Physiological, Epigenetic, and Transcriptome Analyses Provide Insights into the Responses of Wheat Seedling Leaves to Different Water Depths under Flooding Conditions.

Flooding stress, including waterlogging and submergence, is one of the major abiotic stresses that seriously affects the growth and development of plants. In the present study, physiological, epigenetic, and transcriptomic analyses were performed in wheat seedling leaves under waterlogging (WL), half submergence (HS), and full submergence (FS) treatments. The results demonstrate that FS increased the leaves' hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and reduced their chlorophyll contents (SPAD), photosynthetic efficiency (Fv/Fm), and shoot dry weight more than HS and WL. In addition, FS increased catalase (CAT) and peroxidase (POD) activities more than HS and WL. However, there were no significant differences in the contents of H2O2, MDA, SPAD, and Fv/Fm, and the activities of superoxide dismutase (SOD) and POD between the HS and WL treatments. The changes in DNA methylation were related to stress types, increasing under the WL and HS treatments and decreasing under the FS treatment. Additionally, a total of 9996, 10,619, and 24,949 genes were differentially expressed under the WL, HS, and FS treatments, respectively, among which the 'photosynthesis', 'phenylpropanoid biosynthesis', and 'plant hormone signal transduction' pathways were extensively enriched under the three flooding treatments. The genes involved in these pathways showed flooding-type-specific expression. Moreover, flooding-type-specific responses were observed in the three conditions, including the enrichment of specific TFs and response pathways. These results will contribute to a better understanding of the molecular mechanisms underlying the responses of wheat seedling leaves to flooding stress and provide valuable genetic and epigenetic information for breeding flood-tolerant varieties of wheat.

Carbohydrate metabolism and cytology of S-type cytoplasmic male sterility in wheat.

Introduction: Cytoplasmic male sterility (CMS) is an important tool for hybrid heterosis utilization. However, the underlying mechanisms still need to be discovered. An adequate supply of nutrients is necessary for anther development; pollen abortion would occur if the metabolism of carbohydrates were hampered. Methods: In order to better understand the relationship between carbohydrate metabolism disorder and pollen abortion in S-CMS wheat, the submicroscopic structure of wheat anthers was observed using light microscopy and transmission electron microscopy; chloroplast proteome changes were explored by comparative proteomic analysis; sugar measuring and enzyme assays were performed; and the expression patterns of carbohydrate metabolism-related genes were studied using quantitative real-time PCR (qRT-PCR) method. Results: These results indicated that the anther and microspore in S-CMS wheat underwent serious structural damage, including premature tapetum degeneration, nutritional shortage, pollen wall defects, and pollen grain malformations. Furthermore, the number of chloroplasts in the anthers of S-CMS lines decreased significantly, causing abnormal carbohydrate metabolism, and disintegration of osmiophilic granules and thylakoids. Meanwhile, some proteins participating in the Calvin cycle and carbohydrate metabolism were abnormally expressed in the chloroplasts of the S-CMS lines, which might lead to chloroplast dysfunction. Additionally, several key enzymes and genes related to carbohydrate metabolism were significantly inhibited in S-CMS. Discussion: Based on these results, we proposed a carbohydrate metabolism pathway for anther abortion in S-type cytoplasmic male sterility, which would encourage further exploration of the pollen abortion mechanisms for CMS wheat.

Molecular characterization and validation of adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895.

KEY MESSAGE: Adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895 was mapped to the physical interval 710.2-713.2 Mb on the long arm of chromosome 2A. Adult-plant resistance to stripe rust is generally more durable than all-stage resistance. Chinese wheat cultivar Zhongmai 895 showed stable stripe rust resistance at the adult-plant stage. To map the genetic loci underlying its resistance, 171 doubled haploid (DH) lines from a Yangmai 16/Zhongmai 895 cross were genotyped with the wheat 660 K SNP chip. Disease severities of the DH population and parents were assessed in four environments. A major QTL designated QYryz.caas-2AL was mapped to interval 703.7-715.3 Mb on the long arm of chromosome 2A using both chip-based and KASP (kompetitive allele-specific PCR) marker-based methods, explaining 31.5 to 54.1% of the phenotypic variances. The QTL was further validated in an F2 population of cross Emai 580/Zhongmai 895 with 459 plants and a panel of 240 wheat cultivars using KASP markers. Three reliable KASP markers predicted a low frequency (7.2-10.5%) of QYryz.caas-2AL in the test panel and remapped the gene to the physical interval 710.2-713.2 Mb. Based on different physical positions or genetic effects from known genes or QTL on chromosome arm 2AL, the gene was predicted to be a new one for adult-plant stripe rust resistance and was named Yr86. Twenty KASP markers linked to Yr86 were developed in this study based on wheat 660 K SNP array and genome re-sequencing. Three of them are significantly associated with stripe rust resistance in natural population. These markers should be useful for marker-assisted selection and also provide a starting point for fine mapping and map-based cloning of the new resistance gene.

[Expression and Significance of PD-1, PD-L1 and CTLA-4 in the Bone Marrow of Patients with Multiple Myeloma].

OBJECTIVE: To study the expression and significance of PD-1, PD-L1 and CTLA-4 tumor-associated antigens in multiple myeloma. METHODS: Bone marrow specimens from 122 patients with multiple myeloma were collected and divided into new-onset group (NDMM), complete remission group (CRMM) and relapsed and refractory group (RRMM) according to the disease progression stage. The proportion of CD4+ T lymphocytes, CD8+ T lymphocytes, Treg cells and plasma cells in the specimens and the expressions of PD-1, PD-L1 and CTLA-4 were detected by multi-parameter flow cytometry. RESULTS: There was no significant difference in the proportion of CD8+T and Treg cells among the three groups (P>0.05), while the proportions of CD4+T cells and PC in NDMM group were significantly higher than those in the CRMM group (P<0.05), the ratios of CD4+ to CD8+T in the NDMM and RRMM groups were significantly higher than those in the CRMM group (P<0.05). The expressions of PD-1, PD-L1 and CTLA-4 in CD8+ T cells was no significant difference among NDMM, CRMM and RRMM groups (P>0.05). While the expressions of PD-1, PD-L1 and CTLA-4 in CD4+ T cells and PC in the NDMM group were significantly lower than that in the CRMM group (P<0.05). There was significantly difference among the three groups in the expression of PD-1 in Treg cells, of which the NDMM group was significantly lower than that of the CRMM group (all P<0.05). The expressions of PD-1 and CTLA-4 in PC were significantly higher than those in CD8+ T, CD4+ T and Treg cells (P<0.05), the expression of PD-L1 in CD8+ T cells was significantly higher than that in CD4+ T and Treg cells (P<0.05). CONCLUSION: There is a correlation between the immune status of multiple myeloma and the expressions of PD-1, PD-L1 and CTLA-4 in plasma cells and lymphocyte subsets in vivo.

[Application of CD138 Immunomagnetic Bead Sorting Combined with Fluorescence in Situ Hybridization in Multiple Myeloma].

OBJECTIVE: To compare the effects of direct fluorescence in situ hybridization (D-FISH) detection without sorting and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) on cytogenetic analysis of patients with multiple myeloma (MM). METHODS: FISH test results of 229 patients with initial MM were retrospectively analyzed. The patients were divided into two groups, 140 patients were tested with D-FISH and 89 patients with MACS-FISH. The combination probe was designed as P53, D13S319, RB1, 1q21, and IgH. Cytogenetic detection results were compared between the two groups. RESULTS: The total detection rate of cytogenetic abnormalities in D-FISH group was 52.9%, and that in MACS-FISH group was 79.8%. There was a significant difference in the cytogenetic abnormality rate between the two groups (P=0.020). The abnormal genes with the highest detection rate in the two groups were 1q21 and IgH, respectively, while the lowest was P53. There was no significant difference in the percentage of P53 positive cells (positive rate) between the two groups, while D13S319, RB1, 1q21, and IgH showed significant difference in positive cell rate (P=0.0002, P<0.0001, P=0.0033, P=0.0032). There was no significant correlation between the proportion of plasma cells (PC) detected by bone marrow morphology and cytogenetic abnormality rate in the D-FISH group, while there was a correlation between the proportion of PC detected by flow cytometry and cytogenetic abnormality rate (r=0.364). The PC proportion detected by bone marrow morphology and flow cytometry in the MACS-FISH group had no correlation with the cytogenetic abnormality rate and positive cell rate of the 5 genes mentioned above. Additionally, the PC proportion detected by bone marrow morphology and flow cytometry showed significant difference (P<0.0001). CONCLUSION: CD138 immunomagnetic bead sorting combined with FISH technology can significantly improve the abnormality detection rate of MM cytogenetics.

Multiple roles of wheat calmodulin genes during stress treatment and TaCAM2-D as a positive regulator in response to drought and salt tolerance.

Calmodulin (CaM) and calmodulin-like (CML) proteins are the most prominent calcium (Ca2+) sensing proteins involved in Ca2+-signaling processes. However, the function of these calcium sensors in wheat remains unclear. In this study, 15 TaCAMs and 113 TaCMLs were identified from the wheat reference genome. The analysis of cis-acting elements and expression patterns showed that TaCAMs might play an important role in response to abiotic and biotic stresses. TaCAM2-D gene was found to be significantly upregulated under drought and salt stresses, and thus, it was selected to further explore the biological function. Moreover, TaCAM2-D was observed to be localized in the nucleus, membrane and cytoplasm. Overexpression of TaCAM2-D in Arabidopsis conferred greater tolerance to drought and salt. The prediction analysis, the yeast two-hybrid analysis, and bimolecular fluorescence complementation assay indicated that TaCAM2-D interacted with TaMPK8, which is one of the wheat mitogen-activated protein kinases. Thus, the current study provides insights into the understanding of the TaCAM and TaCML genes in wheat.

Effects of three fertilizer application patterns on wheat sowed by machinery in slope cropland of Western Hubei, China.

Unscientific fertilization, unstable grain quality, and low profit are the key problems on wheat production in slope cropland of Western Hubei. To solve these problems, three optimized planting patterns (high nitrogen and potassium reduction, HNPR; medium nitrogen and potassium reductionm, MNPR; low nitrogen and potassium reduction, LNPR) were conducted during two consecutive years to assess their effects on wheat yield, quality, profit, and fertilizer use efficiency in Danjiangkou Reservoir area, a typical slope cropland region with wheat-maize rotation. The results showed that the application of chemical fertilizer significantly increased grain yield (GY) and wet gluten content (WGC) of wheat. Compared with the conventional planting pattern (CK), the partial factor productivity (PFPK) and agricultural fertilizer use efficiency (AFUEK) of potassium were significantly improved in the three optimized planting patterns. The dry matter amount (DMA), GY, and crude protein content (CPC) were the highest under HNPR, which increased by 9.4%, 19.4%, and 7.8% than CK, respectively. Such a result indicated that HNPR benefited wheat to exploit high yield potential. WGC and falling number (FN) were the highest under MNPR, and increased by 3.9%, and 9.3% than CK, respectively, which was suitable for high-efficiency production of medium-gluten wheat. PFPN, AFUEN, PFPK, AFUEK, and net profit were the highest under LNPR, which increased by 15.7%, 134.1%, 131.3%, 368.2%, and 37.3% than CK, respectively, while the CPC and WGC were decreased by 2.1% and 2.6% than CK, respectively, suggesting it was suitable for environment-friendly and simplified production of weak-gluten wheat production. Our results could provide a reference for wheat production in the slope cropland.

Molecular Mapping of Quantitative Trait Loci for Fusarium Head Blight Resistance in a Doubled Haploid Population of Chinese Bread Wheat.

Fusarium head blight (FHB) is a destructive disease of wheat worldwide, particularly in China. To map genetic loci underlying FHB resistance, a doubled haploid (DH) population consisting of 174 lines was developed from a cross between widely grown Chinese cultivars Yangmai 16 and Zhongmai 895. The DH population and parents were evaluated in field nurseries at Wuhan in 2016 to 2017 and 2017 to 2018 crop seasons with both spray inoculation and natural infection, and at Jingzhou in 2017 to 2018 crop season with grain-spawn inoculation. The DH lines were genotyped with a wheat 660K SNP array. The FHB index, plant height, anther extrusion, and days to anthesis were recorded and used for quantitative trait loci (QTL) analysis. Seven QTL for FHB resistance were mapped to chromosome arms 3BL, 4AS, 4BS, 4DS, 5AL, 6AL, and 6BS in at least two environments. QFhb.caas-4BS and QFhb.caas-4DS co-located with semi-dwarfing alleles Rht-B1b and Rht-D1b, respectively, and were associated with anther extrusion. The other five QTL were genetically independent of the agronomic traits, indicating their potential value when breeding for FHB resistance. Based on correlations between FHB indices and agronomic traits in this population, we concluded that increasing plant height to some extent would enhance FHB resistance, that anther extrusion had a more important role in environments with less severe FHB, and that days to anthesis were independent of the FHB response when viewed across years. PCR-based markers were developed for the 3BL and 5AL QTL, which were detected in more than three environments. The InDel marker InDel_AX-89588684 for QFhb.caas-5AL was also validated on a wheat panel, confirming its effectiveness for marker-assisted breeding for improvements in FHB resistance.

Genome-wide association analysis of stripe rust resistance in modern Chinese wheat.

BACKGROUND: Stripe rust (yellow rust) is a significant disease for bread wheat (Triticum aestivum L.) worldwide. A genome-wide association study was conducted on 240 Chinese wheat cultivars and elite lines genotyped with the wheat 90 K single nucleotide polymorphism (SNP) arrays to decipher the genetic architecture of stripe rust resistance in Chinese germplasm. RESULTS: Stripe rust resistance was evaluated at the adult plant stage in Pixian and Xindu in Sichuan province in the 2015-2016 cropping season, and in Wuhan in Hubei province in the 2013-2014, 2016-2017 and 2018-2019 cropping seasons. Twelve stable loci for stripe rust resistance were identified by GWAS using TASSEL and GAPIT software. These loci were distributed on chromosomes 1B, 1D, 2A, 2B, 3A, 3B, 4B (3), 4D, 6D, and 7B and explained 3.6 to 10.3% of the phenotypic variation. Six of the loci corresponded with previously reported genes/QTLs, including Sr2/Yr30/Lr27, while the other six (QYr.hbaas-1BS, QYr.hbaas-2BL, QYr.hbaas-3AL, QYr.hbaas-4BL.3, QYr.hbaas-4DL, and QYr.hbaas-6DS) are probably novel. The results suggest high genetic diversity for stripe rust resistance in this population. The resistance alleles of QYr.hbaas-2AS, QYr.hbaas-3BS, QYr.hbaas-4DL, and QYr.hbaas-7BL were rare in the present panel, indicating their potential use in breeding for stripe rust resistance in China. Eleven penta-primer amplification refractory mutation system (PARMS) markers were developed from SNPs significantly associated with seven mapped QTLs. Twenty-seven genes were predicted for mapped QTLs. Six of them were considered as candidates for their high relative expression levels post-inoculation. CONCLUSION: The resistant germplasm, mapped QTLs, and PARMS markers developed in this study are resources for enhancing stripe rust resistance in wheat breeding.

Genome-Wide Association Analysis of Fusarium Head Blight Resistance in Chinese Elite Wheat Lines.

Fusarium head blight (FHB) is a devastating wheat disease worldwide. To decipher the genetic architecture of FHB resistance in Chinese germplasm, a Wheat Association Panel for Scab Research (WAPS) consisting of 240 leading Chinese wheat cultivars and elite lines was genotyped using the 90K single nucleotide polymorphism (SNP) arrays. The FHB response was evaluated in the field nurseries in Wuhan in Hubei Province over four consecutive years from 2014 to 2017. Five quantitative trait loci (QTL) were consistently identified on chromosome arms 1AS, 2DL, 5AS, 5AL, and 7DS using a mixed linear model (MLM), explaining 5.6, 10.3, 5.7, 5.4, and 5.6% of phenotypic variation, respectively. The QTL on 5AS, 5AL, and 7DS QTL are probably novel. The allelic frequency analysis indicated that cultivars from the Middle and Lower Yangtze River Valleys harbored more favorable alleles and were therefore more resistant than those from other regions. To facilitate in-house germplasm screening and marker-assisted selection (MAS), SNP-derived PCR markers were developed for the QTL regions on 1AS, 5AS, and 5AL QTL. In addition to the above five QTL, the WAPS population had a very low frequency of Fhb1, confirming that the gene is not widely used in Chinese wheat breeding programs. The resistant lines and molecular markers developed in this study are resources and information for enhancing FHB resistance in breeding populations by marker-assisted recurrent selection and gene stacking.

Harnessing Wheat Fhb1 for Fusarium Resistance.

Fusarium head blight (FHB), caused by the fungus Fusarium graminearum, is an economically devastating disease of wheat worldwide. Fhb1, a widely used genetic source of FHB resistance, originated in East Asia. The recent cloning of Fhb1 opens a new avenue to improve FHB resistance in wheat and potentially other crops.

Cytological and Proteomic Analysis of Wheat Pollen Abortion Induced by Chemical Hybridization Agent.

In plants, pollen grain transfers the haploid male genetic material from anther to stigma, both between flowers (cross-pollination) and within the same flower (self-pollination). In order to better understand chemical hybridizing agent (CHA) SQ-1-induced pollen abortion in wheat, comparative cytological and proteomic analyses were conducted. Results indicated that pollen grains underwent serious structural injury, including cell division abnormality, nutritional deficiencies, pollen wall defect and pollen grain malformations in the CHA-SQ-1-treated plants, resulting in pollen abortion and male sterility. A total of 61 proteins showed statistically significant differences in abundance, among which 18 proteins were highly abundant and 43 proteins were less abundant in CHA-SQ-1 treated plants. 60 proteins were successfully identified using MALDI-TOF/TOF mass spectrometry. These proteins were found to be involved in pollen maturation and showed a change in the abundance of a battery of proteins involved in multiple biological processes, including pollen development, carbohydrate and energy metabolism, stress response, protein metabolism. Interactions between these proteins were predicted using bioinformatics analysis. Gene ontology and pathway analyses revealed that the majority of the identified proteins were involved in carbohydrate and energy metabolism. Accordingly, a protein-protein interaction network involving in pollen abortion was proposed. These results provide information for the molecular events underlying CHA-SQ-1-induced pollen abortion and may serve as an additional guide for practical hybrid breeding.