双胚苗水稻单倍体及其杂交后代基因组DNA甲基化特异位点的分析及功能探讨

 采用甲基化敏感性扩增多态性技术对水稻单倍体SARⅡ 628及它与蜀恢527、蜀恢363的杂交后代基因组DNA甲基化位点进行了分析。用16对选择性扩增引物在双亲及杂交后代中共检测到765个DNA甲基化位点,与双亲相比,杂交后代DNA甲基化水平均有不同程度的降低。通过分析两个杂交组合与双亲的甲基化带型差异,发现非单倍体遗传及单倍体独立遗传两种甲基化差异位点。对部分甲基化位点的序列进行功能分析,发现这些位点主要涉及细胞构造、代谢途径、应激反应等生物学功能。推测这类功能基因的甲基化修饰调控着相关基因的开启与关闭,为植物的生存、生长、发育及进化提供动力。     

Natural Homologous Triploidization and DNA Methylation in SARII-628, a Twin-seedling Line of Rice （Oryza sativa）

A total of five pairs of diploid-triploid twin-seedlings (a diploid seedling and a triploid seedling emerged from a grain) were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ‘MSAP (methylation-sensitive AFLP)’ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on M0 generation after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.     

Changes in DNA Methylation Pattern at Two Seedling Stages in Water Saving and Drought-Resistant Rice Variety after Drought Stress Domestication

Recent studies revealed that DNA methylation plays an important role in plant growth and development. In this study, a water-saving and drought-resistant rice variety Huhan 3 was subjected to drought stress from tillering to grain-filling stages in six successive growth cycles. The variations in DNA methylation pattern between the original generation （Go） and the sixth generation （G6） were analyzed by using methylation sensitive amplification polymorphism method. The results revealed that the methylated loci accounted for 34.3% to 34.8% of the total loci. Among these methylated loci, 83.1% to 84.8% were full- and hyper-methylated and 15.2% to 16.9% were hemi-methylated. The DNA methylation level decreased from the three-leaf to four-leaf stages in Huhan 3. Differentially methylated loci （DML） between generations or/and between different developmental stages accounted for 4.0% of the total loci, most of which were only related to plant development （57.9%）. Compared to Go, the DNA methylation pattern of G8 changed after drought domestication, at the three-leaf stage, de-methylation accounting for 59.1%, while at the four-leaf stage, re-methylation for 47.9%. Genome-wide alternations of DNA methylation were observed between the two seedling stages, and DML mainly occurred on the gene＇s promoter and exon region. The genes related to DML involved in a wide range of functional biology and participated in many important biological processes.     

水稻高节位分蘖的QTL定位和互作分析

 高节位分蘖是水稻生产中常见的现象,同时高节位分蘖与水稻的驯化也存在密切的联系。利用来源于窄叶青8号与京系17及春江06与台中本地1号的两个加倍单倍体（DH）群体（分别简称为ZJDH群体和TCDH群体）为材料,对水稻高节位分蘖的遗传特征进行了研究。采用复合区间作图法,在ZJDH群体中共定位到qHOT3、qHOT6-1和qHOT8等3个QTL,分别位于第3、6和8染色体上;对TCDH群体的QTL定位共检测到相关位点2个,分别位于第6和12染色体上。同时,在两个群体中分别检测到4对和7对上位性互作位点。QTL比较分析表明在两个群体中分别定位到的第6染色体上的QTL所在区间可能一致,说明水稻第6染色体对高节位分蘖具有重要的影响。     

干旱驯化后节水抗旱稻苗期不同发育时间DNA甲基化模式变化分析

以一个节水抗旱稻沪旱3号为研究材料,经过连续6代干旱胁迫驯化后,采用甲基化敏感扩增多态性方法在苗期不同发育时间对原始代(G0)和第6代(G6)DNA甲基化变化情况进行分析。结果表明,沪旱3号甲基化位点比例约为34%,其中,全甲基化位点约占84%,半甲基化位点约占16%。在苗期,随着沪旱3号生长发育,甲基化水平下降。不同世代或/和不同发育时间差异甲基化位点占总检测位点的4.0%,其中,大部分仅与发育相关(57.9%),而世代之间无差异。干旱驯化后,G6的DNA甲基化模式发生了改变。G6与G0相比,在3叶期,去甲基化事件占主要部分(59.1%);在4叶期,甲基化事件占主要部分(47.9%)。生长发育涉及全基因组DNA甲基化变化,变化位点主要分布在启动子区域和外显子区域。功能聚类分析表明差异甲基化位点相关基因涉及广泛的功能。     

利用分子标记定位水稻芽期耐冷性基因

 利用籼稻品种南京11和粳稻品种巴利拉的F₁花药进行组织培养,获得67个加倍单倍体植株(Doubled haploid, DH)。 以该群体构建了一张包含131个RFLP标记的水稻分子图谱。 以芽期的死苗率为指标, 评定亲本及各 DH系在低温(4～5℃)条件下的耐冷性。结果表明：死苗率在DH群体中呈双峰连续分布, 表明芽期耐冷性是由主基因控制的数量性状。将死苗率作为数量性状进行QTL的区间作图分析, 发现在第7染色体上G379b-RG4区间存在有与耐冷性有关的基因(Cts7) 。     

Grain Quality and Genetic Analysis of Hybrids Derived from Different Ecological Types in Japonica Rice （Oryza sativa）

The performance and inheritance of 7 quality traits were studied using F2 rice grain derived from 8×8 diallel crossing made by employing 8 parents of different ecological japonica rice types. Differences in each trait among 8 parents were not obvious, but in F2 generation, transgressive phenomena were found in all the traits studied, indicating that the genes controlling these traits among parents were segregated. The inheritance of grain width, grain weight, chalkiness score (CS), gelatinization temperature (GT) and gel consistency (GC) were suitable to additive-dominant model, and dominant effect contributed mainly for the 5 traits. The inheritance of grain length (GL) and amylose content (AC) did not fit into additive-dominant model, existing epistatic interactions. Dominant genes for grain width and grain weight had the efficiency of decreasing effect, and dominant genes for CS, GT and GC had the efficiency of enhancing effect. Koshihikari contained more recessive genes for gelatinization temperature than other varieties. Zhendao 88 had more dominant genes in grain width and grain weight than other varieties. Xiushui 04 possessed more dominant genes for GL and GC, and more recessive genes for CS than other varieties.     

水稻籼粳交DH群体幼苗中胚轴长度的QTLs定位和上位性分析

应用籼粳交IR64/Azucena的DH群体及其构建的分子标记遗传图谱,在遮光条件下,通过适温和低温逆境下发芽,测定中胚轴长度。采用QTL Mapper 基因定位软件检测控制中胚轴长度的加性效应QTLs和加性×加性上位性QTLs,在第1、3、6、7、8、12等6条染色体上定位了8个控制中胚轴长度的QTLs,其中在第1、3、7、8染色体上定位了4个具有加性效应的QTLs,位于第7染色体的1个加性效应QTL的增长等位基因来自于父本Azucena,它能使中胚轴伸长0.26 cm,其贡献率达17.5%,其余3个加性效应QTLs的增长等位基因来自于母本IR64,能使中胚轴伸长0.10～0.21 cm,在第3、7、12等3条染色体中共检测到2对加性×加性上位性效应,其贡献率分别为21.62%和2.27%,同时各检测到2对加性效应×环境的互作效应和上位性与环境的互作效应。对应用分子标记辅助育种选育中胚轴伸长的矮秆水稻的可能性进行了讨论。     

杂草稻和栽培稻氮代谢对镉胁迫反应的差异

在水培条件下,以杂草稻和粳稻越光为材料,研究了Cd胁迫对水稻氮吸收和氮代谢酶活性的影响。Cd处理设0、01、10和50 μmol/L 4个水平,以硝酸铵为氮源。结果表明,两种水稻的植株Cd含量和积累量均随溶液Cd处理水平的提高而显著增加,但增加程度两水稻材料存在着明显的差异,在较高Cd水平（10和50 μmol/L）下,杂草稻根系Cd含量和积累量低于越光,但地上部呈相反的趋势,表明杂草稻根系吸收的Cd转运至地上部的量高于越光,揭示出杂草稻地上部具有较强的解毒能力。植株氮含量、积累量以及氮代谢有关酶,如硝酸还原酶、谷草转氨酶和谷丙转氨酶等活性随着Cd处理水平的提高而下降,但Cd胁迫对这些参数的抑制程度两水稻材料之间存在着明显差异,总体上对杂草稻的抑制作用较弱。表明杂草稻对Cd的耐性要强于越光,且几种氮代谢酶活性变化与Cd耐性存在着一定的相关。     

测定水稻硅含量的一种简易方法

 介绍了一种简易的测量水稻硅含量的方法，其主要特点为准确、方便、能批量化测定水稻硅含量。以珍汕97B/密阳46重组自交系群体，两次重复，共498个株系的谷壳、剑叶和茎秆为材料，验证了该方法的可靠性。每份材料测定2份样品，两次测定结果达极显著正相关（P<0.01），线性相关系数分别为0.97954、0.97026和0.98848。取10份代表性样品进行了高温碱熔解法的测定，两种方法测定结果高度一致， r = 0.9993。     

水稻籼粳交DH群体白叶枯病抗性的QTL定位

以中抗白叶枯病的粳稻品种春江06、感病品种TN1及其DH群体为材料,接种白叶枯病菌浙173后,考察了该DH群体的白叶枯病抗性,并进行了数量性状座位(QTL)分析。共检测到控制白叶枯病的3个QTL（qBBR1、qBBR3 1和qBBR7）,分别位于第1、3和第7染色体上,其中第3染色体上的qBBR3 1和第7染色体上的qBBR7加性效应为正值;而位于第1染色体上的qBBR1为负值,3个QTL的总效应达54.4%。春江06中不存在白叶枯病抗性主基因,它的抗性由微效多基因控制,其中在第3和第7染色体上分别有1个QTL。     

水稻出苗顶土动力源研究

在自然条件下,通过设计不同的出苗阻力（覆土深度2 cm、4 cm和6 cm）来测定中胚轴伸长潜力不同的水稻种质的出苗速度、成苗率和芽鞘节及中胚轴的长度,以分析影响水稻种子顶土出苗的动力源。结果表明,水稻种子的出苗动力主要来源于芽鞘节间和中胚轴的伸长。在不同阻力处理条件下,不同种质出苗动力差异较大,其中覆土2 cm的不同种质出苗率相差不明显,与室内发芽率结果比较接近,覆土4 cm和覆土6 cm的两个处理不同种质间出苗率差异明显,长胚轴种质出苗速度快,出苗率高,出苗率与室内发芽率相差不大;而短胚轴种质出苗速度慢,出苗率低,其中春江683的出苗率与室内发芽率相比分别下降了6.5和86.2个百分点。研究表明水稻中胚轴的伸长对覆土较深的种子顶土出苗起到关键作用,长胚轴种质顶土出苗动力来自于芽鞘节与中胚轴的共同作用,出苗动力强;而短胚轴顶土出苗动力主要来源于芽鞘节的伸长,出苗动力较弱。还对通过选育长胚轴直播稻品种解决直播稻易倒伏和出苗差等难题的可能性进行了探讨。     

水稻根系活力的遗传分析

 以典型籼粳交(窄叶青8号/京系17) F1代花培加倍的DH群体为材料，在抽穗期用TTC法考察根系活力。利用已构建的分子连锁图谱，采用区间作图法对根系活力进行QTL分析，在第4染色体的RG449和RG809之间检测到一个QTL。用Epistat软件分析影响根系活力的单个位点和位点间的互作，结果在RG809附近检测到一个单基因，并检测到2个条件型互作和4对互适型互作，这些位点分布在第1、2、4、7、8、9和11染色体上。     

杂交水稻种子穗萌遗传效应的研究

 以穗萌抗性水平不一的6个不育系和7个恢复系为亲本材料，采用不完全双列杂交的试验设计，并运用包括胚乳、细胞质和母体植株三套遗传体系的种子遗传模型及估算遗传方差分量和预测遗传效应值的统计分析方法，对杂交水稻种子穗萌特性的遗传效应进行了研究。结果表明，杂交水稻种子的穗发芽率同时受种子直接加性、种子直接显性和母体加性效应的影响，且以种子直接效应为主，不受细胞质和母体显性效应的影响。在选择育种中，田间穗上发芽率以单粒选为好。而穗萌指数同时受制于种子直接遗传效应、母体效应和细胞质效应，但母体植株中只测到显著的母体加性效应。     

水稻根系育种的意义与前景

 针对目前水稻育种的理论与实践中，主要考虑地上部分的形态、生理性状，而对根系这一重要的营养吸收、物质合成器官的有关性状未能开展明确的育种改良的现状，论述了开展根系育种对改良和提高水稻的品种特性，提高产量和品质的重要性、迫切性以及前景。     

Transgenic Expression of the Recombinant Phytase in Rice （Oryza sativa）

In many plants, phytic acid (phytate, 1, 2, 3, 4, 5, 6-hexakisphosphate) is one of the main storage forms of phosphate. About 80% of phosphorus (P) in cereal plants, including rice is stored as phytic acid [1-2]. P in phytic acid can’t be utilized by monogastric animals including human, while it was estimated that only 1/3 of the total P in most of the vegetal feedstuff could be efficiently utilized by the livestock. Therefore, for animal feed with P supplementation is expected to meet the d…     

水稻柠檬酸合酶编码基因的初步研究

 以粳稻cDNA为模板，设计一对特异性引物，获得编码水稻柠檬酸合酶基因的cDNA序列，全长为1470 bp，ORF区含475个氨基酸，推定蛋白序列与红葡萄、烟草、甜菜、拟南芥和柑橘等物种都有较高的同源性（>70％），氨基末端含一线粒体靶信号。Southern分析表明此基因在水稻基因组中具有多个拷贝数。经0.4 mmol/L IPTG诱导,根据Western印迹分析获得预测的蛋白大小为59 kD。     

农杆菌介导的籼稻转化研究

系统研究了影响农杆菌转化水稻的主要理化因子,以建立一个农杆菌介导法获得大量转基因籼稻植株的体系。农杆菌EHA101(pIG12Hm)感染后的籼稻Pusa Basmati 1(南亚优质米推广品种)悬浮细胞系经3周选择,其Gus阳性的抗性愈伤组织占感染细胞团总数的比例可达10%左右,最高达21.1%,转化频率有显著提高。用另外两个农杆菌菌株/质粒,EHA105(pCAMBIA1201)、EHA105(pCAMBIA1301)转化该悬浮细胞系的频率均在5%~10%之间。以转化植株的克隆数计算,Basmati 1的转化频率为1%~5%。     

Quantitative Trait Loci for Resistance to Stripe Disease in Rice （Oryza sativa）

In order to map the quantitative trait loci for rice stripe resistance, a molecular linkage map was constructed based on the F2 population derived from a cross between Zhaiyeqing 8 and Wuyujing 3. Reactions of the two parents, F1 individual and 129 F2:3 lines to rice stripe were investigated by both artificial inoculation at laboratory and natural infection in the field, and the ratios of disease rating index were scored. The distribution of the ratios of disease rating index in Zhaiyeqing 8/Wuyujing 3 F2:3 population ranged from 0 to 134.08 and from 6.25 to 133.6 under artificial inoculation at laboratory and natural infection in the field, respectively, and showed a marked bias towards resistant parent (Zhaiyeqing 8), indicating that the resistance to rice stripe was controlled by quantitative trait loci (QTL). QTL analysis showed that the QTLs detected by the two inoculation methods were completely different. Only one QTL, qSTV7, was detected under artificial inoculation, at which the Zhaiyeqing 8 allele increased the resistance to rice stripe, while two QTLs, qSTV5 and qSTV1, were detected under natural infection, in which resistant alleles came from Zhaiyeqing 8 and Wuyujing 3, respectively. These results showed that resistant parent Zhaiyeqing 8 carried the alleles associated with the resistance to rice stripe virus and the small brown planthopper, and susceptible parent Wuyujing 3 also carried the resistant allele to rice stripe virus. In comparison with the results previously reported, QTLs detected in the study were new resistant genes to rice stripe disease. This will provide a new resistant resource for avoiding genetic vulnerability for single utilization of the resistant gene Stvb-i.