Association genetics in Corymbia citriodora subsp variegata identifies single nucleotide polymorphisms affecting wood growth and cellulosic pulp yield

Wood is an important biological resource which contributes to nutrient and hydrology cycles through ecosystems, and provides structural support at the plant level. Thousands of genes are involved in wood development, yet their effects on phenotype are not well understood. We have exploited the low genomic linkage disequilibrium (LD) and abundant phenotypic variation of forest trees to explore allelic diversity underlying wood traits in an association study. Candidate gene allelic diversity was modelled against quantitative variation to identify SNPs influencing wood properties, growth and disease resistance across three populations of Corymbia citriodora subsp. variegata, a forest tree of eastern Australia. Nine single nucleotide polymorphism (SNP) associations from six genes were identified in a discovery population (833 individuals). Associations were subsequently tested in two smaller populations (130160 individuals), validating our findings in three cases for actin 7 (ACT7) and COP1 interacting protein 7 (CIP7). The results imply a functional role for these genes in mediating wood chemical composition and growth, respectively. A flip in the effect of ACT7 on pulp yield between populations suggests gene by environment interactions are at play. Existing evidence of gene function lends strength to the observed associations, and in the case of CIP7 supports a role in cortical photosynthesis.

Increasing anthocyanin content in Queen Garnet plum and correlations with in-field measures

While plums are traditionally bred for fresh fruit traits such as size, sweetness, yield and disease resistance the Queensland Government breeding program for Japanese plum ( Prunus salicina Lindl.) also selected for anthocyanin content to develop a new plum selection named 'Queen Garnet'. When ripe or overripe, it has a near black skin and deep red flesh colour, which when combined, result in exceptionally high anthocyanin content, reaching up to 277 mg/100 g fruit. The skin fraction contributes 36-66% of the total anthocyanin content of fruit. The plum is now being commercially grown to be processed into a range of functional products from food colourants to premium health products. These are sold on the basis of anthocyanin and antioxidant content. Protocols for increasing anthocyanin content have therefore been researched to maximise the total anthocyanin yield rather than fresh fruit weight and taste. The principal approach is through selective harvest of overripe plums high in colour, although post-harvest storage at 21°C results in further anthocyanin synthesis. Modified processing is also required to ensure recovery of anthocyanins from the skin fraction. The plum products have entered testing for assessing health properties beginning with an initial proof of in vivo bioavailability of the anthocyanins.

Predictive modelling to aid the regional-scale management of a vertebrate pest

Extensive resources are allocated to managing vertebrate pests, yet spatial understanding of pest threats, and how they respond to management, is limited at the regional scale where much decision-making is undertaken. We provide regional-scale spatial models and management guidance for European rabbits (Oryctolagus cuniculus) in a 260,791 km(2) region in Australia by determining habitat suitability, habitat susceptibility and the effects of the primary rabbit management options (barrier fence, shooting and baiting and warren ripping) or changing predation or disease control levels. A participatory modelling approach was used to develop a Bayesian network which captured the main drivers of suitability and spread, which in turn was linked spatially to develop high resolution risk maps. Policy-makers, rabbit managers and technical experts were responsible for defining the questions the model needed to address, and for subsequently developing and parameterising the model. Habitat suitability was determined by conditions required for warren-building and by above-ground requirements, such as food and harbour, and habitat susceptibility by the distance from current distributions, habitat suitability, and the costs of traversing habitats of different quality. At least one-third of the region had a high probability of being highly suitable (support high rabbit densities), with the model supported by validation. Habitat susceptibility was largely restricted by the current known rabbit distribution. Warren ripping was the most effective control option as warrens were considered essential for rabbit persistence. The anticipated increase in disease resistance was predicted to increase the probability of moderately suitable habitat becoming highly suitable, but not increase the at-risk area. We demonstrate that it is possible to build spatial models to guide regional-level management of vertebrate pests which use the best available knowledge and capture fine spatial-scale processes.

Breeding programs on Atlantic salmon in Norway: lessons learned

An early establishment of selective breeding programs on Atlantic salmon has been crucial for the success of developing efficient and sustainable salmon farming in Norway. A national selective breeding program was initiated by AKVAFORSK at the beginning of the 1970s, by collecting fertilized eggs from more than 40 Norwegian river populations. Several private selective breeding programs were also initiated in the 1970s and 1980s. While these private programs were initiated using individual selection (i.e. massselection) to genetically improve growth, the national program was designed to gradually include all economically important traits in the breeding objective (i.e. growth, age at sexual maturation, disease resistance and quality traits) using a combined family and within-family selection strategy. Independent of which selection strategy and program design used, it is important to secure and maintain a broad genetic variation in the breeding populations to maximize selection response. It has been documented that genetically improved salmon from the national selective breeding program grow twice as fast as wild Atlantic salmon and require 25 per cent less feed, while salmon representing the private breeding programs all show an intermediate growth performance. As a result of efficient dissemination of genetically improved Atlantic salmon, the Norwegian salmon farming industry has reduced its feed costs by more than US$ 230 million per year! The national selective breeding program on Atlantic salmon was commercialized into a breeding company (AquaGen) in 1992. Five years later, several private companies and the AKVAFORSK Genetics Center (AFGC) established a second breeding company (SalmoBreed) using breeding candidates from one of the private breeding programs. These two breeding companies have similar products, but different strategies on how to organize the breeding program and to disseminate the genetically improved seed to the Norwegian salmon industry. Greater competition has increased the necessity to document the genetic gain obtained from the different programs and to market the economic benefits of farming the genetically improved breeds. Both breeding companies have organized their dissemination to get a sufficient share of the economic benefits in order to sustain and improve their breeding programs.

Perspectives from agriculture: advances in livestock breeding - implications for aquaculture genetics

In this paper we present livestock breeding developments that could be taken into consideration in the genetic improvement of farmed aquaculture species, especially in freshwater fish. Firstly, the current breeding objective in aquatic species has focused almost exclusively on the improvement of body weight at harvest or on growth related traits. This is unlikely to be sufficient to meet the future needs of the aquaculture industry. To meet future demands breeding programs will most likely have to include additional traits, such as fitness related ones (survival, disease resistance), feed efficiency, or flesh quality, rather than only growth performance. In order to select for a multi-trait breeding objective, genetic variation in traits of interest and the genetic relationships among them need to be estimated. In addition, economic values for these traits will be required. Generally, there is a paucity of data on variable and fixed production costs in aquaculture, and this could be a major constraint in the further expansion of the breeding objectives. Secondly, genetic evaluation systems using the restricted maximum likelihood method (REML) and best linear unbiased prediction (BLUP) in a framework of mixed model methodology could be widely adopted to replace the more commonly used method of mass selection based on phenotypic performance. The BLUP method increases the accuracy of selection and also allows the management of inbreeding and estimation of genetic trends. BLUP is an improvement over the classic selection index approach, which was used in the success story of the genetically improved farmed tilapia (GIFT) in the Philippines, with genetic gains from 10 to 20 per cent per generation of selection. In parallel with BLUP, optimal genetic contribution theory can be applied to maximize genetic gain while constraining inbreeding in the long run in selection programs. Thirdly, by using advanced statistical methods, genetic selection can be carried out not only at the nucleus level but also in lower tiers of the pyramid breeding structure. Large scale across population genetic evaluation through genetic connectedness using cryopreserved sperm enables the comparison and ranking of genetic merit of all animals across populations, countries or years, and thus the genetically superior brood stock can be identified and widely used and exchanged to increase the rate of genetic progress in the population as a whole. It is concluded that sound genetic programs need to be established for aquaculture species. In addition to being very effective, fully pedigreed breeding programs would also enable the exploration of possibilities of integrating molecular markers (e.g., genetic tagging using DNA fingerprinting, marker (gene) assisted selection) and reproductive technologies such as in-vitro fertilization using cryopreserved spermatozoa.

The review of recent advances in fish genetics and biotechnology

Great advances have been, and are being made in our knowledge of the genetics and molecular biology (including genomics, proteomics and structural biology). Global molecular profiling technologies such as microassays using DNA or oligonucleotide chip, and protein and lipid chips are being developed. The application of such biotechnological advances are inevitable in aquaculture in the areas of improvement of aquaculture stocks where many molecular markers such as RFLPs, AFLDs and RAPD are now available for genome analysis, finger printing and genetic linkage mapping. Transgenic technology has been developed in a number of fish species and research is being pursed to produce transgenic fish carrying genes that encode antimicrobial peptides such as lysozyme thereby achieving disease resistance in fish. Also it is a short cut to achieving genetic change for fast growth and other desirable traits like early sexual maturity, temperature tolerance and feed conversion efficiency. KEYWORDS: Fish genetics, transgenesis, monoploidy, diploidy, polyploidy,gynogenesis, androgenesis, cryopreservation.

Effects of dietary supplementation of commercial human probiotic and antibiotic on the growth rate and content of intestinal microflora in ornamental fishes

The effects of dietary supplementation of commercial human probiotic, Lactobacil and antibiotic, oxytetracycline on the growth, survival, disease resistance and content of intestinal microflora in two ornamental fishes, viz., goldfish, Carassius auratus and swordtail, Xiphophorus helleri were studied. The total wet weight gain, food conversion ratio and specific growth rate of C. auratus did not vary significantly (p>0.05) among treatments. While in X. helleri, significant differences existed in the total wet weight gain, survival, food conversion ratio and specific growth rate among treatment groups (p<0.05). The counts of antibiotic resistant bacteria in fish gut increased with days of culture in all the treatments and the increase was more in antibiotic fed fishes. A reduction in the development of antibiotic resistance among the bacterial flora of fish gut was noticed in probiotic fed groups of C auratus and X. helleri. The results of the present study revealed that the effects of human probiotic on the growth, survival and disease resistance of ornamental fishes are variable and difficult to reproduce the similar effect on different species.

小麦—偏凸山羊草6Mv/6B代换系的研究

小麦条锈病（Puccinia striiformis f. sp. tritici）是世界性小麦病害，可导致受害小麦减产30%以上，甚至绝收。小麦条锈病在我国西南、华北麦区危害严重，四川麦区是小麦条锈病发病最重的地区之一，每年因条锈病流行造成小麦产量损失巨大。利用抗条锈病品种是控制该病害最安全、经济的有效途径，因此挖掘利用抗病新基因，开展抗病遗传基础研究是当前育种工作中面临的重要任务。 偏凸山羊草（Aegilops ventricosa，DDMvMv，2n=28）是一年生草本植物，起源于地中海西部沿岸地区，具有对小麦白粉病、锈病等高抗或免疫、耐盐、抗寒、蛋白质含量高等优良性状，是小麦遗传育种很好的种质资源。本研究以高抗条锈病的小麦—偏凸山羊草6Mv/6B代换系（Moisson 6Mv/6B）为材料，对其含有的带条锈病抗性基因的偏凸山羊草6Mv染色体在四川小麦背景中的传递情况、与小麦—簇毛麦双端体附加系所具有的白粉病抗性的聚合以及对Moisson 6Mv/6B进行电离辐射诱变筛选抗条锈病的小麦—偏凸山羊草易位系三个方面进行了研究。取得的主要研究结果如下： 1. Moisson 6Mv/6B与高感条锈病的四川地区普通小麦品种绵阳26、绵阳93-124和SW3243的杂种F1与其普通小麦亲本分别作为父、母本回交，通过对其BC1和F2的结实率、根尖细胞有丝分裂中期染色体的观察以及对条锈病抗性的鉴定，发现含6Mv染色体的F1植株作母本时的回交结实率（83.10%）普遍高于含6Mv染色体的F1植株作父本（48.61%），结实率与普通小麦基因型密切相关（χ2=34.15>>χ20.05=5.99（df=2））；6Mv染色体在三种四川小麦中通过雌、雄配子传递的传递方式与其传递率间没有显著相关性，其传递率与普通小麦基因型呈显著相关性（χ2=6.42>χ20.05=5.99（df=2））。 2. Moisson 6Mv/6B与高抗白粉病的小麦—簇毛麦双端体附加系Pana（2n=42+2t）正反杂交，希望在聚合两者抗性的同时观察不同受体背景下的抗性反应。对Moisson 6Mv/6B和Pana正反杂交的结实率、杂交后代的农艺性状进行观察，并对杂交后代进行基因组荧光原位杂交（GISH）分析及条锈病和白粉病的抗性鉴定。结果表明Moisson 6Mv/6B作母本时杂交结实率（80.56%）高于Pana作母本时（58.33%），结实率与杂交方式间紧密相关（χ2=4.96>χ20.05=3.84（df=1））；Moisson 6Mv/6B和Pana杂交后代株高比最高亲本高约10cm，成熟期也较两亲本提前两个星期左右；正反杂交后代中具有偏凸山羊草6Mv染色体的植株具有条锈病抗性，具有簇毛麦端体的植株具有白粉病抗性，同时筛选到4株含有偏凸山羊草和簇毛麦遗传物质并对条锈病和白粉病兼抗的材料，证明来自偏凸山羊草6Mv染色体的条锈病抗性与来自簇毛麦端体的白粉病抗性已经聚合在一起，且没有产生相互抑制的作用，暗示通过这两个抗性基因的聚合是完全能获得兼抗条锈病和白粉病的小麦新种质。 3. 对Moisson 6Mv/6B在减数分裂时期的成株进行总剂量为6Gy、辐射频率为120rad/min的60Co-γ射线辐射，对辐射植株自交后代进行农艺性状及根尖细胞有丝分裂中期染色体形态观察和条锈病抗性鉴定。结果为辐射植株自交结实率为2.22%，根尖细胞有丝分裂中期的染色体存在明显碎片，辐射自交后代植株对条锈病具有成株期抗性。 小麦—偏凸山羊草6Mv/6B代换系对条锈病抗性稳定，是培育条锈病抗性品种的良好供体。本研究证明在四川小麦背景中要利用该品种抗性，在结实数满足需要时，可将其作父本，亦可作母本，但关键是要选择好一个优良的受体基因型；同时其条锈病抗性与来自簇毛麦的白粉病抗性没有相互抑制作用，可将两者抗性有效聚合用于小麦育种中。 Wheat stripe rust (Puccinia striiformis f. sp. Tritici) is a worldwide disease of wheat, and could lead to victims of 30 percent or even total destruction of wheat production. Wheat stripe rust harms badly in China's southwest and North China. Sichuan province is one of the regions damaged by wheat stripe rust heavily. The use of resistant varieties is the most secure and economical way to control the wheat stripe rust. Therefore, it is essential to identify new disease-resistant genes and genetically research of disease resistance. Aegilops ventricosa (DDMvMv, 2n = 28) is an annual herbaceous plant, originating in the coastal areas of the western Mediterranean, with good characters such as resistance of wheat powdery, rust, salt, cold and high protein content. It is a good germplasm resource. In this study, the wheat- Aegilops ventricosa 6Mv/6B substitution line Moisson 6Mv/6B (highly resistant to the wheat stripe rust) was used to study on the transmission of chromosome 6Mv of Aegilops ventricosa in different genetic background of Sichuan wheat varieties, hybridization with wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew) and screening of wheat- Aegilops ventricosa translocation line by exposuring Moisson 6Mv/6B under ionizing radiation. The main results are as following: 1. Moisson 6Mv/6B was crossed with Sichuan wheat varieties mianyang26, mianyang93-124 and SW3243 (highly susceptible to stripe rust), respectively. Their F1 hybrids were further backcrossed as male and female to corresponding wheat varieties. The seed-setting rate, chromosomes confirmation in the mitotic metaphase of root tip cells, and resistance to stripe rust of the subsequent BC1 and F2 plants were investigated. The average seed-setting rate of backcross via 6Mv as female donor (83.10%) was higher than that of backcross via 6Mv as male donor (48.61%), suggesting that the seed-setting rate was associated with the wheat genotypes（χ2=34.15>>χ20.05=5.99（df=2））. In all analyzed populations, transmission frequencies of chromosome 6Mv were not correlated with the ways of 6Mv through male or through female. However, transmission frequencies of chromosome 6Mv were significantly correlated with Sichuan wheat genotypes（χ2=6.42>χ20.05=5.99（df=2））. 2. To aggregating the resistances to stripe rust and powdery mildew, as well as research on the resistance reactions in different genetic background, Moisson 6Mv/6B was reciprocally hybrided with the wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew). The seed-setting rate, agronomic characters, genomic in situ hybridization (GISH) of hybrid progenies，and resistances to stripe rust and powdery mildew were investigated. The results showed that the seed-setting rate of hybridization via Moisson 6Mv/6B as female donor (80.56%) was significant higher than that via Pana as female donor (58.33%). The seed-setting rate was associated with the hybrid methods (χ2 = 4.96> χ20.05 = 3.84 (df = 1)). The plant height of hybrid progenies was about 10 cm higher than Pana, the parent with maximum height. And the maturity of hybrid progenies was about two weeks earlier than that of the parents. In the hybrid progenies, the plants with the 6Mv chromosome have the resistance to stripe rust and the plants with the telosome from Haynaldia villosa have the resistance to powdery mildew. It was found that four plants with both the 6Mv chromosome and the telosome from Haynaldia villosa were resistant to stripe rust and powdery mildew. It indicated that the resistance to stripe rust and powdery mildew aggregated, and no mutual inhibition was found. It implied that the aggregation of the two resistance genes was able to provide the new wheat germplasm with the resistances to stripe rust and powdery mildew. 3. Moisson 6Mv/6B was irradiated with 60Co-γ rays of 6Gy (120rad/min) during meiosis. The agronomic characters and chromosomes confirmation in the mitotic metaphase of root tip cells，as well as resistance to stripe rust were investigated. The seed-setting rate of irradiated plants was only 2.22%. The chromosomes in mitotic metaphase had clear fragments. The resistance to stripe rust of progeny of irradiated plants was the adult-plant resistance. The wheat- Aegilops ventricosa 6Mv/6B substitution line is a good stripe rust resistance donor for its stabile resistance. Our study demonstrated that the key for use the resistance is to choose a good receptor. There is no difference between Moisson 6Mv/6B be the female and be the male if the seed number meets the requirement. At the same time, the stripe rust resistance of Moisson 6Mv/6B did not have the mutual inhibition with the powdery mildew resistance from Haynaldia villosa. It is able to aggregate the two resistances for wheat breeding.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

Electrochemical measurement of hydrogen peroxide in plasma: Evaluation of freshwater prawn (Macrobrachium rosenbergii) and crucian carp (Cyprinus carpio) phagocytes under natural conditions

An electrochemical technique for the real-time detection of hydrogen peroxide (H2O2) was employed to describe respiratory burst activity (RBA) of phagocytes in plasma which can be used to evaluate the ability of immune system and disease resistance. The method is based upon the electric current changes, by redox reaction on platinum electrode of extracellular hydrogen peroxide (H2O2) released from phagocytes stimulated by the zymosan at 680 mV direct current (d.c.). Compared with the control, activation of respiratory burst by zymosan particles results in a high amperometric response, and a current peak was obtained during the whole monitoring process. The peak current was proved by addition Of Cu2+ and other controls, to be the result of intense release of H2O2 from phagocytes. The peak area was calculated and used to evaluate the quantity of effective H2O2, which represents the quantity of H2O2 beyond the clearance of related enzymes in plasma. According to Faraday's law, the phagocytes' ability of prawns to generate effective H2O2 was evaluated from 1.253 x 10(-14) mol/cell to 6.146 x 10(-14) mol/cell, and carp from 1.689 x 10(-15) Mol/Cell to 7.873 x 10(-1)5 mol/cell. This method is an acute and quick detection of extracellular effective H2O2 in plasma and reflects the capacity of phagocytes under natural conditions, which could be applied for selecting species and parents with high immunity for breeding in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis

In order to observe the effect of salinity on disease resistance and white spot syndrome virus (WSSV) proliferation in Fenneropenaeus chinensis, shrimps with latent WSSV were subjected to two acute salinity changes from the original salinity of 22 ppt to 18 and 14 ppt in an hour, respectively. The total haemocyte count (THC) of the challenged group showed no evident change under salinity adjustments, but the phenoloxidase (PO) index declined significantly (P<0.05) corresponding to continuing acute salinity changes from the 24th to the 72nd hour. According to the WSSV load detected by quantitative real-time PCR method, it was found that WSSV carried by the challenged group and control group were significantly different (P<0.05); acute salinity change from 22 to 14 ppt led to the WSSV carried in the challenged group being significantly higher (P<0.05) than that of those surviving in 22 ppt, but salinity change from 22 to 18 ppt had no such effect. At the end of the 72-h experiment, the challenged group subjected to salinity change from 22 to 14 ppt had nearly 3 times the WSSV load as the control group with no salinity change. Therefore, salinity changes over a particular range could result in a decrease of immunocompetence and obvious WSSV proliferation in the shrimps, leading to white spot syndrome developing from a latent infection to an acute outbreak. (C) 2005 Elsevier B.V All rights reserved.

Índice de cruzabilidade entre espécies de passifloras nas condições do Distrito Federal.

2006

BRS Cometa: nova cultivar de feijoeiro comum com grão do tipo comercial carioca para a região do Distrito Federal.

ABSTRACT: During the years 2003 and 2004, four experiments were conducted in the winter season, under irrigation and in the raining season in the experimental area of the Embrapa Cerrados Research Center at Planaltina, Federal District. The objective was to identify cultivars that presented the following characteristics: resistance to diseases, high yield, resistance to lodging, desirable plant height and good market acceptance, to be indicated for cultivation in the Federal District. It was concluded that the cultivar BRS Cometa is indicated for this region, due to its high yield characteristics, resistance to diseases and lodging, excellent cooking qualities and erect type.

Phenotypical characterization in "Plasmopara halstedii" (sunflower downy mildew) isolates of several races

Phenotypic variation (morphological and pathogenic characters), and genetic variability were studied in 50 isolates of seven Plasmopara halstedii (sunflower downy mildew) races 100, 300, 304, 314, 710, 704 and 714. There were significant morphological, aggressiveness, and genetic differences for pathogen isolates. However, there was no relationship between morphology of zoosporangia and sporangiophores and pathogenic and genetic characteristics for the races used in our study. Also, our results provided evidence that no relation between pathogenic traits and multilocus haplotypes may be established in P. halstedii. The hypothesis explaining the absence of relationships among phenotypic and genetic characteristics is discussed.