青藏高原东缘几种树苗对增强紫外线-B和氮供应的响应

大气臭氧的损耗导致了地球表面具有生物学效应的紫外线-B(UV-B)辐射增强。同时，大气成分变化中除了UV-B辐射增强外，氮沉降是一个新近出现而又令人担忧的环境问题，其来源和分布正在迅速扩展到全球范围，并不断向陆地和水生生态系统沉降。本试验在四川省境内的中国科学院茂县生态站内进行，以云山、冷杉、色木槭和红椋子幼苗为模式植物，从生长形态、光合作用、抗氧化能力和矿质营养等方面研究了青藏高原东缘4种树苗对全球变化－增强UV-B辐射和氮供应(氮沉降)的响应。该试验为室外盆栽试验，包括四个处理：(1)大气UV-B辐射+无额外的氮供应(C)；(2)大气UV-B辐射+额外的氮供应(N)；(3)增强UV-B辐射+无额外的氮供应(UV-B)；(4)增强UV-B辐射+额外的氮供应(UV-B+N)。其目的：一方面有助于丰富我国对全球变化及区域响应研究的全面认识，进一步完善在全球气候变化条件下臭氧层削减和氮沉降对陆地生态系统影响的内容；另一方面，在一定程度上有助于我们更好的理解在全球变化下森林更新的早期过程。具体结果如下： 增强的UV-B辐射在生长形态、光合、抗氧化能力、活性氧和矿质营养方面对4种幼苗都有显著的影响。UV-B辐射增强对幼苗的影响不仅与物种有关，而且，还与氮营养水平相关。总体表现为，高的UV-B辐射导致了色木槭和红椋子幼苗叶片的皱缩和卷曲，并降低了色木槭幼苗的叶片数和叶重，在额外的氮供应下，云杉、冷杉和红椋子的叶重也显著地降低了；色木槭和红椋子幼苗叶片的解剖结构受到了增强的UV-B辐射的影响，增强的UV-B辐射显著地降低了色木槭叶片的栅栏组织厚度，提高了红椋子叶片的厚度；增强的UV-B辐射显著地降低了4种幼苗的单株总生物量、植物地下部分的生长、总叶绿素含量 [Chl (a + b)]、净光合速率和最大量子产量(Fv/Fm)，提高了4种幼苗叶片的膜脂过氧化(MDA含量)，改变了植物体不同器官中的矿质元素含量；增强的UV-B辐射提高了冷杉、色木槭和红椋子叶片中的过氧化氢含量(H2O2)、超氧负离子(O2-)生成速率，在额外的氮供应下，云杉叶片中的活性氧含量也显著地提高了；在无额外的氮供应条件下，增强的UV-B辐射显著地提高了4种幼苗叶片中的UV-B吸收物质、脯氨酸含量和抗氧化酶的活性(SOD、POD、CAT、GR和APX)。在额外的氮供应条件下，UV-B辐射的增强却显著地降低了冷杉叶片中脯氨酸含量和红椋子叶片中UV-B吸收物质含量，但是，在4种幼苗叶片中，5种抗氧化酶的活性对UV-B辐射的增强没有明显的规律性，增强的UV-B辐射显著地提高了云杉叶片中的POD、SOD和GR的活性，提高了冷杉叶片中的POD和GR活性，提高了色木槭叶片中的POD、SOD和CAT活性和红椋子幼苗叶片中的POD和SOD活性。从这些结果可知，植物在遭受高的UV-B辐射导致的过氧化胁迫时，植物体内形成了一定的保护机制，但是，这种保护不能抵抗高的UV-B辐射对植物的伤害。 额外的氮供应在生长形态、光合、抗氧化能力、活性氧和矿质营养方面对4种幼苗都有一定的影响，不同幼苗对额外的氮供应响应不同，并且受到UV-B辐射水平的影响。在当地现有的UV-B辐射水平下，额外的氮供应显著地提高了幼苗的单株总生物量、植物地下部分的生长、Chl (a + b)、净光合速率(红椋子除外)、UV-B吸收物质(冷杉除外)、脯氨酸含量(红椋子除外)和部分抗氧化酶的活性，降低了H2O2的含量、O2-的生成速率和MDA含量(红椋子除外)，改变了植物体内部分矿质元素含量，显著地提高了云杉和冷杉叶片中的Fv/Fm。这些指标总体表明，在当地现有大气UV-B辐射水平下，额外的氮供应对植物的生长和发育是有利的。在增强的UV-B辐射水平下，4种幼苗的生长形态和光合大部分指标都没有受到额外氮供应的影响，额外的氮供应提高了红椋子幼苗的单株总生物量和Chl (a + b)含量，提高了冷杉和色木槭叶片中的活性氧含量和MDA含量，却降低了红椋子叶片中的活性氧含量；额外的氮供应也提高了云杉、色木槭和红椋子叶片中UV-B吸收物质和脯氨酸含量，降低了冷杉叶片中UV-B吸收物质和脯氨酸含量；在抗氧化酶活性方面，额外的氮供应降低了云杉、冷杉叶片中5种抗氧化酶的活性和红椋子叶片中POD和GR的活性，提高了色木槭叶片中的POD和SOD的活性；4种幼苗植物体内的矿质元素含量对额外的氮供应没有显著的规律性。从这些结果可知，在高的UV-B辐射下，额外的氮供应提高了云杉、冷杉和色木槭幼苗对高的UV-B辐射的敏感性，然而，额外的氮供应却促进了红椋子幼苗的生长，原因可能是，在高的UV-B辐射下，额外的氮供应增加了红椋子叶片的厚度、叶重和叶片数，降低了叶片中活性氧含量的结果。表明在高的UV-B辐射水平下，额外的氮供应降低了红椋子幼苗对高的UV-B辐射的敏感性。 在全球变化的趋势下，UV-B辐射增强和氮沉降可能同时存在，我们的研究表明，与大气UV-B辐射+无额外的氮供应处理相比，增强UV-B辐射+额外的氮供应处理显著地降低了幼苗的单株总生物量(红椋子除外)、Chl (a + b)、净光合速率、Fv/Fm(冷杉除外)和MDA含量(红椋子除外)，提高了活性氧含量 (云杉除外)、UV-B紫外吸收物质含量(冷杉除外)、脯氨酸含量和部分抗氧化酶的活性，改变了植物体不同器官中的矿质元素含量。结果表明，在当地现有条件下，全球变化(UV-B辐射增强和氮沉降)对云杉、冷杉和色木槭幼苗的生长是不利，尽管植物体内一些抗氧化性指标提高了，然而，却对红椋子幼苗的单株总生物量的累积没有显著的影响。 The depletion of the ozone led to the increase of ultraviolet-B (UV-B) with biological effects in the earth’s surface. At the same time, except for enhanced UV-B radiation, nitrogen deposition was an anxious environmental problem at present, rapidly expanding to the global scope and continuously depositing to land and aquatic ecosystem. The experiment was conducted in Maoxian Ecological Station of Chinese Academy of Sciences, Sichuan province, China. Picea asperata, Abies faxoniana, Acer mono Maxim and Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings were selected as model plants to assess the effects of enhanced UV-B radiation and supplemental nitrogen supply on growth, morphological, photosynthesis, antioxidant and mineral nutrient traits of 4 species seedlings in east Qinghai-Tibetan Plateau. The experiment was potted outdoor, including 4 treatments: (1) ambient UV-B without supplemental nitrogen (control, C); (2) ambient UV-B with supplemental nitrogen (N); (3) enhanced UV-B without supplemental nitrogen (UV-B); (4) enhanced UV-B with supplemental nitrogen (UV-B+N). One hand, it was helpful for enriching our country to comprehensive understanding of the researches in the global change and the region response, further perfecting the effects of the depleted ozone layer and nitrogen deposition on land ecosystem under the global change; the other hand, it was favorable for us to better understanding of the early process of forest renews under the global change. The results were as follows: Enhanced UV-B radiation had significant effects on 4 species seedlings in growth, morphological, photosynthesis, antioxidant and mineral nutrient traits of 4 species seedlings. The effects of enhanced UV-B on plants were not only related with species, but also related with nitrogen nutrient level. Generally, the increase of UV-B radiation led to the shrinkage and curl of leaves in Acer mono Maxim and Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings, and reduced the number of leaf and leaf weight of Acer mono Maxim seedlings, under supplemental nitrogen supply, leaf weight of Picea asperata, Abies faxoniana and Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings significantly also reduced; the anatomical features of leaf in Acer mono Maxim and Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings affected by enhanced UV-B radiation, the increase of UV-B radiation markedly reduced the palisade tissue thickness of Acer mono Maxim leaf and enhanced the leaf thickness of Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings; the enhanced UV-B radiation significantly reduced total biomass per plant of 4 species seedlings, the growth of the underground parts, Chl (a + b), net photosynthetic rate and maximum potential quantum yield of photosynthesis (Fv/Fm), and increased the degree of lipid peroxidation (MDA content) and changed the content of mineral elements in different parts of plants; the enhanced UV-B radiation also increased the rate of superoxide radical (O2-) production and hydrogen peroxide (H2O2) content in leaves of Abies faxoniana, Acer mono Maxim, Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings, under supplemental nitrogen supply, the reactive oxygen species in leaves of Picea asperata seedlings also significantly increased by enhanced UV-B radiation; under without supplemental nitrogen supply, enhanced UV-B radiation evidently induced an increase in UV-B absorbing compounds, proline content and the activities of antioxidant enzymes (SOD, POD, CAT, GR and APX) of leaves in 4 species seedlings. Under supplemental nitrogen supply, enhanced UV-B radiation induced a decrease in proline content of leaves in Abies faxoniana seedlings and UV-B absorbing compounds of leaves in Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings, but, there were no obvious rules in the activities of five antioxidant enzymes of 4 species seedling leaves to enhanced UV-B radiation, enhanced UV-B radiation significantly increased the activities of POD, SOD and GR in Picea asperata leaves, the activities of POD and GR in Abies faxoniana leaves and the activities of POD, SOD and CAT in Acer mono Maxim leaves. The results indicated that some protective mechanism was formed when plants were exposed to enhanced UV-B radiation, but the protection could not counteract the harm of high UV-B radiation on plants. Supplemental nitrogen supply had some effects on 4 species seedlings in growth, morphological, photosynthesis, antioxidant and mineral nutrient traits. The response of 4 species seedlings was different to supplemental nitrogen supply, and was affected by UV-B levels. Under local ambient UV-B radiation, supplemental nitrogen supply significantly increased the total biomass per plant, the growth of underground parts, Chl (a + b), net photosynthetic rate (except for Acer mono Maxim seedlings), UV-B absorbing compounds (except for Abies faxoniana seedlings), proline content (except for Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings) and the activities of some antioxidant enzymes, and reduced H2O2 content, the rate of O2- production and MDA content (except for Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings) and changed the content of mineral elemental in different parts; supplemental nitrogen supply also evidently increased Fv/Fm in Picea asperata and Abies faxoniana seedlings. These results indicated that supplemental nitrogen supply was favorable for the growth of plants under local ambient UV-B radiation. Under enhanced UV-B radiation, mostly parameters in growth and morphology of 4 species seedlings were not affected by supplemental nitrogen supply. Supplemental nitrogen supply increased the total biomass per plant and Chl (a + b) of Swida hemsleyi (Schneid. et Wanger.) Sojak seedling, increased the reactive oxygen species and MDA content in Abies faxoniana and Acer mono Maxim leaves, and reduced the reactive oxygen species in Swida hemsleyi (Schneid. et Wanger.) Sojak leaves; supplemental nitrogen supply also increased UV-B absorbing compounds and proline content in Picea asperata, Acer mono Maxim and Swida hemsleyi (Schneid. et Wanger.) Sojak leaves, decreased UV-B absorbing compounds and proline content in Abies faxoniana leaves; in the activities of antioxidant enzymes, supplemental nitrogen supply significantly reduced the activities of antioxidant enzymes in Picea asperata and Abies faxoniana leaves and the activities of POD and GR in Swida hemsleyi (Schneid. et Wanger.) Sojak leaves, and increased the activities of POD and SOD in Acer mono Maxim leaves; the content of mineral elements in 4 species seedlings was no significantly rule to supplemental nitrogen supply. We knew from the results, under enhanced UV-B radiation, supplemental nitrogen supply made Picea asperata, Acer faxoniana and Acer mono Maxim seedlings more sensitivity to enhanced UV-B radiation, however, accelerated the growth of Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings. The reason was probably that supplemental nitrogen supply increased the leaf thickness, leaf weight and leaf number, reduced the reactive oxygen content of leaf in Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings grown under high UV-B radiation. This showed that supplemental nitrogen supply reduced the sensitivity of Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings to high UV-B radiation. Under the tendency of the global change, enhanced UV-B radiation and nitrogen deposition may probably coexist. The results showed, compared with the treatment of ambient UV-B radiation without supplemental nitrogen supply, the treatment of enhanced UV-B radiation with supplemental nitrogen supply significantly reduced the total biomass per plants (except for Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings), Chl (a + b), net photosynthetic rate, Fv/Fm and MDA content (except for Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings), and increased reactive oxygen content (except for Picea asperata seedlings), UV-B absorbing compounds (except for Abies faxoniana seedlings), proline content and part antioxidant enzymes, and changed the content of mineral elements of different parts. The results indicated that the global change (enhanced UV-B and nitrogen deposition) were not favorable for the growth of plants under local ambient UV-B radiation and nitrogen nutrient level,, though increased some antioxidant indexes, however, the treatment of enhanced UV-B with supplement nitrogen supply did not significantly affect on the biomass accumulation of Swida hemsleyi (Schneid. et Wanger.) Sojak seedlings.

CO2浓度升高对红桦幼苗生理与生长的影响

大气CO2浓度的增加已经成为不可争议的事实。预计本世纪末大气CO2浓度将增加到约700µmol mol-1。森林年光合产量约占陆地生态系统年光合产量的70%。森林树木是一个巨大的生物碳库，约占全球陆地生物碳库的85%。森林树木对CO2的固定潜力是缓解由大气CO2浓度升高引起的未来全球气候变化问题的决定性因子之一。红桦（Betula albosinensis Burk.）是川西亚高山采伐迹地自然或人工恢复的重要树种。本研究以1a红桦幼苗为模式植物，采用人工模拟的方法，研究CO2浓度升高对不同种内竞争强度（种群水平）下红桦幼苗的生理特征、生长、干物质积累及其分配的影响，探讨在种内竞争生长条件下红桦幼苗的“光合适应机理”与生长特征，为西南亚高山森林生产力对未来全球变化的预测提供重要参考。 本研究的主要结果如下： 1）在种内竞争生长条件下红桦幼苗经过CO2浓度升高熏蒸4个月后，叶片出现“光合适应”现象。与对照相比，低种植密度（28株m-2）和高种植密度（84株m-2）条件下的红桦幼苗净光合速率（A）、气孔导度（gs）、蒸腾速率（E）、表观量子产量（AQY）和羧化速率（CE）显著降低，而水分利用效率（WUE）则显著提高。CO2浓度升高处理的红桦幼苗叶片Rubisco活性、单位叶面积N浓度、叶绿素a、叶绿素b和类胡萝卜素浓度都显著降低。但CO2浓度对红桦幼苗的叶绿素a与叶绿素b的比值没有显著影响。CO2浓度升高显著增加红桦幼苗单位叶面积的非结构性碳水化合物（TNC）浓度，结果是红桦幼苗的比叶面积（SLA，cm2 g-1）显著降低。 2）与对照相比，CO2浓度升高处理的红桦幼苗高、基径、单叶面积和侧枝的相对生长速率（R GR）显著提高，尤其在试验处理的早期。CO2浓度升高既增加单株红桦幼苗总叶片数量又增加单叶面积，结果是单株红桦幼苗的总叶面积比对照显著增加。 3）CO2浓度升高处理显著增加红桦幼苗干物质积累（尤其是细根生物量），改变了红桦幼苗生物量的分配格局。与对照相比，CO2浓度升高处理的红桦幼苗叶重比（LWR）、叶面积比（LAR）、叶根重比（Wl/Wr）和源汇重比（leaf weight to non-leaf weight ratio, Wsource/Wsink）显著下降（高种植密度的LWR除外），而根冠比（R/S）则显著增加。在两种种植密度条件下，CO2浓度升高显著增加红桦幼苗根生物量的分配比率，显著降低叶片的生物量分配比率，对主茎、侧枝以及地上生物量的分配比率不变或约有下降。 总之，长期生长在CO2浓度升高条件下的红桦幼苗光合能力下降，并伴随Rubisco活性、叶N浓度、光合色素浓度的显著降低以及TNC浓度的显著增加。支持树木光合速率下降与Rubisco活性、叶N浓度下降以及TNC浓度增加紧密相关的假设。CO2浓度升高处理红桦幼苗的早期相对生长速率大大高于对照，而后期迅速下降，说明红桦幼苗生物量的显著增加主要归功于CO2浓度升高的早期促进作用和叶面积的显著增加。CO2浓度升高显著增加红桦幼苗根系生物量和根冠比，表明红桦幼苗“额外”固定的C向根系转移。 The steady increae of atmospheric CO2 concentration（[CO2]）has been inevitable fact. Models predict that the atmospheric [CO2] will increase to about 700µmol mol-1 at the end of the twenty-first century. As trees constitute a majoor carbon reservoir–85% of total plant carbon is found in forest, and their ability to sequester carbon is a key determinant of future global change problems caused by increases in atmospheric CO2. In addition to the role of forests in the global carbon cycle, inceased growth could be of economic benefit, for example, offsetting deleterious effects of climatic changes. Betula albosinensis (Burk.) usually emerges as the pioneer species in initial stage and as constructive species in later stages of forest community succession of mountain forest area, and also is one of important tree species for afforestation in logged area, in southwesten China. In this experinment, Betula albosinensis seedling (one-year-old) was used as the model plant. B. albosinensis seedlings were grown under two all-day [CO2], ambient (about 350 µmol·mol-1) and elevated [CO2] (about 700 µmol·mol-1), and two planting densities of 28 plants per m2 and 84 plants per m2. The objectives were to characterize birch mature leaf photosynthesis, growth, mass accumulation and allocation responses to long-tern elevated growth [CO2] under the influences of neighbouring plants, and to assess whether elevated [CO2] regulated birch mature leaf photosynthetic capacity, in terms of leaf nitrogen concentration (leaf [N]), activity of ribulose bisphosphate carboxygenase (Rubisco), Rubisco photosynthetic efficiency, and total nonstructural carbohydrates (TNC) concentration, and also to provide a strong reference to predict the productivity of subalpine forests under the future global changes. The results are as follows: 1) B.albosinensis seedlings exposed to elevated [CO2] for 120 days, photosynthetic acclimation phenomena occurred. At two planting densities, leaves of birch seedlings grown under elevated [CO2] had lower net photosynthetic rate (A), stomatal conductance (gs), transpiration (E), apparent quantum yield (AQY) and carboxylated efficiency (CE) and higher water use efficiency (WUE), compared to those of B.albosinensis seedlings grown under ambient [CO2]. Based on the leaf area, leaf [N], Rubisco activity and photosynthetic pigments concentrations of B. albosinensis seedlings grown under elevated [CO2] were significantly lower than those grown under ambient [CO2]. The ratio of chlorophyll a to chlorophyll b concentration was not affected by elevated [CO2]. Under elevated [CO2], the TNC concentration per unit leaf area significantly increased, resulting in significant decrease in specific leaf area. Thus leaf photosynthetic capacity of B. albosinensis seedlings would perform worse under rising atmospheric [CO2] and the influences of neighbouring plants. 2) Under elevated [CO2], the relative growth rate (RGR) of B. albosinensis seedlings height, basal diameter, a leaf area and branch length significantly increased, especially at the initial stage of exposure to elevated [CO2], and a leaf area and leaf numbers per B. albosinensis seedling also significantly increased. Thus the total leaf area per B. albosinensis seedling was significantly increased under elevated [CO2]. 3) As the increase of RGR and total leaf area, biomass of B. albosinensis seedling grown elevated [CO2] was higher, compared to that of B.albosinensis seedlings grown at ambient [CO2]. Elevated [CO2] changed the biomass allocation pattern of B. albosinensis seedling. At two planting densities, B. albosinensis seedlings grown elevated [CO2] had lower leaf weight to total weight ratio (LWR), leaf area to total weight ratio (LAR) and leaf weight to non-leaf weight ratio (Wsource/Wsink), but higher root weight to shoot weight ratio (R/S), compared to those of B.albosinensis seedlings grown at ambient [CO2]. Under elevated [CO2], roots biomass to total biomass ratio was signigicantly increased, leaves biomass to total biomass ratio was significantly decreased. The main stem and branch biomass to total biomass ratio were not affected by elevated [CO2]. In conclusion, our results supported the hypothesis that the decline in photosynthetic capacity of C3 plants will appear after long-term exposure to elevated [CO2], accompanying with the significant decrease in Rubisco activity, leaf N concentration, photosynthetic pigments concentration, and significant increase in total non-structural carbohydrates concentration. Our results also have shown that the increase of biomass of B. albosinensis seedlings should be attributed to initial stimulation on RGR and total leaf area resulted from elevated [CO2]. Under elevated [CO2], the extra carbon sequestered by B.albosinensis seedlings transferred into under-ground part because of increase in root biomass and R/S.

遮荫与外源脱落酸喷施对粗枝云杉(Picea asperata Mast.)不同种群抗旱性的影响

本研究通过粗枝云杉不同种群进行的温室半控制试验，采用植物生态学、生理学和生物化学的研究方法，系统地研究了粗枝云杉不同种群抗旱性的生长、形态、生理和生化机理，并结合有关研究进行综合分析，得出主要研究结论如下： 1.粗枝云杉对干旱胁迫的综合反应 粗枝云杉在干旱胁迫下的适应机制为：（1）相对生长速率及植株结构的调整：干旱胁迫下虽然植株相对生长速率显著降低，且有相对较多的生物量向根部分配，但并未发现细根/总根比增加。（2）粗枝云杉对干旱胁迫的光合作用表现为：干旱胁迫显著地降低了控制的理想条件下的气体交换，但干旱胁迫对PSII最大光化学效率（Fv/Fm）没有影响，表明干旱并未影响到光合机构。（3）干旱还影响了很多生理生化过程，包括渗透调解物质（游离脯氨酸）、膜脂过氧化产物、脱落酸（ABA）含量的增加，以及保护酶活性的升高。这些结果证明植物遭受干旱胁迫后发生了一系列的形态、生理和生化响应，这些变化能提高干旱时期植物的存活和生长能力。 2.粗枝云杉不同种群对干旱胁迫反应的种群差异 粗枝云杉三个种群－干旱种群（四川丹巴和甘肃迭部）和湿润种群（四川黑水）对干旱适应不同，这种不同应归因于它们采用的用水策略不同：在水分良好和干旱胁迫条件下，受试种群在相对生长速率和水分利用效率（WUE）方面都表现出显著的种群间差异。与湿润种群相比，干旱种群在两种水分条件下有更高的WUE。粗枝云杉不同种群的碳同位素组分（δ13C）只在干旱胁迫下有显著差异，并且这种差异在水分良好时比干旱胁迫条件下小，说明生理响应和干旱适应性之间的关系受植物内部抗旱机制和外部环境条件（如水分可利用性）或两者互作效应的影响。这些结果说明干旱种群和湿润种群所采用的用水策略不同。干旱种群有更强的抗旱能力，采用的是节水型的用水策略，而湿润种群抗旱能力较弱，采用的是耗水型的用水策略。 3. 遮荫对粗枝云杉不同种群抗旱性影响 干旱胁迫显著降低了全光条件下叶相对含水量（RWC）、相对生长速率、气体交换参数、PSII的有效量子产量（Y），提高了非光化学猝灭效率（qN）、水分利用效率、脯氨酸（PRO）积累、脱落酸（ABA）含量及保护酶活性。然而这种变化在遮荫条件下不明显。我们得出结论适度遮荫降低了干旱对植物的胁迫作用。另一方面，在干旱条件下，与湿润种群相比，干旱种群抗旱性更强，表现在干旱种群净光合速率与单位重量上叶氮含量（Nmass）降低较少。另外，干旱种群表现出更为敏感的气孔导度，更高的热耗散能力（qN）能力、用水效率、ABA积累、保护酶活性，以及更低的总用水量、相对生长速率。这一结果表明这两种群采用不同的生理策略对干旱和遮荫做出反应。许多生长和生理反应差异与这两个种群原产地气候条件相适应。 4. 外源脱落酸(ABA)喷施对粗枝云杉不同种群抗旱性影响 外源ABA喷施在干旱和水分良好条件下均不同程度地提高了根/茎比，表明根和茎对ABA敏感程度不同。实验结果还表明，外源ABA喷施对这两个种群在干旱胁迫期间影响不同。干旱胁迫期间，伴随着ABA喷施，湿润种群净光合速率(A)显著降低，而干旱种群净光合速率变化不明显。另一方面，外源ABA喷施显著提高了干旱条件下干旱种群的单位叶面积重（LMA）、根/茎比、细根/总根（Ft）比、水分利用效率（WUE）、ABA含量, 以及保护酶活性。然而，外源ABA喷施对湿润种群的上述测定指标没有显著影响。这一结果表明干旱种群对外源ABA喷施更为敏感, 反应在更大的气孔导度降低，更高的生物量可塑性，及更高的水分利用效率、ABA含量和保护酶活性。综上所述，我们得出结论，粗枝云杉对外源ABA敏感性因种群的不同而不同。该研究结果可为两个明显不同种群在适应分化方面提供强有力的证据。 Arid or semi-arid land covers more than half of China's land territory. In arid systems, severe shortages of soil water often coincide with periods of high temperatures and high solar radiation, producing multiple stresses on plant performance. Protection from high radiation loads in shaded microenvironments during drought may compensate for a loss of productivity due to reduced irradiance when water is available. Additionally, ABA, a well-known stress-inducible plant hormone, has long been studied as a potential mediator for induction of drought tolerance in plants. Picea asperata Mast., which is one of the most important tree species used for the production of pulp wood and timber, is a prime reforestation species in western China. In this experiment, different population of P. asperata were used as experiment material to study the adaptability to drought stress and population differences in adaptabiliy, and the effects of shade and exogenous abscisic acid (ABA) application on the drought tolerance. Our results cold provide a strong theoretical evidence and scientific direction for the afforestation, and rehabilitation of ecosystem in the arid and semi-arid area, and provide a strong evidence for adaptive differentiation of different populations, and so may be used as criteria for species selection and tree improvement. The results are as follows: 1. A large set of parallel response to drought stress Drought stress caused pronounced inhibition of the growth and increased relatively dry matter allocation into the root; drought stress also caused pronounced inhibition of photosynthesis, while drought showed no effects on the maximal quantum yield of PSII photochemistry (Fv/Fm) in dark-adapted leaves, indicating that drought had no effects on the primary photochemistry of PSII. However, in light-adapted leaves, drought reduced the quantum yield of PSII electron transport (Y) and increased the non-photochemical quenching (qN). Drought also affected many physiological and biochemical processes, including increases in superoxide dismutase (SOD), ascorbate peroxidase (APX) activities, malondialdehyde and ABA content. These results demonstrate that there are a large set of parallel changes in the morphological, physiological and biochemical responses when plants are exposed to drought stress; these changes may enhance the capability of plants to survive and grow during drought periods. 2. Difference in adaptation to drought stress between contrasting populations of Picea asperata There were significant population differences in growth, dry matter allocation and water use efficiency. Compared with the wet climate population (Heishui), the dry climate population (Dan ba and Jiebu) showed higher LMA, fine root/total root ratio and water use efficiency under drought-stressed treatments. The results suggested that there were different water-use strategies between the dry population and the wet population. The dry climate population with higher drought tolerance may employ a conservative water-use strategy, whereas the wet climate population with lower drought tolerance may employ a prodigal water-use strategy. These variations in drought responses may be used as criteria for species selection and tree improvement. 3. The effects of shade on the drought tolerance For both populations tested, drought resulted in lower needle relative water content (RWC), relative growth rate (RGR), gas exchange parameters and effective PSII quantum yield (Y), and higher non-photochemical quenching (qN), water use efficiency (WUE), proline (PRO) and abscisic acid (ABA) accumulation, superoxide dismutase (SOD), ascorbate peroxidase (APX) activities as well as malondialdehyde (MDA) levels and electrolyte leakage in sun plants, whereas these changes were not significant in shade plants. Our study results implied that shade, applied together with drought, ameliorated the detrimental effects of drought. On the other hand, compared with the wet climate population, the dry climate population was more tolerant to drought in the sun treatment, as indicated by less decreases in A and mass-based leaf nitrogen content (Nmass), more responsive stomata, greater capacity for non-radiative dissipation of excitation energy as heat (analysed by qN), and higher WUE，higher level of antioxidant enzyme activities，higher ABA accumulation as well as lower MDA content and electrolyte leakage. Many of the differences in growth and physiological responses reported here are consistent with the climatic differences between the locations of the populations of P. asperata. 4. The effects of exogenous abscisic acid (ABA) application on the drought tolerance For both populations tested, exogenous ABA application increased root/shoot ratio (Rs) under well-watered and drought-stressed conditions, indicating that there was differential sensitivity to ABA in the roots and shoots. However, it appeared that ABA application affected the two P. asperata populations very differently during drought. CO2 assimilation rate (A) was significantly decreased in the wet climate population, but only to a minor extent in the dry climate population following ABA application during soil drying. On the other hand, ABA application significantly decreased stomatal conductance (gs), transpiration rate (E) and malondialdehyde (MDA) content, and significantly increased leaf mass per area (LMA), Rs, fine root/total root ratio (Ft), water use efficiency (WUE), ABA contents, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities under drought condition in the dry climate population, whereas ABA application did not significantly affect these parameters in the wet population plants. The results clearly demonstrated that the dry climate population was more responsive to ABA application than the wet climate population, as indicated by the strong stomata closure and by greater plasticity of LMA and biomass allocation, as well as by higher WUE, ABA content and anti-oxidative capacity to defense against oxidative stress, possibly predominantly by APX. We concluded that sensitivity to exogenous ABA application is population dependent in P. asperata. Our results provide strong evidence for adaptive differentiation between populations of P. asperata.

生姜化感作用机理及其栽培模式研究

近年来，随着对作物重茬（连年种植）障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。而作为重要调料和药用植物的生姜，其连作障碍也备受关注，系统地研究生姜化感作用将有助于理解和最终解决生姜连作障碍问题。本文通过研究生姜不同部位、不同浓度的水浸液对与其间作的两个物种（大豆和四季葱）种子的萌发及幼苗生长的影响，从而证明生姜化感作用的存在；并通过温室盆栽实验研究了生姜的自毒作用（即研究生姜不同部位、不同浓度的水浸液对其幼苗的形态、生理生化、光合作用、土壤酶、土壤微生物多样性及土壤养分的影响），从而揭示生姜退化和衰老的机制，并为生姜筛选出合适的间作物种提供科学依据，对生姜连作障碍提出科学的解决方法。主要研究结果如下： 1. 与对照相比，生姜所有部位（根茎、茎、叶）、所有浓度（10、20、40、 80 g l-1）的水浸液均抑制了大豆种子和葱籽的萌发率、幼苗生长、水分吸收和脂肪酶活性，并且其抑制程度随着水浸液浓度的增加而增强，其生姜各部位水浸液抑制效应的强弱顺序为茎＞叶＞根茎。这一结果表明生姜根茎、茎、叶含有能够抑制大豆种子和葱籽种子萌发和幼苗生长的水溶性化感物质。根茎是生姜的主要收获部位，而生姜的残株（主要是茎和叶）应该从大田中处理掉以减轻其抑制效应。生姜水浸液中主要化感成分包括：根茎水浸液中主要是丁香酸和伞花内脂；茎水浸液中主要是阿魏酸，且其含量最高为73.4 ug/g；叶水浸液中除了阿魏酸，其他六种物质均检测出来，但含量较高的主要有丁香酸、伞花内脂和香豆酸。 2. 生姜茎和叶不同浓度的水浸液均显著抑制了生姜幼苗的株高、每株叶片数和叶面积，其抑制程度随着水浸液浓度的增加而有所增强，而生姜幼苗每株分枝数差异不显著；同时生姜水浸液也极大程度地影响了生姜幼苗的生物量（包括地下生物量、地上生物量和总生物量，均为鲜重）。在同一浓度下，茎水浸液对生姜幼苗形态指标及生物量指标均显示出最强的抑制作用，叶水浸液次之，根茎水浸液最弱。与对照相比，低浓度的生姜根茎水浸液提高了生姜幼苗叶片内四种抗氧化酶（SOD、POD、CAT、APX）活性，高浓度的根茎水浸液抑制了四种抗氧化酶活性，而茎和叶水浸液均随着浓度的增加而抑制了四种抗氧化酶活性，三种水浸液均随着浓度的增加降低了生姜幼苗叶片内叶绿素的含量，而增加了生姜幼苗叶片的相对电导率和丙二醛含量。同时，三种水浸液均随着浓度的增加降低了生姜幼苗的光合参数（包括胞间CO2浓度、气孔导度、蒸腾速率及净光合速率）。 3. 三种生姜水浸液对所测六种土壤酶活性均产生了不同程度的影响，其中影响最大的是酸性磷酸酶和蔗糖酶，在10 g l-1 时就达到了显著水平，并且所有酶均有随着水浸液浓度增加而增大的趋势；相同部位的水浸液随着浓度的增加，细菌和真菌的数量呈增加趋势，而放线菌的数量呈减少趋势；三种生姜水浸液均随着浓度的增加降低了土壤中有机质的含量，加剧了土壤中硝态氮含量的积累，根茎水浸液对土壤有效磷、速效钾和铵态氮均显示出低浓度提高其含量而高浓度降低其含量的趋势，而茎和叶水浸液则随着浓度的增加均降低了其含量。 4. 与生姜单作相比，所有间作系统均在旺盛生长期和收获期不同程度地提高了土壤酶活性，同时也增加了土壤细菌数量及土壤微生物总数但不显著；所有间作系统在旺盛生长期和收获期均不同程度地影响了土壤真菌及放线菌数量（增加或减少），所有间作系统间的多样性指数差异不显著，除了旺盛生长期四种作物（生姜-大豆-四季葱-大蒜）的间作模式显著降低了多样性指数，其值仅为生姜单作的33.18%；生姜与大豆间作不仅提高了19.6%的生姜产量而且获得了较好的经济效益，并且，所有间作系统均显著抑制了生姜姜瘟病的发生。 5. 不同栽培模式不同程度地影响了收获期生姜的株高、分枝数、根茎产量及内在品质。其中处理2显著地促进了生姜的分枝（10.5%），同时处理2、3和4也促进了生姜的生长（株高分别增加了15.0%、11.4%和14.0%），并且这三个处理提高了生姜的产量；处理2和3能有效提高生姜块茎中维生素C（分别较单作生姜显著提高了3.29%和4.05%）、处理3显著提高了可溶性糖（8.2%）、姜辣素（4.6%）和蛋白质等有益物质的含量，降低硝酸盐有害物质的含量（处理2显著降低了14.0%），改善了姜块的外观和内在品质。并且，生姜与大豆间作具有最高的纯收入和产投比，分别较生姜单作提高了24.80%和8.8%。Recently, allelopathy has been more and more paid attentions by national and foreign scholars with profound research on reasons of crop replanted (continuous planted) obstacle. Ginger rhizome is valuable all over the world either as a spice or herbal medicine and ginger replanted obstacle is also paid attentions. Systematic research on ginger allelopathy will contribute to understanding and ultimate solving problem of ginger replanted obstacle. The effects of ginger aqueous extracts with different parts and concentrations on seed germination and early seedling growth of soybean and chive were studied in this article to testify that ginger existed allelopathy. Furthermore, ginger autotoxicity was also studied by pot experiment in greenhouse (namely research on effects of ginger aqueous extracts with different parts and concentrations on morphological indexes, physiological and biochemical indexes, photosynthesis, soil enzymes, soil microbial diversity and soil nutrients) to reveal mechanism of ginger degeneration and senescence, provide scientific basis for selecting appropriate intercropping species and put forward scientific resolvent for ginger replanted obstacle. The main results were as follows: 1. All aqueous extracts at all concentrations inhibited seed germination, seedling growth, water uptake and lipase activity of soybean and chive compared with the control, and the degree of inhibition increased with the incremental extracts concentration. The degree of toxicity of different ginger plant parts can be classified in order of decreasing inhibition as stem＞leaf＞rhizome. The results of this study suggested that rhizome, stem and leaf of ginger contained water soluble allelochemicals which could inhibit seed germination and seedling growth of soybean and chive. The rhizome is the main harvested part of ginger. The residue (mainly stems and leaves) of the ginger plant should be removed from the field so as to diminish its inhibitory effect. The main allelopathic components of three kind of aqueous extracts were as follows: Rhizome extract chiefly contained syringic acid and vmbelliferone and stem extract mainly contained frulic acid whose content was the highest (73.4 ug/g). The other six substances were detected except of frulic acid, but only contents of syringic acid, vmbelliferone and p-coumaric acid were higher. 2. Stem and leaf aqueous extracts of ginger with different concentrations significantly inhibited plant height, leaf numbers per plant and leaf area, and the degree of inhibition increased with the incremental extracts concentration. However, tiller number per plant of ginger seedling showed no significant difference. At the same time, ginger aqueous extracts also influenced biomass including under-ground biomass, above-ground biomass and total biomass (fresh weight) to a large extent. Under the same concentration, stem aqueous extract showed the mostly inhibitory effect on morphological indexes and biomass indexes of ginger seedling. Rhizome aqueous extract showed the leastly inhibitory effect and leaf aqueous extract was intervenient. Enhanced concentration of ginger aqueous extracts significantly reduced total chlorophyll content, accompanying with increases in memberane permeability (REL) and lipid peroxidation (MDA). Compared with the control, rhizome ginger aqueous extract of lower concentration (10 g l-1) increased the activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT; ascorbate peroxidase, APX) of ginger leaf tissue and higher concentration inhibited the activities of four antioxidant enzymes. However, stem and leaf aqueous extract inhibited the activities of four antioxidant enzymes with increase in concentration. Meanwhile, enhanced concentration of ginger aqueous extracts significantly reduced photo-parameters of ginger seedling (including CO2 concentration, stoma conductivity, net photosynthesis rate and transpiration rate). 3. Rhizome, stem and leaf ginger aqueous extract showed different effect on six soil enzyme activities, and acid phosphatase and invertase showed significant effect when aqueous extract concentration got 10 g l-1. Furthermore, six soil enzyme activities increased with increase in aqueous extract concentration. Bcterial and fungi number tended to increase while antinomyces tented to decrease with the increase in aqueous extract concentration of identical part. Ginger aqueous extracts reduced soil organic matter content with increased concentration, accompanying with NO3-—N accumulation in soil. Rhizome aqueous extract showed the same tendency for available P, available K and NH4+—N, namely lower concentration increased their contents in soil and higher concentration reduced their contents. While stem and leaf aqueous extracts reduced their contents with the increamental concentration. 4. All intercropping systems increased soil enzyme activities to different extent both at VGS and at HS compared to solo ginger. All intercropping systems increased the colony numbers of soil bacteria and total of soil microbe but not significantly either at VGS or at HS. All intercropping systems increased the colony numbers of soil fungi and actinomytes to a different extent (increase or decrease) both at VGS and at HS. For DI, difference between all cultivation patterns and S-G was not significant either at VGS or at HS except that G-S-C-G whose value was only 33.18% of S-G at VGS significantly decreased. G-S not only increased ginger yield by 19.6% but also obtained better economic benefit. Furthermore, all intercropping systems significantly inhibited occurrence of bacterial wilt of ginger. 5. Different cultivated pattern influenced plant height, tiller numbers, rhizome yields and intrinsic quality of ginger. Treatment 2 significantly facilitated tiller occurring (10.5%). Treatment 2, 3 and 4 promoted ginger growth (plant height respectively increased 15.0%、11.4% and 14.0%) and enhanced rhizome yields. Treatment 2 and 3 effectively increased vitamin C content (significantly increased 3.29% and 4.05% compared to solo ginger). Treatment 3 significantly increased contents of beneficial substances such as soluble sugar (8.2%), gingerols (4.6%) and protein. Treatment 2 significantly decreased contents of deleterious substance namely nitrate (14.0%) and improved appearance and intrinsic quality of ginger rhizome. Furthermore, treatment 2 (ginger/soybean intercropping) could obtain better economic benefit and showed the highest net income and ratio of benefit and cost whose values respectively increased by 24.80% and 8.8% compared to solo ginger.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

水稻同源四倍体三系法杂种优势利用研究

水稻是重要的粮食作物，其产量的增加和品质的改良都是关系国计民生的大事。就我国现阶段的国情而言，水稻产量在现有水平上稳步提升仍是未来十几年甚至几十年农业生产最重要的目标之一。尽管根据“超级杂交水稻育种”的战略设想和水稻育种实践，通过不断地改进育种技术可望在更高的产量水平上进行水稻杂种优势利用，在稻属植物内还具有很大的产量潜力可以挖掘。然而，仅仅从现有的种质基础出发，要更大幅度提高水稻单产，实现“超级杂交稻”的目标也存在一些困难：现有的推广品种是二倍体，尽管种类众多，但是其基因组的来源相对单一；同时，水稻基因组DNA含量也是作物中最少的，基因组内寻求开发潜力有一定困难；水稻作为C3植物，光合利用效率不高也是制约水稻产量提高的因素之一。因此，寻求常规手段以外的技术突破或者方法创新，是实现“超级杂交稻”的目标的迫切需求。本研究利用秋水仙素能抑制细胞分裂中纺锤丝的收缩、使细胞染色体加倍的作用，对水稻幼穗诱导的愈伤组织细胞进行加倍，并分化出再生植株；创制出水稻同源四倍体新的种质材料，在此基础上选育水稻同源四倍体雄性不育三系材料，并实现水稻同源四倍体的三系配套，开展水稻同源四倍体杂种优势利用和四倍体杂交水稻选育研究，建立水稻同源四倍体杂种优势利用的新技术体系。这不仅有助于倍性水平杂种优势的开拓和利用，同时也将为我国新世纪“超级稻”育种研究开辟一条新的技术途径。 水稻幼穗诱导愈伤组织并分化成苗是一项成熟、简单的组织培养技术。本研究以普通二倍体水稻亲本为材料，用秋水仙素进行水稻的多倍体化诱导，创制同源四倍体水稻三系亲本材料并对其进行鉴定。多倍体化以秋水仙素诱导的愈伤组织培养为基础，研究不同秋水仙素浓度梯度和愈伤组织诱导培养基组合对诱导四倍体植株的影响。结果表明在MS+2,4 D 1.0mg/L+ KT0.2mg/L+ IAA0.2mg/L 和500mg/L的秋水仙素处理下，水稻愈伤组织染色体加倍（有最高的效率）效果较好，平均加倍频率可达25.26%，其中，材料CDR22和IR26诱导较易成功，加倍频率分别达到75%和26.5%；相对材料94109 1.3%加倍频率和冈46B 10.8%加倍频率，诱导率差异极显著。 对水稻四倍体材料进行了形态学鉴定结果表明，与二倍体水稻对照相比其株高、穗长、花粉育性等主要农艺性状，确定四倍体材料在穗长和千粒重两方面极显著提高，种子的长度和宽度也显著增长。对花粉育性鉴定，确认水稻四倍体不育系材料仍为不育，保持系材料自交和杂交可育，恢复系材料自交和杂交可育。对四倍体材料进行细胞形态、染色体数目等方面进行细胞学鉴定，经核型分析表明水稻四倍体材料具有48条染色体，是二倍体水稻的两倍。水稻四倍体材料根尖分生组织细胞与二倍体的根尖分生组织细胞相比，细胞体积、细胞核和核仁显著增大。四倍体三系材料在细胞有丝分裂中期均可规则排列在赤道板，并能均等地移向两极；后期观察中没有发现染色体分离滞后现象，分裂末期细胞能够形成大小相对均一的子细胞。水稻同源四倍体三系材料细胞分裂未见异常，植株生长发育正常。 从1996年至2006年，针对结实率、有效分蘖、着粒数和穗长等主要农艺性状，通过系谱选育的方法，对培育的同源四倍体水稻亲本材料进行了连续选择和改良，取得较好成效。表现为结实率的改良效果极佳，所有改良材料的平均结实率均呈上升趋势，如D237（29.70％→72.70％）、DTB（19.55％→53.21％）等。有效分蘖总体呈现上升趋势，但在不同的年份，如1998和2002存在较大的负向波动。部分材料改良效果明显，如D19B（5.87→13.50）、D什香 (7.00→12.00)等；同时一些材料如DTB和D明恢63虽然总体略有提高，但在不同的年份波动很大，因此存在较大改良阻力，原因还有待进一步研究。着粒数的改良上升趋势比较显著，除保持系的DTB之外，其余材料的平均着粒数有显著提高。穗长的改良阻力较大，虽然不同材料总体上有所提高，但效果并不显著，并且不同年份有较大负向波动（2001）。此外还对株高、剑叶长等性状也进行了选择，但效果不显著，原因有待进一步提高。同源四倍体材料产量相关性状遗传改良幅度不一致，保持系和恢复系间的遗传改良效果也存在差异。这为同源四倍体水稻的进一步利用打下了良好的基础。 籼稻和粳稻亚种间杂交及杂种优势利用的主要障碍就是其低的结实率。而同源四倍体杂交水稻的研究为提高杂交水稻的杂种优势利用创造了新的途径。本研究通过随机区组设计方案，挑选性状优良的二倍体水稻材料，包括雄性不育系，保持系和恢复系进行秋水仙素诱导加倍，从而获得同源四倍体水稻对应的三系材料。利用选育的优良水稻同源四倍体三系材料，配制7个杂交组合，杂交F1代与其恢复系亲本进行比较，用于计算超亲优势（HB）；而杂交F1代与生产上大面积推广的二倍体杂交品种汕优63进行比较，用于计算杂种优势。结果显示，同源四倍体杂交水稻的超亲优势表现为：每株有效穗变化幅度为1.4%至105.9%，总粒数为0.5%至74.3%，每穗实粒数为17.6%至255.7%，结实率为9.6%至130.4%。这些农艺性状的改良使得这7个杂种F1的理论产量的超亲优势高达64.8%至672.7%。小区试验中四倍体杂交水稻组合T461A/T4002和T461A/T4193分别比二倍体对照汕优63提高46.3%和38.3%以上，除一个品种以外所有品种产量均接近或高于汕优63的产量。同源四倍体水稻强大的杂种优势表明，亚种间杂交育性低的问题可通过四倍体化及强化选择来解决。此外，同源四倍体杂交水稻器官的巨大性也是其产量提高的有利因素，水稻同源四倍体三系杂种优势利用研究具有一定的理论价值和商业生产潜力。 Rice is one of the major food crops, the improvement of the production and quality of it is an important thing related to the people's livelihood. On China's current national conditions, steadily increase of the rice yield based on the current level is still one of the most important goals in the next decade or even decades of agricultural production. According to the "super hybrid rice breeding" the strategic and rice breeding practice, improvement of the use of hybrid rice heterosis through continuous improvements in breeding technology is expected to get a higher level of rice yield, there are also a great yield potential can be exploited. However, there are also some difficulties to increase rice yield obviously and implement the goal of "super hybrid rice" based on the existing germplasm: Rice varieties in promotion are diploid, although there are many varieties, but their genome are from a comparatively single source; Meanwhile, the rice genome DNA are the least among the crops, it is difficult to exploit the development potential within the genome; Rice as C3 plants, photosynthetic efficiency is not high, it is one of the factors constraint rice yield. Therefore, seeking technological breakthroughs or innovative methods different from conventional means is the urgent needs to reach the target of "super hybrid rice". Using colchicine inhibit spindle contraction during cell division, double the cell chromosome, we induced callus cells from rice panicle to be doubled, and differentiated regeneration; we created a new autotetraploid rice germplasm material, and on that basis we bred male sterility three line autotetraploid rice materials, and the achieved the three line rice autotetraploid matchmaking, researched in autotetraploid rice heterosis usage and tetraploid hybrid rice breeding, constituted a new technology system of autotetraploid hybrid rice heterosis utilization. This not only helps the tetraploid rice heterosis exploration and use, but also inaugurates a new technical means for China in the new century "super rice" breeding research. We chose ordinary diploid rice as materials, using colchicine to induce the polyploidization, created the autotetraploid rice three-line materials and identified them. The polyploidization was based on the colchicine-induced callus tissue culture, and we experimented different colchicine concentrations and culture mediums to induce tetraploid plants, confirmed that the optimal concentration for inducement was 500 mg/L, the average induce rate was 25.26 %. Among all the materials, CDR22 and IR26 had higher induced rate; in contrary, 94109 and GANG46B had lower induced rate, the difference was significant. Autotetraploid materials was identified of both morphological and cytological, compared plant height, length of pollen sterility, and other major agronomic traits with a diploid rice as the control plant, identified that the autotetraploid materials had very significant advantages in ear length and thousand-grain weight, as well as the size of the seeds. Cytology identification included observation of the cell morphology, the number of chromosomes, and karyotype analysis on the autotetraploid materials confirmed that their chromosome number was 48, twice of the diploid rice. Mitoses in the three lines were common: chromosomes arrayed normally in metaphase and separated balanced into the two poles, chromosome moved without lagging in anaphase and daughter cells normally formed in telophase except one. It has been proved that tetraploid rice has normal meiosis as their diploid relatives, which usually including series of sub-phases as interphase, prophase I (five sub-phases), prophase II, metaphase I, II, anaphase I, II and telophase I, II. However, abnormal phenomena, such as formation of tetravalent, trivalent and univalent, chromosome lagging and so on, which would finally block meiosis. Configurations of chromosome in metaphaseⅠwere versatile in structure and form accept the bivalent. That condition varied in different strain, suggesting more complex paring configurations and more versatile genetic characters in tetraploid rice. All these abnormalities in meiosis contributed to low fertility of gamete and might consequently resulted in low seed setting. Successive selection and improvement on seed set, productive tiller per plant, total grains per panicle, panicle length and so on had been carried out from 1996 to 2006. The raise of seed sets was significant in both restorers and maintainers. Seed sets of some strains were improved more significantly than others, for example D237（29.70％→72.70％）、DTB（19.55％→53.21％）and et al.. Productive tiller per plant was improved to some extant. The tendency of improvement was rising on the whole but changed in some years such as 1998 and 2002. Part of the stains increased greatly, such as D19B（5.87→13.50）、Dshixiang (7.00→12.00) and so on, but some strains including DTB and Dminghui63 only increased little and decreased in some years by unknown reason. Total grains per panicle increased significantly and all strains except DTB increased. Improvement of panicle length termed to be hard. Different strains showed different capacities for improvement and floating existed in different years for example 2001. It has been proved that other agronomical traits including plant length, flag leaf length and so on could be improved but not significantly by selection also. In a word, agronomical traits could be raised by successive selection that is prerequisite for further utility of autotetraploid rice. Poor fertility is the main barrier for utilizing heterosis between the two rice (Oryza stiva L.) sub-species, indica and japonica. Recently, the development of autotetraploid hybrids (2n=4x=48) has been suggested as a new method for increasing heterosis in hybrid rice. Using standard experimental protocols, the elite diploid rice male sterile, maintainer, and restorer lines were colchine-doubled and autotetraploid counterparts were obtained. Seven resulting hybrids were analyzed for heterobeltiosis (HB), where the F1 was compared to the male parent, and the degree of heterosis, where the F1 was compared to the diploid commercial hybrid, Shanyou 63. The HB among the autotetraploid hybrids ranged from 1.4 to 105.9% for the productive panicles per plant, 0.5 to 74.3% for total kernels per panicle, 17.6 to 255.7% for filled kernels per panicle, and 9.6 to 130.4% for seed set. Improvements in these yield components resulted in the HB for kernel yield ranging from 64.8 to 672.7% among the seven hybrids. Hybrids T461A/T4002 and T461A/T4193 yielded 46.3 and 38.3% more, respectively than Shanyou 63, and all other hybrids but one yielded the same or more than Shanyou 63. The high heterosis for yield suggests that hybrid sterility between two rice sub-species may be overcome by using tetraploid lines followed by intensive selection. Also, the gigantic features of the autotetraploid hybrids may establish a plant structure able to support the higher yield.

糯小麦杂交后代的基因型鉴定及Wx基因的效应研究

糯小麦在食品加工业、淀粉加工业及其它工业上有着重要用途，是近年来许多国家小麦研究的重要课题。国外糯小麦选育尚未突破高产与糯性相结合的难点，国内目前还没有培育出高蛋白强筋型的糯小麦品种，这在一定程度上与缺乏合适的育种方法和高效、实用的糯小麦分子标记辅助育种技术有关。国内外对Wx基因效应的研究主要利用缺体－四体系、重组自交系或近等基因系，还未见有利用遗传背景相同的BC5F2代回交改良群体的报道。 糯性位点近等基因系是小麦淀粉品质育种的重要材料，而我国目前还没有一套中国栽培小麦遗传背景的糯性位点近等基因系。为了选育部分糯小麦、全糯小麦和中国栽培小麦遗传背景的糯性位点近等基因系，我们利用Wx蛋白电泳和高效实用的分子标记技术体系来鉴定糯小麦杂交后代的基因型，结果证明该体系能有效地用于糯小麦的分子标记辅助育种。以中国春糯性位点全套近等基因系为研究材料，对小麦Wx基因的6个STS标记和1个CAPS标记进行了筛选，改良PCR扩增条件以及产物检测方式后，从这些标记中筛选出3个标记，包括鉴定Wx-A1、Wx-D1位点的2个共显性STS标记和Wx-B1位点的1个显性STS标记。利用上述3个分子标记从BC5F2 代回交改良群体中筛选出了8种Wx基因型，经卡方检验，其分离比符合3对基因的分离比例，其中基因型为aabbdd的植株有2株，直链淀粉含量分别为1.81%和0.82%，为全糯小麦；基因型为AAbbdd，aabbDD的部分糯性植株各有1株，直链淀粉含量分别为15.24%和17.57%。以上4株植株的农艺性状和品质性状接近回交亲本“川育12”，并明显优于全糯材料“98Y1441”，表明采用回交法与Wx基因分子标记辅助选择相结合，有助于培育高产、优质的全糯和部分糯小麦。同时，本研究中建立的分子标记技术体系，也为选育具有中国栽培小麦遗传背景的糯性位点近等基因系奠定了基础。 在基因型鉴定的基础上，利用糯小麦杂交后代BC5F2代回交改良群体研究了各基因缺失降低直链淀粉含量的效果和各基因合成直链淀粉的能力，以及直链淀粉含量与农艺性状、品质性状、淀粉糊化特性等的相关性。缺失不同Wx基因的8种基因型，直链淀粉含量差异显著。研究单缺失基因型发现，减少效应最大的是Wx-B1b基因，Wx-B1b和Wx-D1b基因没有显著差异，减少效应最小的是Wx-A1b基因。研究双缺失基因型发现，Wx基因合成直链淀粉的能力，Wx-B1a基因最高，Wx-A1a基因最低，而Wx-B1a和Wx-D1a基因差异很小；直链淀粉含量与株高、穗长、小穗数、穗粒数、千粒重等农艺性状相关不显著，表明淀粉品质育种可以与高产育种实现有机结合；直链淀粉含量与SDS-沉降值呈显著负相关(r=-0.726)，说明直链淀粉含量降低在一定程度上有利于提高小麦营养与加工品质，这一结果至今未见有文献报道；全糯类型的淀粉糊化特性与其他类型显著不同，具有最高的峰值粘度和稀懈值，最低的低谷粘度、最终粘度、反弹值、峰值时间、糊化温度、起始糊化温度，表明糯小麦淀粉在食品和工业上具有特殊用途；稀懈值与直链淀粉含量呈极显著负相关(r=-0.969)，其他粘度参数与直链淀粉含量呈显著正相关(最终粘度r=0.797，低谷粘度r=0.910、反弹值r=0.954、峰值时间r=0.970、糊化温度r=0.962、起始糊化温度r=0.932)。以上结论可为糯小麦品种选育和淀粉品质改良提供理论依据。 Waxy wheat is very important in food processing industry, starch processing industry and the other industries, so it is a focus of wheat research in many countries these years. Foreign wheat breeders have not conquered the difficulty of high yield combined with waxy character, and there is no waxy wheat variety with high protein and strong gluten in China at present，all of which were caused by lacking proper breeding methods and effective, applied molecular markers-assisted selection technique at a certain extent. Until now, research about the effect of waxy genes mainly depended on nullisomic-tetrasomic lines, recombinant lines or near-isogenic lines, and it is lacking the reports of using improved BC5F2 backcross progenies under the common genetic background. Near-isogenic lines at the Wx loci are important materials for wheat starch quality breeding. However, there are no such lines under the Chinese cultivated wheat genetic background. To develop partial waxy wheats, waxy wheats and near-isogenic lines at the Wx loci under the Chinese cultivated wheat genetic background, we use SDS-PAGE of waxy proteins and effective, applied molecular marker-assisted selection technical system to identify the genotype of waxy wheat’s progenies. The results indicated that such a system is applicable in waxy wheat’s molecular marker-assisted selection effectively. A series of Chinese Spring Wx loci near-isogenic lines were used to identify the specific bands of six STS markers and one CAPS marker of Wx genes. After optimizing PCR amplification and separating of PCR products, three co-dominant and dominant STS-markers were identified at the Wx-A1, Wx-D1 and Wx-B1 loci, respectively, which were used to identify the genotype of waxy wheat’s progenies. Eight Wx genotypes were developed from the improved BC5F2 backcross progenies, which follows Mendelian segregation. Among them, there were two aabbdd waxy plants whose amylose content in the BC5F3 seeds were 1.81% and 0.82%, respectively. In addition, there were partial waxy plants (AAbbdd and aabbDD) whose amylose content in the BC5F3 seeds were 15.24 % and 17.57%, respectively. Most agronomic and quality traits of these four plants resembled those of the recurrent parent “Chuanyu 12”, and superior to waxy wheat parent “98Y1441”. This shows that backcross approach in combination with molecular marker-assisted selection of waxy genes is helpful to develop partial and full waxy wheat with good traits in the waxy wheat breeding program. At the same time, molecular marker-assisted selection technical system in this paper, also establish the base for breeding the near-isogenic lines at the Wx loci under the Chinese cultivated wheat genetic background. According to the results of genotype identification, we use waxy wheat’s improved BC5F2 backcross progenies to verify the effects of null alleles on reducing amylose content and determine the amylose synthesis capacity of each Wx gene independently, and investigate the relativity among amylose content with agonomic traits, quality traits, starch pasting properties respectively. There was significant difference in amylose content of the eight genotypes carrying different null Wx alleles. The reducing effect of the single null alleles was the most significant in Wx-B1b, followed by Wx-D1b and Wx-A1b, and there was no significant difference between Wx-B1b and Wx-D1b. The results of the double null lines further demonstrated that for the capacity of amylose synthesis, Wx-B1a was the highest, followed by Wx-D1a and Wx-A1a, and there was no significant difference between Wx-B1a and Wx-D1a. Amylose contents of the eight genotypes were not significantly correlated with plant height, spike length, spikelets per spike, grains per spike, 1000-grain weight, which showed that starch quality breeding could integrate with high yield breeding. Amylose contents of the eight genotypes were negatively significantly correlated with SDS-sedimentation value(r=-0.726), which suggested that reduction in amylose content is propitious to improve quality at a certain extent. Starch pasting properties of the full waxy type was significantly different from the other seven types, with the highest peak viscosity and breakdown, and lowest valley viscosity, final viscosity, setback, peak time, pasting temperature and starting pasting temperature. It indicated that waxy wheat starch has special use in food and industry. Breakdown was negatively significantly correlated with amylose contents (r=-0.969), and the other parameters were positively significantly correlated with amylose contents (r=0.797 for final viscosity, r=0.910 for trough viscosity, r =0.954 for setback, r =0.970 for peak time, r=0.962 for pasting temperature and r=0.932 for starting pasting temperature ). The results of the study are very useful for waxy wheat variety breeding and starch quality improvement.

同源四倍体与二倍体水稻的遗传差异及产量、品质性状研究

本文以中国科学院成都生物研究所培育的同源四倍体水稻和二倍体水稻为材料，进行遗传差异及产量、品质性状的研究：1、以二倍体水稻为对照，研究了同源四倍体水稻在2004 年和2005 年的结实情况。结果同源四倍体的花粉育性、结实率均不同程度下降，尤其低代材料更是大幅度下降。F 检验表明，同源四倍体不同个体间的各产量性状均差异显著，说明其具有很大的遗传改良潜力。从1996 年到2005 年对部分同源四倍体水稻进行了连续选择改良，T 检验表明经过9 年的选择改良，其结实率显著提高。本文还对同源四倍体水稻各产量性状间的相关性进行了分析,结果结实率与花粉育性、穗着粒数、穗实粒数极显著相关；理论产量与花粉育性、有效穗数、穗着粒数、穗实粒数、结实率及千粒重极显著相关。i2、用(CT)n 微卫星标记和PCR-Acc Ι分子标记对40 份同源四倍体和14 份二倍体水稻Wx 基因进行研究。结果，(CT)n 微卫星标记检测，Wx 基因呈Wx1、Wx2 和Wx3 3 种多态性；PCR-Acc Ι 检测，Wx 基因表现为G-型和T-型。测定稻米直链淀粉含量(AC)、胶稠度(GC)和糊化温度(GT)，并探讨其与Wx 基因的关系，结果，二倍体和同源四倍体水稻均存在：Wx 基因型相同，AC 差异较小，Wx 基因型不同时，AC 差异较大，Wx1 基因型品种AC 最高， Wx2 基因型品种AC 次之，Wx3 基因型品种AC 最低；基因型相同时，同源四倍体AC 低于二倍体；同源四倍体与对应二倍体间，Wx 基因型相同时，AC 差异很小；而Wx 基因型发生变异时，AC 差异很大。同时，进行相关性分析，结果二倍体和四倍体水稻均存在AC、GC 与Wx 基因密切相关；而GT 与Wx 基因相关不显著。综合分析，(CT)n微卫星标记与PCR-Acc Ι 分子标记检测的相关系数为0.842，呈极显著正相关，可以将其结合起来进行同源四倍体新品种的选育和改良。3、利用RAPD 技术，对同源四倍体和二倍体水稻的遗传差异进行分析。17条引物在同源四倍体中扩增出178 条带（PPB＝81.5%），在二倍体中扩增出173条带（PPB＝76.3%）；同源四倍体和二倍体的Shannon 遗传多样性指数分别为0.4848 和0.4679，多态信息量分别为0.3301 和0.3216，遗传距离分别为0.3572和0.3460；同源四倍体与其对应二倍体间遗传距离为0.1914-0.4633，平均为0.2914。表明同源四倍体的遗传多样性高于二倍体，且同源四倍体与其二倍体之间存在较大的遗传差异，这些将为水稻品种改良和新品种选育提供科学依据。上述产量、品质性状及遗传差异分析的结果，不仅有利于加快同源四倍体水稻的遗传改良进程，而且为进一步研究、利用同源四倍体水稻奠定了初步基础。 AbstractStudy on genetic diversity, yield characters and quality traitsof autotetraploid and diploid riceLiu Yuhua (Botany)Directed by Associate Prof. Tu ShengbinIn this study, diploid and autotetraploid rice, which were cultivated in ChengduInstitute of Biology were analyzed in genetic diversity, yield characters and qualitytraits.In the study, 2 diploid and 29 autotetraploid rice(2n=4x=48) materials, including4 preliminary and 25 advanced generation, were investigated for yield characters.Compared with diploid check, the pollen fertility and seed set of autotetraploiddeclined dramatically, especially in preliminary generation. F-test indicated that therewere remarkable differences among different varieties, showing that autotetraploidmaterials had strong potential for improvement. From 1996 to 2005, someautotetraploid rice had been selected and improved. T-test showed that seed setincreased obviously. The relationships among yield characters of autotetraploid ricewere analyzed. Seed set was strongly correlated with pollen fertility, total grainnumber per panicle and productive grain number per panicle; theoretical yield wasstrongly correlated with pollen fertility, productive panicle number per plant, totalgrain number per panicle, productive grain number, seed set and 1000-grain weight.Wx genotypes of 40 autotetraploid rice and 14 diploid rice were tested by usingthe (CT)n microsatellite marker and a cleaved amplified polymorphic sequence(CAPS) molecular marker named PCR-Acc Ι. Three microsatellite alleles wereproduced, i.e. Wx1, Wx2 and Wx3 both in autotetraploid and in diploid rice.Comparatively, PCR- Acc Ι molecular marker produced two genotypes, G-type andT-type for both autotetraploid and diploid rice. In this study, amylose content (AC), gel of consistency (GC) and gelatinization temperature (GT) of rice grain weremeasured and their relationships with Wx alleles were analyzed. The results showedthat variation of AC between autotetraploid and diploid rice was small when they hadthe same Wx genotype. However, variation of AC turned to be large when the Wxgenotypes were different. Actually, AC met the maximum value in Wx1 varieties andWx2 varieties the middle and Wx3 varieties the minimum. And AC was lower inautotetraploid than in diploid. Correlation analysis was done in this experiment. ACand GC of rice grain were probably controlled by Wx gene or other gene whoselocation was strictly linked to Wx gene, while GT of rice was not. The correlationcoefficient between Wx genotypes which revealed by (CT)n microsatellite marker andPCR-Acc Ι molecular marker was 0.842 with significant level. That revealed aconsistent result between the two types of markers. So it was possible to utilize boththe two types of markers to select and promote germplasm of autotetraploid rice.RAPD molecular markers were used to analyze the genetic diversity betweendiploid and autotetraploid rice. 178 repeatable bands were detected through 17 RAPDprimers with percentage of polymorphic bands was 81.5% in autotetraploid rice while173 repeatable bands were detected with percentage of polymorphic bands was 76.3%in diploid rice. According to the measurement of Shannon index, polymorphicinformation content and genetic distance, genetic diversity of autotetraploid was on ahigher level, genetic variation between autotetraploid and diploid rice was relativelyhigh. All these contributed to the genetic selection and improvement in rice breeding.As mentioned above, the results are not only helpful to promote the process ofrice improvement, but also to confirm the basic for further study of autotetraploid rice.