Effects of plant density on alfalfa (Medicago sativa L.) seed yield in western Heilongjiang areas.

Alfalfa (Medicago sativa L.) is known as the "king of forages". The aim of the current study is to determine the optimum planting density as the key cultivation technique for high yield of alfalfa seed. Alfalfa variety (Longmu 801) was planted in experimental fields from 2014 to 2017. In the planting density test, the row spacing was 65, 80, and 95 cm, and the plant spacing was 30, 45, 60, 75, and 90 cm. The seed yield and yield components in the row spacing and plant spacing tests were measured. On the basis of 3 years average of the experimental data, the highest seed yield of 225.49 kg ha-1 was obtained with row spacing vs plant spacing of 65 and 60 cm, respectively. Correlation analysis showed a significant positive correlation between the racemes per stem, pods per raceme, pods per stem, seeds per pod, and the seed yield. These results suggested that Longmu 801 should be cultivated with 65 cm row spacing and 60 cm plant spacing to maximize seed yields in western Heilongjiang areas.

Global analysis of seed transcriptomes reveals a novel PLATZ regulator for seed size and weight control in soybean.

Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.

GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean.

Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.

OsBBX11 on qSTS4 links to salt tolerance at the seeding stage in Oryza sativa L. ssp. Japonica.

Rice has been reported to be highly sensitive to salt stress at the seedling stage. However, the lack of target genes that can be used for improving salt tolerance has resulted in several saline soils unsuitable for cultivation and planting. To characterize new salt-tolerant genes, we used 1,002 F2:3 populations derived from Teng-Xi144 and Long-Dao19 crosses as the phenotypic source to systematically characterize seedlings' survival days and ion concentration under salt stress. Utilizing QTL-seq resequencing technology and a high-density linkage map based on 4,326 SNP markers, we identified qSTS4 as a major QTL influencing seedling salt tolerance, which accounted for 33.14% of the phenotypic variation. Through functional annotation, variation detection and qRT-PCR analysis of genes within 46.9 Kb of qSTS4, it was revealed that there was one SNP in the promoter region of OsBBX11, which resulted in a significant response difference between the two parents to salt stress. Transgenic plants using knockout-based technology and demonstrated that Na+ and K+ in the roots of the functional-loss-type OsBBX11 were translocated largely to the leaves under 120 mmol/L NaCl compared with the wild-type, causing osbbx11 leaves to die after 12 days of salt stress due to an imbalance in osmotic pressure. In conclusion, this study identified OsBBX11 as a salt-tolerance gene, and one SNPs in the OsBBX11 promoter region can be used to identify its interacting transcription factors. This provides a theoretical basis for finding the molecular mechanism of OsBBX11 upstream and downstream regulation of salt tolerance and molecular design breeding in the future.

Zinc-finger protein GmZF351 improves both salt and drought stress tolerance in soybean.

Abiotic stress is one of the most important factors reducing soybean yield. It is essential to identify regulatory factors contributing to stress responses. A previous study found that the tandem CCCH zinc-finger protein GmZF351 is an oil level regulator. In this study, we discovered that the GmZF351 gene is induced by stress and that the overexpression of GmZF351 confers stress tolerance to transgenic soybean. GmZF351 directly regulates the expression of GmCIPK9 and GmSnRK, leading to stomata closing, by binding to their promoter regions, which carry two CT(G/C)(T/A)AA elements. Stress induction of GmZF351 is mediated through reduction in the H3K27me3 level at the GmZF351 locus. Two JMJ30-demethylase-like genes, GmJMJ30-1 and GmJMJ30-2, are involved in this demethylation process. Overexpression of GmJMJ30-1/2 in transgenic hairy roots enhances GmZF351 expression mediated by histone demethylation and confers stress tolerance to soybean. Yield-related agronomic traits were evaluated in stable GmZF351-transgenic plants under mild drought stress conditions. Our study reveals a new mode of GmJMJ30-GmZF351 action in stress tolerance, in addition to that of GmZF351 in oil accumulation. Manipulation of the components in this pathway is expected to improve soybean traits and adaptation under unfavorable environments.

Isolation of a Novel QTL, qSCM4, Associated with Strong Culm Affects Lodging Resistance and Panicle Branch Number in Rice.

Rice breeders are now developing new varieties with semi-high or even high plant height to further increase the grain yield, and the problem of lodging has re-appeared. We identified a major quantitative trait locus (QTL), qSCM4, for resistance to lodging by using an F2 segregant population and a recombinant self-incompatible line population from the cross between Shennong265 (SN265) and Lijiangxintuanheigu (LTH) after multiple years and multiple environments. Then, the residual heterozygous derived segregant population which consisted of 1781 individual plants, and the BC3F2 segregant population which consisted of 3216 individual plants, were used to shorten the physical interval of qSCM4 to 58.5 kb including 11 genes. DNA sequencing revealed the most likely candidate gene for qSCM4 was Os04g0615000, which encoded a functional protein with structural domains of serine and cysteine. There were 13 DNA sequence changes in LTH compared to SN265 in this gene, including a fragment deletion, two base changes in the 3' UTR region, six base changes in the exons, and four base changes in the introns. A near-isogenic line carrying qSCM4 showed that it improved the lodging resistance through increasing stem thickness by 25.3% and increasing stem folding resistance by 20.3%. Furthermore, it was also discovered that qSCM4 enhanced the primary branch per panicle by 16.7%, secondary branch by per panicle 9.9%, and grain number per panicle by 14.7%. All the above results will give us a valuable genetic resource for concurrently boosting culm strength and lodging resistance, and they will also provide a basis for further research on the lodging resistance mechanism of rice.

The genetic basis of high-latitude adaptation in wild soybean.

In many plants, flowering time is influenced by daylength as an adaptive response. In soybean (Glycine max) cultivars, however, photoperiodic flowering reduces crop yield and quality in high-latitude regions. Understanding the genetic basis of wild soybean (Glycine soja) adaptation to high latitudes could aid breeding of improved cultivars. Here, we identify the Tof4 (Time of flowering 4) locus, which encodes by an E1-like protein, E1La, that represses flowering and enhances adaptation to high latitudes in wild soybean. Moreover, we found that Tof4 physically associates with the promoters of two important FLOWERING LOCUS T (FT2a and FT5a) and with Tof5 to inhibit their transcription under long photoperiods. The effect of Tof4 on flowering and maturity is mediated by FT2a and FT5a proteins. Intriguingly, Tof4 and the key flowering repressor E1 independently but additively regulate flowering time, maturity, and grain yield in soybean. We determined that weak alleles of Tof4 have undergone natural selection, facilitating adaptation to high latitudes in wild soybean. Notably, over 71.5% of wild soybean accessions harbor the mutated alleles of Tof4 or a previously reported gain-of-function allele Tof5H2, suggesting that these two loci are the genetic basis of wild soybean adaptation to high latitudes. Almost no cultivated soybean carries the mutated tof4 allele. Introgression of the tof4-1 and Tof5H2 alleles into modern soybean or editing E1 family genes thus represents promising avenues to obtain early-maturity soybean, thereby improving productivity in high latitudes.

Combination of Water-Saving Irrigation and Nitrogen Fertilization Regulates Greenhouse Gas Emissions and Increases Rice Yields in High-Cold Regions, Northeast China.

Increased rice production, which benefitted from cropping areas expansion and continuous N applications, resulted in severe increases in greenhouse gases (GHG) emissions from 1983 to 2019 in Heilongjiang Province, China. Therefore, field trials were performed in the high-cold Harbin region, Northeast China, to determine the efficiency of incorporating water regimes with N fertilization in minimizing the impact of rice production on GHG emissions. Two water-saving irrigation strategies, intermittent irrigation (W1) and control irrigation (W2), were used relative to continuous flooding (W0), and we combined them with six fertilized treatments. Our results demonstrated that W1 and W2 significantly decreased seasonal CH4 emissions by 19.7-30.0% and 11.4-29.9%, enhanced seasonal N2O emissions by 77.0-127.0% and 16.2-42.4%, and increased significantly yields by 5.9-12.7% and 0-4.7%, respectively, compared with W0. Although trade-offs occurred between CH4 and N2O emissions, W1 and W2 resulted in significant reductions in global warming potential (GWP). Moreover, low N rates (<120 kg N ha-1) performed better in GWP than high N rates. N fertilization and irrigation regimes had remarkable effects on rice yields and GWP. In conclusion, the incorporation of W1 and a N application under 120 kg N ha-1 could simultaneously mitigate GWP while enhancing production in black soils in high-cold Northeast China.

Genome-wide association study uncovers major genetic loci associated with flowering time in response to active accumulated temperature in wild soybean population.

Flowering time and active accumulated temperature (AAT) are two key factors that limit the expanded production especially for soybean across different regions. Wild soybean provides an important germplasm for functional genomics study in cultivar soybean. However, the studies on genetic basis underlying flowering time in response to AAT especially in wild soybean were rarely reported. In this study, we used 294 wild soybean accessions derived from major soybean production region characterized by different AAT in Northeast of China. Based on genome-wide association study (GWAS), we identified 96 SNPs corresponded to 342 candidate genes that significantly associated with flowering time recorded in two-year experiments. Gene Ontology enrichment analysis suggests that the pathways of photosynthesis light reaction and actin filament binding were significantly enriched. We found three lead SNPs with -log10(p-value) > 32 across the two-year experiments, i.e., Chr02:9490318, Chr04:8545910 and Chr09:49553555. Linkage disequilibrium block analysis shows 28 candidate genes within the genomic region centered on the lead SNPs. Among them, expression levels of three genes (aspartic peptidase 1, serine/threonine-protein kinase and protein SCAR2-like) were significantly differed between two subgroups possessing contrasting flowering time distributed at chromosome 2, 4 and 9, respectively. There are 6, 7 and 3 haplotypes classified on the coding regions of the three genes, respectively. Collectively, accessions with late flowering time phenotype are typically derived from AAT zone 1, which is associated with the haplotypic distribution and expression levels of the three genes. This study provides an insight into a potential mechanism responsible for flowering time in response to AAT in wild soybean, which could promote the understanding of genetic basis for other major crops.

Genome-Wide Association Study of Partial Resistance to P. sojae in Wild Soybeans from Heilongjiang Province, China.

Phytophthora root rot (PRR) is a destructive disease of soybeans (Glycine max (L.) Merr) caused by Phytophthora sojae (P. sojae). The most effective way to prevent the disease is growing resistant or tolerant varieties. Partial resistance provides a more durable resistance against the pathogen compared to complete resistance. Wild soybean (Glycine soja Sieb. & Zucc.) seems to be an extraordinarily important gene pool for soybean improvement due to its high level of genetic variation. In this study, 242 wild soybean germplasms originating from different regions of Heilongjiang province were used to identify resistance genes to P. sojae race 1 using a genome-wide association study (GWAS). A total of nine significant SNPs were detected, repeatedly associated with P. sojae resistance and located on chromosomes 1, 10, 12, 15, 17, 19 and 20. Among them, seven favorable allelic variations associated with P. sojae resistance were evaluated by a t-test. Eight candidate genes were predicted to explore the mechanistic hypotheses of partial resistance, including Glysoja.19G051583, which encodes an LRR receptor-like serine/threonine protein kinase protein, Glysoja.19G051581, which encodes a receptor-like cytosolic serine/threonine protein kinase protein. These findings will provide additional insights into the genetic architecture of P. sojae resistance in a large sample of wild soybeans and P. sojae-resistant breeding through marker-assisted selection.

Intelligent Identification and Features Attribution of Saline-Alkali-Tolerant Rice Varieties Based on Raman Spectroscopy.

Planting rice in saline-alkali land can effectively improve saline-alkali soil and increase grain yield, but traditional identification methods for saline-alkali-tolerant rice varieties require tedious and time-consuming field investigations based on growth indicators by rice breeders. In this study, the Python machine deep learning method was used to analyze the Raman molecular spectroscopy of rice and assist in feature attribution, in order to study a fast and efficient identification method of saline-alkali-tolerant rice varieties. A total of 156 Raman spectra of four rice varieties (two saline-alkali-tolerant rice varieties and two saline-alkali-sensitive rice varieties) were analyzed, and the wave crests were extracted by an improved signal filtering difference method and the feature information of the wave crest was automatically extracted by scipy.signal.find_peaks. Select K Best (SKB), Recursive Feature Elimination (RFE) and Select F Model (SFM) were used to select useful molecular features. Based on these feature selection methods, a Logistic Regression Model (LRM) and Random Forests Model (RFM) were established for discriminant analysis. The experimental results showed that the RFM identification model based on the RFE method reached a higher recognition rate of 89.36%. According to the identification results of RFM and the identification of feature attribution materials, amylum was the most significant substance in the identification of saline-alkali-tolerant rice varieties. Therefore, an intelligent method for the identification of saline-alkali-tolerant rice varieties based on Raman molecular spectroscopy is proposed.

The bZIP Transcription Factor Family in Adzuki Bean (Vigna Angularis): Genome-Wide Identification, Evolution, and Expression Under Abiotic Stress During the Bud Stage.

Adzuki bean (Vigna angularis) is an important dietary legume crop that was first cultivated and domesticated in Asia. Currently, little is known concerning the evolution and expression patterns of the basic leucine zipper (bZIP) family transcription factors in the adzuki bean. Through the PFAM search, 72 bZIP members of adzuki bean (VabZIP) were identified from the reference genome. Most of them were located on 11 chromosomes and seven on an unknown chromosome. A comprehensive analysis, including evolutionary, motifs, gene structure, cis-elements, and collinearity was performed to identify VabZIP members. The subcellular localization results showed VabZIPs might locate on the nuclear. Quantitative real-time PCR (qRT-PCR) analysis of the relative expression of VabZIPs in different tissues at the bud stage revealed that VabZIPs had a tissue-specific expression pattern, and its expression was influenced by abiotic stress. These characteristics of VabZIPs provide insights for future research aimed at developing interventions to improve abiotic stress resistance.

A functionally divergent SOC1 homolog improves soybean yield and latitudinal adaptation.

Soybean (Glycine max) grows in a wide range of latitudes, but it is extremely sensitive to photoperiod, which reduces its yield and ability to adapt to different environments. Therefore, understanding of the genetic basis of soybean adaptation is of great significance for breeding and improvement. Here, we characterized Tof18 (SOC1a) that conditions early flowering and growth habit under both short-day and long-day conditions. Molecular analysis confirmed that the two SOC1 homologs present in soybeans (SOC1a and SOC1b) underwent evolutionary functional divergence, with SOC1a having stronger effects on flowering time and stem node number than SOC1b due to transcriptional differences. soc1a soc1b double mutants showed stronger functional effects than either of the single mutants, perhaps due to the formation of SOC1a and SOC1b homodimers or heterodimers. Additionally, Tof18/SOC1a improves the latitudinal adaptation of cultivated soybeans, highlighting the functional importance of SOC1a. The Tof18G allele facilitates adaptation to high latitudes, whereas Tof18A facilitates adaptation to low latitudes. We demonstrated that SOC1s contribute to floral induction in both leaves and shoot apex through inter-regulation with FTs. The SOC1a-SOC1b-Dt2 complex plays essential roles in stem growth habit by directly binding to the regulatory sequence of Dt1, making the genes encoding these proteins potential targets for genome editing to improve soybean yield via molecular breeding. Since the natural Tof18A allele increases node number, introgressing this allele into modern cultivars could improve yields, which would help optimize land use for food production in the face of population growth and global warming.

Effect of straw retention on carbon footprint under different cropping sequences in Northeast China.

Inappropriate farm management practices can lead to increased agricultural inputs and changes in atmospheric greenhouse gas (GHG) emissions, impacting climate change. This study was initiated in 2012 to assess the potential for straw retention to mitigate the negative environmental impact of various cropping systems on the Songnen Plain using the life cycle assessment (LCA) method combined with field survey data. Straw retention (STR) and straw removal (STM) treatments were established in continuous corn (CC) and corn-soybean rotation (CS) systems in a split-plot experiment. The effects of straw retention on the carbon footprint (CF) of cropland under different cropping systems were compared. The CF under CC was 2434-2707 kg CO2 ha-1 year-1, 49-57% higher than that under CS. Nitrogen fertilizer produced the most CO2, accounting for 66-80% of the CF. The carbon balances of the CC and CS systems with STR were positive, with annual carbon sequestrations of 9633 and 2716 kg CO2 ha-1 year-1, respectively. The carbon balance (CB) of CC-STR was 255% higher than that of CS-STR. This study demonstrates that STR under CC cultivation is an environmentally friendly practice for agricultural production, can help achieve high-yield and low-carbon production in rainfed cropland, and can support the sustainable development of grain production in Northeast China.

A transcriptional regulatory module controls lipid accumulation in soybean.

Soybean (Glycine max) is one of the most important oilseed crops. However, the regulatory mechanism that governs the process of oil accumulation in soybean remains poorly understood. In this study, GmZF392, a tandem CCCH zinc finger (TZF) protein which was identified in our previous RNA-seq analysis of seed-preferred transcription factors, was found to function as a positive regulator of lipid production. GmZF392 promotes seed oil accumulation in both transgenic Arabidopsis and stable transgenic soybean plants by binding to a bipartite cis-element, containing TG- and TA-rich sequences, in promoter regions, activating the expression of genes in the lipid biosynthesis pathway. GmZF392 physically interacts with GmZF351, our previously identified transcriptional regulator of lipid biosynthesis, to synergistically promote downstream gene expression. Both GmZF392 and GmZF351 are further upregulated by GmNFYA, another transcription factor involved in lipid biosynthesis, directly (in the former case) and indirectly (in the latter case). Promoter sequence diversity analysis showed that the GmZF392 promoter may have been selected at the origin of the Glycine genus and further mildly selected during domestication from wild soybeans to cultivated soybeans. Our study reveals a regulatory module containing three transcription factors in the lipid biosynthesis pathway, and manipulation of the module may improve oil production in soybean and other oilseed crops.

Genome-scale analyses and characteristics of putative pathogenicity genes of Stagonosporopsis cucurbitacearum, a pumpkin gummy stem blight fungus.

Outbreaks of gummy stem blight (GSB), an emerging seed pumpkin disease, have increased in number and have become more widespread in recent years. Previously we reported that Stagonosporopsis cucurbitacearum (Sc.) is the dominant fungal cause of pumpkin seedling GSB in Northeast China, where it has greatly reduced crop yields in that region. Here, high-throughput whole-genome sequencing and assembly of the Sc. genome were conducted toward revealing pathogenic molecular regulatory mechanisms involved in fungal growth and development. Zq-1 as representative Sc. strain, DNA of Zq-1was prepared for genomic sequencing, we obtained 5.24 Gb of high-quality genomic sequence data via PacBio RS II sequencing. After sequence data was processed to filter out low quality reads, a hierarchical genome-assembly process was employed that generated a genome sequence of 35.28 Mb in size. A total of 9844 genes were predicted, including 237 non-coding RNAs, 1024 genes encoding proteins with signal peptides, 2066 transmembrane proteins and 756 secretory proteins.Transcriptional identification revealed 54 differentially expressed secretory proteins. Concurrently, 605, 130 and 2869 proteins were matched in the proprietary databases Carbohydrate-Active EnZymes database (CAZyme), Transporter Classification Database (TCDB) and Pathogen-Host Interactions database (PHI), respectively. And 96 and 36 DEGs were identified form PHI database and CAZyme database, respectively. In addition, contig00011.93 was an up-regulated DEG involving ATP-binding cassette metabolism in the procession of infection. In order to test relevance of gene predictions to GSB, DEGs with potential pathogenic relevance were revealed through transcriptome data analysis of Sc. strains pre- and post-infection of pumpkin. Interestingly, Sc. and Leptosphaeria maculans (Lm.) exhibited relatively similar with genome lengths, numbers of protein-coding genes and other characteristics. This work provides a foundation for future exploration of additional Sc. gene functions toward the development of more effective GSB control strategies.

Identification of a Major QTL and Candidate Gene Analysis of Salt Tolerance at the Bud Burst Stage in Rice (Oryza sativa L.) Using QTL-Seq and RNA-Seq.

BACKGROUND: Salt stress is one of the main abiotic stresses that limits rice production worldwide. Rice salt tolerance at the bud burst stage directly affects the seedling survival rate and the final yield in the direct seeding cultivation model. However, the reports on quantitative trait locus (QTL) mapping and map-based cloning for salt tolerance at the bud burst stage are limited. RESULTS: Here, an F2:3 population derived from a cross between IR36 (salt-sensitive) and Weiguo (salt-tolerant) was used to identify salt-tolerant QTL interval at the bud burst stage using a whole-genome sequencing-based QTL-seq containing 40 extreme salt-tolerant and 40 extreme salt-sensitive individuals. A major QTL, qRSL7, related to relative shoot length (RSL) was detected on chromosome 7 using ΔSNP index algorithms and Euclidean Distance (ED) algorithms. According to single nucleotide polymorphisms (SNPs) between the parents, 25 Kompetitive allele-specific PCR (KASP) markers were developed near qRSL7, and regional QTL mapping was performed using 199 individuals from the F2:3 population. We then confirmed and narrowed down qRSL7 to a 222 kb genome interval. Additionally, RNA sequencing (RNA-seq) was performed for IR36 and Weiguo at 36 h after salt stress and control condition at the bud burst stage, and 5 differentially expressed genes (DEGs) were detected in the candidate region. The qRT-PCR results showed the same expression patterns as the RNA-seq data. Furthermore, sequence analysis revealed a 1 bp Indel difference in Os07g0569700 (OsSAP16) between IR36 and Weiguo. OsSAP16 encodes a stress-associated protein whose expression is increased under drought stress. CONCLUSION: These results indicate that OsSAP16 was the candidate gene of qRSL7. The results is useful for gene cloning of qRSL7 and for improving the salt tolerance of rice varieties by marker assisted selection (MAS).

Identification of key genes and modules in response to Cadmium stress in different rice varieties and stem nodes by weighted gene co-expression network analysis.

Soil cadmium (Cd) pollution threatens food safety. This study aimed to identify genes related to Cd accumulation in rice. Low- (Shennong 315, short for S315) and high- (Shendao 47, short for S47) Cd-accumulative rice cultivars were incubated with CdCl2·2.5H2O. RNA-seq and weighted gene co-expression network analysis (WGCNA) were performed to identify the modules and genes associated with Cd-accumulative traits of rice. After Cd stress treatment, the Cd content in various tissues of S315 was significantly higher than that of S47. In the stem nodes, the Cd distribution results of the two varieties indicated that the unelongated nodes near the root (short for node A) had a stronger ability to block Cd transfer upwards than the panicle node (short for node B). Cd stress induced huge changes in gene expression profiles. After analyzing the differentially expressed genes (DEGs) in significantly correlated WGCNA modules, we found that genes related to heavy metal transportation had higher expression levels in node A than that in node B, such as Copper transporter 6 (OS04G0415600), Zinc transporter 10 (OS06G0566300), and some heavy-metal associated proteins (OS11G0147500, OS03G0861400, and OS10G0506100). In the comparison results between S315 and S47, the expression of chitinase (OS03G0679700 and OS06G0726200) was increased by Cd treatment in S315. In addition, OsHSPs (OS05G0460000, OS08G0500700), OsHSFC2A (OS02G0232000), and OsDJA5 (OS03G0787300) were found differentially expressed after Cd treatment in S315, but changed less in S47. In summary, different rice varieties have different processes and intensities in response to Cd stress. The node A might function as the key tissue for blocking Cd upward transport into the panicle via vigorous processes, including of heavy metal transportation, response to stress, and cell wall.

An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants.

OBJECTIVES: In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. RESULTS: We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). CONCLUSION: Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.