Major and stably expressed QTL for traits related to the mature wheat embryo independent of kernel size.

KEY MESSAGE: Two major and stably expressed QTL for traits related to mature wheat embryo independent of kernel size were identified and validated in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. As the juvenile of a highly differentiated plant, mature wheat (Triticum aestivum L.) embryos are highly significant to agricultural production. To understand the genetic basis of traits related to wheat embryo size, the embryo of mature kernels in a recombination inbred line that contained 126 lines from four environments was measured. The genetic loci of embryo size, including embryo length (EL), embryo width (EW), embryo area (EA), embryo length/kernel length (EL/KL), embryo width/kernel width (EW/KW), and EL/EW, were identified based on a genetic linkage map constructed based on PCR markers and the Wheat 55 K single nucleotide polymorphism (SNP) array. A total of 50 quantitative trait loci (QTL) for traits related to wheat embryo size were detected. Among them, QEL.sicau-2SY-4A for EL and QEW.sicau-2SY-7B for EW were major and stably expressed and were genetically independent of KL and KW, respectively. Their effects were further verified in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. Further analysis showed that TraesCS4A02G343300 and TraesCS7B02G006800 could be candidate genes for QEL.sicau-2SY-4A and QEW.sicau-2SY-7B, respectively. In addition, significant positive correlations between EL and kernel-related traits and the 1,000-grain weight were detected. Collectively, this study broadens our understanding of the genetic basis of wheat embryo size and will be helpful for the further fine-mapping of interesting loci in the future.

Quick mapping and characterization of a co-located kernel length and thousand-kernel weight-related QTL in wheat.

KEY MESSAGE: A co-located KL and TKW-related QTL with no negative effect on PH and AD was rapidly identified using BSA and wheat 660 K SNP array. Its effect was validated in a panel of 218 wheat accessions. Kernel length (KL) and thousand-kernel weight (TKW) of wheat (Triticum aestivum L.) contribute significantly to kernel yield. In the present study, a recombinant inbred line (RIL) population derived from the cross between the wheat line S849-8 with larger kernels and more spikelets per spike and the line SY95-71 was developed. Further, of both the bulked segregant analysis (BSA) and the wheat 660 K single nucleotide polymorphism (SNP) array were used to rapidly identify genomic regions for kernel-related traits from this RIL population. Kompetitive Allele Specific PCR markers were further developed in the SNP-enriched region on the 2D chromosome to construct a genetic map. Both QKL.sicau-SSY-2D for KL and QTKW.sicau-SSY-2D for TKW were identified at multiple environments on chromosome arm 2DL. These two QTLs explained 9.68-23.02% and 6.73-18.32% of the phenotypic variation, respectively. The effects of this co-located QTL were successfully verified in a natural population consisting of 218 Sichuan wheat accessions. Interestingly, the major QTL was significantly and positively correlated with spike length, but did not negatively affect spikelet number per spike (SNS), plant height, or anthesis date. These results indicated that it is possible to synchronously improve kernel weight and SNS by using this QTL. Additionally, several genes associated with kernel development and filling rate were predicted and sequenced in the QTL-containing physical intervals of reference genomes of 'Chinese spring' and Aegilops tauschii. Collectively, these results provide a QTL with great breeding potential and its linked markers which should be helpful for fine mapping and molecular breeding.

A novel, major, and validated QTL for the effective tiller number located on chromosome arm 1BL in bread wheat.

KEY MESSAGE: A novel and major QTL for the effective tiller number was identified on chromosomal arm 1BL and validated in two genetic backgrounds The effective tiller number (ETN) substantially influences plant architecture and the wheat yield improvement. In this study, we constructed a genetic map of the 2SY (20828/SY95-71) recombinant inbred line population based on the Wheat 55K array as well as the simple sequence repeat (SSR) and Kompetitive Allele Specific PCR (KASP) markers. A comparison between the genetic and physical maps indicated the marker positions were consistent in the two maps. Additionally, we identified seven tillering-related quantitative trait locus (QTLs), including Qetn-sau-1B.1, which is a major QTL localized to a 6.17-cM interval flanked by markers AX-89635557 and AX-111544678 on chromosome 1BL. The Qetn-sau-1B.1 QTL was detected in eight environments and explained 12.12-55.71% of the phenotypic variance. Three genes associated with the ETN were detected in the physical interval of Qetn-sau-1B.1. We used a tightly linked KASP marker, KASP-AX-110129912, to further validate this QTL in two other populations with different genetic backgrounds. The results indicated that Qetn-sau-1B.1 significantly increased the ETN by up to 23.5%. The results of this study will be useful for the precise mapping and cloning of Qetn-sau-1B.1.

Identification and characterization of mRNAs and lncRNAs of a barley shrunken endosperm mutant using RNA-seq.

Barley shrunken endosperm mutants have been extensively reported. However, knowledge of the underlying molecular mechanisms of these mutants remains limited. Here, a pair of near isogenic lines (normal endosperm: Bowman and shrunken endosperm: sex1) was subjected to transcriptome analysis to identify mRNAs and lncRNAs related to endosperm development to further dissect its mechanism of molecular regulation. A total of 2123 (1140 up- and 983 down-regulated) unique differentially expressed genes (DEGs) were detected. Functional analyses showed that these DEGs were mainly involved in starch and sucrose metabolism, biosynthesis of secondary metabolites, and plant hormone signal transduction. A total of 343 unique target genes were identified for 57 differentially expressed lncRNAs (DE lncRNAs). These DE lncRNAs were mainly involved in glycerophospholipid metabolism, starch and sucrose metabolism, hormone signal transduction, and stress response. In addition, key lncRNAs were identified by constructing a co-expression network of the target genes of DE lncRNAs. Transcriptome results suggested that mRNA and lncRNA played a critical role in endosperm development. The shrunken endosperm in barley seems to be closely related to plant hormone signal transduction, starch and sucrose metabolism, and cell apoptosis. This study provides a foundation for fine mapping, elucidates the molecular mechanism of shrunken endosperm mutants, and also provides a reference for further studies of lncRNAs during the grain development of plants.

The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function.

Chloroplasts are key players in photosynthesis and immunity against microbial pathogens. However, the precise and timely regulatory mechanisms governing the control of photosynthesis-associated nuclear genes (PhANGs) expression in plant immunity remain largely unknown. Here we report that TaPIR1, a Pst-induced RING-finger E3 ubiquitin ligase, negatively regulates Pst resistance by specifically interacting with TaHRP1, an atypical transcription factor histidine-rich protein. TaPIR1 ubiquitinates the lysine residues K131 and K136 in TaHRP1 to regulate its stability. TaHRP1 directly binds to the TaHRP1-binding site elements within the PhANGs promoter to activate their transcription via the histidine-rich domain of TaHRP1. PhANGs expression induces the production of chloroplast-derived ROS. Although knocking out TaHRP1 reduces Pst resistance, TaHRP1 overexpression contributes to photosynthesis, and chloroplast-derived ROS production, and improves disease resistance. TaPIR1 expression inhibits the downstream activation of TaHRP1 and TaHRP1-induced ROS accumulation in chloroplasts. Overall, we show that the TaPIR1-mediated ubiquitination and degradation of TaHRP1 alters PhANGs expression to disrupt chloroplast function, thereby increasing plant susceptibility to Pst.

Loss of ADP-glucose transporter in barley sex1 mutant caused shrunken endosperm but with elevated protein and ß-glucan content in whole meal.

Grain shape and plumpness affect barley yield. Despite numerous studies on shrunken endosperm mutants in barley, their molecular mechanism and application potential in the food industry are largely unknown. Here, map-based cloning, co-segregation analyses, and allelic variant validation revealed that the loss of HORVU6Hr1G037950 encoding an ADP-glucose transporter caused the shrunken endosperm in sex1. Haplotype analysis suggested that hap4 in the promoter sequence was positively related to the hundred-grain weight showing a breeding potential. A pair of near-isogenic lines targeting HORVU6Hr1G037950 was produced and characterized to investigate molecular mechanisms that SEX1 regulates endosperm development. Results presented that the absence of the SEX1 gene led to the decrease of starch content and A-type granules size, the increase of ß-glucan, protein, gelatinization temperature, soluble sugar content, amylopectin A chains, and B1 chains. Enzymatic activity, transcriptome and metabolome analyses revealed the loss of SEX1 results in an impaired ADP-glucose-to-starch conversion process, consequently leading to higher soluble sugar contents and lower starch accumulation, thereby inducing a shrunken-endosperm phenotype in sex1. Taken together, this study provides new insights into barley grain development, and the elevated protein and ß-glucan contents of the whole meal in sex1 imply its promising application in the food industry.

Genetic Mapping and Validation of Loci for Kernel-Related Traits in Wheat (Triticum aestivum L.).

Kernel size (KS) and kernel weight play a key role in wheat yield. Phenotypic data from six environments and a Wheat55K single-nucleotide polymorphism array-based constructed genetic linkage map from a recombinant inbred line population derived from the cross between the wheat line 20828 and the line SY95-71 were used to identify quantitative trait locus (QTL) for kernel length (KL), kernel width (KW), kernel thickness (KT), thousand-kernel weight (TKW), kernel length-width ratio (LWR), KS, and factor form density (FFD). The results showed that 65 QTLs associated with kernel traits were detected, of which the major QTLs QKL.sicau-2SY-1B, QKW.sicau-2SY-6D, QKT.sicau-2SY-2D, and QTKW.sicau-2SY-2D, QLWR.sicau-2SY-6D, QKS.sicau-2SY-1B/2D/6D, and QFFD.sicau-2SY-2D controlling KL, KW, KT, TKW, LWR, KS, and FFD, and identified in multiple environments, respectively. They were located on chromosomes 1BL, 2DL, and 6DS and formed three QTL clusters. Comparison of genetic and physical interval suggested that only QKL.sicau-2SY-1B located on chromosome 1BL was likely a novel QTL. A Kompetitive Allele Specific Polymerase chain reaction (KASP) marker, KASP-AX-109379070, closely linked to this novel QTL was developed and used to successfully confirm its effect in two different genetic populations and three variety panels consisting of 272 Chinese wheat landraces, 300 Chinese wheat cultivars most from the Yellow and Huai River Valley wheat region, and 165 Sichuan wheat cultivars. The relationships between kernel traits and other agronomic traits were detected and discussed. A few predicted genes involved in regulation of kernel growth and development were identified in the intervals of these identified major QTL. Taken together, these stable and major QTLs provide valuable information for understanding the genetic composition of kernel yield and provide the basis for molecular marker-assisted breeding.

Synthesis and anti-cancer cell activity of pseudo-ginsenoside Rh2.

ß-d-Glucopyranoside,(3ß,12ß,20E)-12,25-dihydroxydammar-20(22)-en-3-yl (pseudo-ginsenoside Rh2) and its 20Z-isomer were synthesized from ginsenoside Rh2 under a mild condition, via a simple three-step called acetylation, elimination-addition and saponification. In addition, their activities were evaluated by eight different human tumor cells, compared with ginsenoside Rh2 group. Results indicated that the reaction in the side chain might greatly enhance the anti-proliferative activity of ginsenosides.

pseudo-G-Rh2 induces mitochondrial-mediated apoptosis in SGC-7901 human gastric cancer cells.

This study was designed to investigate the effect of pseudo-G-Rh2, a novel metabolite of ginsenoside Rh2, on the apoptosis of SGC-7901 human gastric cancer cells. Pseudo-G-Rh2 demonstrated antitumor activity and significantly inhibited the proliferation of SGC-7901 cells in a concentration-dependent manner. After treatment with pseudo-G-Rh2, SGC-7901 cells showed typical apoptotic morphological features, such as chromatin condensation and DNA fragmentation. Pseudo-G-Rh2 could induce mitochondrial membrane potential loss, which led to the release of cytochrome c (Cyt c), Smac/Diablo and apoptosis-inducing factor (AIF) to the cell cytoplasm. Furthermore, pseudo-G-Rh2 exposure not only decreased the expression of the Bcl-2 protein but also increased the expression of the Bax protein and the activities of caspase-9 and caspase-3 in SGC-7901 cells. These results demonstrated that pseudo-G-Rh2 inhibited the proliferation of SGC-7901 cells by initiating apoptosis. Pseudo-G-Rh2-induced apoptosis was associated with a drop in the mitochondrial transmembrane potential, down-regulation of Bcl-2, up-regulation of Bax and activation of caspase-9 and caspase-3.

Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway.

Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities including anti-tumor. Here, for the first time, we studied the molecular mechanism of Juglone-induced apoptosis in human leukemia HL-60 cells. In the present study, HL-60 cells were incubated with Juglone at various concentrations. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry. Expression of Bcl-2 and Bax mRNA was determined by quantitative polymerase chain reaction (qPCR). The results showed that Juglone inhibits the growth of human leukemia HL-60 cells in dose- and time-dependent manner. Topical morphological changes of apoptotic body formation after Juglone treatment were observed by Hoechst 33342 staining. The percentages of Annexin V-FITC-positive/PI negative cells were 7.81%, 35.46%, 49.11% and 66.02% with the concentrations of Juglone (0, 0.5, 1.0 and 1.5 microg/ml). Juglone could induce the mitochondrial membrane potential (DeltaPsim) loss, which preceded release of cytochrome c (Cyt c), Smac and apoptosis inducing factor (AIF) to cell cytoplasm. A marked increased of Bax mRNA and protein appeared with Juglone treatment, while an evidently decreased of Bcl-2 mRNA and protein appeared at the same time. These events paralleled with activation of caspase-9, -3 and PARP cleavage. And the apoptosis induced by Juglone was blocked by z-LEHD-fmk, a caspase-9 inhibitor. Those results of our studies demonstrated that Juglone-induced mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent apoptosis pathways and the elevated ratio of Bax/Bcl-2 was also probably involved in this effect.