水稻耐储藏特性三年动态鉴定与QTL分析

种子耐储藏特性是粮食作物的特殊农艺性状之一, 耐储藏性能对种子生产和种质资源保存有重要意义。以粳型超级稻龙稻5 (LD5)和高产籼稻中优早8 (ZYZ8)杂交衍生的重组自交系(RILs)群体(共180个株系)为实验材料, 自然高温高湿条件下放置1年、2年和3年后, 对不同储藏时段种子发芽率进行比较, 并利用223个分子标记的遗传图谱进行动态QTL鉴定。结果表明, 不同储藏时段龙稻5的发芽率均显著低于中优早8, 株系间耐储性存在较大差异; 不同储藏时段发芽率显著相关, 相邻存储时段发芽率关系紧密。共检测到17个耐储性相关的QTLs, 3个老化时段分别检测到5、4和3个, 检测到5个动态条件QTLs, 单一QTL解释5.60%-32.76%的表型变异, 加性效应在-16.78%-16.95%范围内。主效QTL簇qSSC2、qSSC6、qSSC7和qSSC8能调控不同储藏时段的发芽率, qSSC6具有明显降低发芽率的效应。共检测到26对上位性互作位点, 主效QTL qSS1和qSS4参与上位性互作, 这表明上位性互作是调控耐储藏性状的重要遗传组成。研究结果为水稻(Oryza sativa)耐储性相关QTL的精细定位奠定基础, 同时丰富了耐储性分子标记辅助选择育种的基因资源。     

DELAYED HEADING DATE1 interacts with OsHAP5C/D,delays flowering time and enhances yield in rice

Heading date is an important agronomic trait affecting crop yield. The GRAS protein family is a plant‐specific super family extensively involved in plant growth and signal transduction. However, GRAS proteins are rarely reported have a role in regulating rice heading date. Here, we report a GRAS protein DHD1 (Delayed Heading Date1) delays heading and enhances yield in rice. Biochemical assays showed DHD1 physically interacts with OsHAP5C/D both in vitro and in vivo. DHD1 and OsHAP5C/D located in the nucleus and showed that rhythmic expression. Both DHD1 and OsHAP5C/D affect heading date by regulating expression of Ehd1. We propose that DHD1 interacts with OsHAP5C/D to delay heading date by inhibiting expression of Ehd1.     

A metabolome‐based core hybridisation strategy for the prediction of rice grain weight across environments

Marker‐based prediction holds great promise for improving current plant and animal breeding efficiencies. However, the predictabilities of complex traits are always severely affected by negative factors, including distant relatedness, environmental discrepancies, unknown population structures, and indeterminate numbers of predictive variables. In this study, we utilised two independent F₁ hybrid populations in the years 2012 and 2015 to predict rice thousand grain weight (TGW) using parental untargeted metabolite profiles with a partial least squares regression method. A stable predictive model for TGW was built based on hybrids from the population in 2012 (r = 0.75) but failed to properly predict TGW for hybrids from the population in 2015 (r = 0.27). After integrating hybrids from both populations into the training set, the TGW of hybrids could be predicted but was largely dependent on population structures. Then, core hybrids from each population were determined by principal component analysis and the TGW of hybrids in both environments were successfully predicted (r > 0.60). Moreover, adjusting the population structures and numbers of predictive analytes increased TGW predictability for hybrids in 2015 (r = 0.72). Our study demonstrates that the TGW of F₁ hybrids across environments can be accurately predicted based on parental untargeted metabolite profiles with a core hybridisation strategy in rice. Metabolic biomarkers identified from early developmental stage tissues, which are grown under experimental conditions, may represent a workable approach towards the robust prediction of major agronomic traits for climate‐adaptive varieties.     

Bioimaging of multiple elements by high‐resolution LA‐ICP‐MS reveals altered distribution of mineral elements in the nodes of rice mutants

Inter‐vascular transfer in rice (Oryza sativa) nodes is required for delivering mineral elements to developing tissues, which is mediated by various transporters in the nodes. However, the effect of these transporters on distribution of mineral elements in the nodes at a cellular level is still unknown. Here, we established a protocol for bioimaging of multiple elements at a cellular level in rice node by laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS), and compared the mineral distribution profile between wild‐type (WT) rice and mutants. Both relative comparison of mineral distribution normalized by endogenous ¹³C and quantitative analysis using spiked standards combined with soft ablation gave valid results. Overall, macro‐nutrients such as K and Mg were accumulated more in the phloem region, while micro‐nutrients such as Fe and Zn were highly accumulated at the inter‐vascular tissues of the node. In mutants of nodal Zn transporter OsHMA2, Zn localization pattern in the node tissues did not differ from that of WT; however, Zn accumulation in the inter‐vascular tissues was lower in uppermost node I but higher in the third upper node III compared with the WT. In contrast, Si deposition in the mutants of three nodal Si transporters Lsi2, Lsi3 and Lsi6 showed different patterns, which are consistent with the localization of these transporters. This improved LA‐ICP‐MS analysis combined with functional characterization of transporters will provide further insight into mineral element distribution mechanisms in rice and other plant species.     

Targeted exome sequencing of unselected heavy‐ion beam‐irradiated populations reveals less‐biased mutation characteristics in the rice genome

Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M₂ lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M₂ lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations.     

Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice.