric fluorescence measurement of F1 individual and parents revealed that DH individuals can be distinguished on the basis of waxiness trait. The results of phenotypic identification and flow cytometry can identify the homozygotes or heterozygotes of F1 generation individuals. The data of SNP genotyping coupled with phenotypic waxiness trait revealed that the genetic distance between W1A and F1 homozygotes were smaller as compared to their heterozygotes. It was found that compared with allo-octoploids, aneuploidy from allo-octoploid segregation did not significantly increase the DH induction rate, but reduced male infiltration rate and heterozygous site rate of induced F1 generation. The ploidy, SNP genotyping and flow cytometry results cumulatively shows that DH induction is attributed to the key genes regulation from the parents of Y3560 and Y3380, which significantly increase the induction efficiency as compared to ploidy. CONCLUSION: Based on our findings, we hypothesize that genetic characteristics and aneuploidy play an important role in the induction of DH individuals in Brassca napus, and the induction process has been explored. It provides an important insight for us to locate and clone the genes that regulate the inducibility in the later stage.

Maternal karyogene and cytoplasmic genotype affect the induction efficiency of doubled haploid inducer in Brassica napus.

BACKGROUND: Artificial synthesis of octoploid rapeseed double haploid (DH) induction lines Y3380 and Y3560 was made possible by interspecific hybridization and genome doubling techniques. Production of pure lines by DH induction provides a new way to achieve homozygosity earlier in B.napus. Previously, the mechanism of induction, and whether the induction has obvious maternal genotypic differences or not, are not known so far. RESULTS: In this study, different karyogene and cytoplasmic genotype of B.napus were pollinated with the previously reported DH inducers e.g. Y3380 and Y3560. Our study presents a fine comparison of different cytoplasmic genotypes hybridization to unravel the mechanism of DH induction. Ploidy identification, fertility and SSR marker analysis of induced F1 generation, revealed that ploidy and phenotype of the induced F1 plants were consistent with that type of maternal, rather than paternal parent. The SNP chip analysis revealed that induction efficiency of DH inducers were affected by the karyogene when the maternal cytoplasmic genotypes were the same. However, DH induction efficiency was also affected by cytoplasmic genotype when the karyogenes were same, and the offspring of the ogura cytoplasm showed high frequency inducer gene hybridization or low-frequency infiltration. CONCLUSION: The induction effect is influenced by the interaction between maternal karyogene and cytoplasmic genotype, and the results from the partial hybridization of progeny chromosomes indicate that the induction process may be attributed to the selective elimination of paternal chromosome. This study provides a basis for exploring the mechanism of DH inducer in B.napus, and provides new insights for utilization of inducers in molecular breeding.

Leaf area and photosynthesis of newly emerged trifoliolate leaves are regulated by mature leaves in soybean.

Leaf anatomy and the stomatal development of developing leaves of plants have been shown to be regulated by the same light environment as that of mature leaves, but no report has yet been written on whether such a long-distance signal from mature leaves regulates the total leaf area of newly emerged leaves. To explore this question, we created an investigation in which we collected data on the leaf area, leaf mass per area (LMA), leaf anatomy, cell size, cell number, gas exchange and soluble sugar content of leaves from three soybean varieties grown under full sunlight (NS), shaded mature leaves (MS) or whole plants grown in shade (WS). Our results show that MS or WS cause a marked decline both in leaf area and LMA in newly developing leaves. Leaf anatomy also showed characteristics of shade leaves with decreased leaf thickness, palisade tissue thickness, sponge tissue thickness, cell size and cell numbers. In addition, in the MS and WS treatments, newly developed leaves exhibited lower net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (E), but higher carbon dioxide (CO 2 ) concentration in the intercellular space (Ci) than plants grown in full sunlight. Moreover, soluble sugar content was significantly decreased in newly developed leaves in MS and WS treatments. These results clearly indicate that (1) leaf area, leaf anatomical structure, and photosynthetic function of newly developing leaves are regulated by a systemic irradiance signal from mature leaves; (2) decreased cell size and cell number are the major cause of smaller and thinner leaves in shade; and (3) sugars could possibly act as candidate signal substances to regulate leaf area systemically.

Shade Inhibits Leaf Size by Controlling Cell Proliferation and Enlargement in Soybean.

To gain more insight into the physiological function of shade and how shade affects leaf size, we investigated the growth, leaf anatomical structure, hormones and genes expressions in soybean. Soybean seeds were sown in plastic pots and were allowed to germinate and grow for 30 days under shade or full sunlight conditions. Shade treated plants showed significantly increase on stem length and petiole length, and decrease on stem diameters, shoot biomass and its partition to leaf also were significantly lower than that in full sunlight. Smaller and thinner on shade treated leaves than corresponding leaves on full sunlight plants. The decreased leaf size caused by shade was largely attributable to cell proliferation in young leaves and both cell proliferation and enlargement in old leaves. Shade induced the expression of a set of genes related to cell proliferation and/or enlargement, but depended on the developmental stage of leaf. Shade significantly increased the auxin and gibberellin content, and significantly decreased the cytokinin content in young, middle and old leaves. Taken together, these results indicated that shade inhibited leaf size by controlling cell proliferation and enlargement, auxin, gibberellin and cytokinin may play important roles in this process.

Transcriptome analysis of shade-induced inhibition on leaf size in relay intercropped soybean.

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.