Breeding for increased grain protein and micronutrient content in wheat: Ten years of the GPC-B1 gene

To provide food and nutrition security for a growing world population, continued improvements in the yield and nutritional quality of agricultural crops will be required. Wheat is an important source of calories, protein and micronutrients and is thus a priority to breed for improvements in these traits. The GRAIN PROTEIN CONTENT-B1 (GPC-B1) gene is a positive regulator of nutrient translocation which increases protein, iron and zinc concentration in the wheat grain. In the ten years since it was cloned, the impacts of GPC-B1 allelic variation on quality and yield traits have been extensively analyzed in diverse genetic backgrounds in field studies spanning forty environments and seven countries. In this review, we compile data from twenty-five studies to summarize the impact of GPC-B1 allelic variation on fifty different traits. Taken together, the results demonstrate that the functional copy of the GPC-B1 gene is associated with consistent positive effects on grain protein, Fe and Zn content with only marginally negative impacts on yield. We conclude that the GPC-B1 gene has the potential to increase nutritional and end use quality in a wide range of modern cultivars and environments and discuss the possibilities for its application in wheat breeding.     

Leaching characteristics of homologues of benzalkonium chloride from wood treated with ammoniacal copper quaternary wood preservative

In Japan, ammoniacal copper quaternary wood preservatives type-1 (ACQ-1), which contains copper and benzalkonium chloride as its active ingredients, is among the most widely used wood preservatives in the pressure treatment of wood. Benzalkonium chloride (BAC) in commercial ACQ-1 products mainly comprises C12 and C14 homologues. In the present study, the leaching characteristics of these BAC homologues were investigated using the heartwood and sapwood portions of Japanese cedar, Japanese larch, and Sakhalin fir treated with ACQ-1 and 1% monoethanolamine (MEA) solution containing equimolar amounts of homologues. Distilled water (DW) and artificial seawater (SW) were used as leaching media. Consequently, it was observed that the leaching rate of the C12 homologue tended to be higher than that of the C14 homologue in DW. The leaching of C12 homologues was accelerated by using SW, resulting in a significantly higher leaching rate than the C14 homologue using SW. It was thought that the difference in the hydrophobicities based on alkyl chain lengths resulted in these phenomena. However, when the heartwood portion of Japanese larch was treated with the homologues in MEA, the leaching rate of the C14 homologue was significantly higher than that of the C12 homologue.     

南瓜基因组DNA的细菌人工染色体(BAC)文库的构建

[目的]构建南瓜基因组DNA的细菌人工染色体(BAC)文库。[方法]以南瓜幼芽为材料,利用HindⅢ酶切体系,初步构建南瓜基因组DNA的BAC文库。[结果]研究成功构建了南瓜基因组DNA的BAC文库。[结论]该技术为南瓜相关基因的克隆、功能验证、物理图谱的构建和基因组测序等研究工作奠定了基础。     

大白菜抗霜霉病同源基因的筛选与亚克隆文库的构建

根据植物抗霜霉病基因保守区设计简并引物,利用三维PCR方法对大白菜(Brassica campestris ssp.pekiensis)‘85-1'BAC文库进行筛选,从19 200个BAC克隆中筛选到含有目的基因的10个阳性克隆.利用HindⅡ对其中的94一G-17克隆进行酶切,回收1～5 kb的DNA片段并连接到载...     

绒山羊Y染色体Amelogenin基因BAC筛选与鉴定

Amelogenin基因是控制牙釉蛋白的表达基因.它不仅在动物牙齿发育过程中起重要的调控作用,而且可用于动物性别鉴定.为了获得绒山羊Amelogenin基因全序列、研究绒山羊牙齿的进化过程、实施牙齿保护,本研究根据GenBank收录的绵羊Y染色体基因Amelogenin-intron4序列设计引物,以绒山羊基因组DNA为模板,通过PCR技术,进行绒山羊BAC文库Amelogenin筛选与克隆.测序后得到259bp的目的序列.此片段与其他物种进行核苷酸序列比对,结果显示,与绵羊、牛同源性分别达98％和96％,而与人、黑猩猩、老鼠无同源性.属于GT-AG型内含子,具有增强Amelogenin基因转录的作用,并且通过实验筛选出该片段所在的BAC为山羊Y染色体的BAC.     

奶山羊细菌人工染色体基因文库构建的初步研究

细菌人工染色体(BAC)文库是进行动物基因组学研究的一个重要手段,为配合山羊乳腺生物反应器的研究,对构建山羊基因组BAC文库进行了初步研究。用H indⅢ酶对莎能奶山羊基因组进行部分酶切,用CHEF(箝位匀强电场)二次电泳法回收150 kb大小条带,电透析回收高分子量(HMW)DNA;同时以BAC载体pB eloBAC 11为材料,分别采用限制性内切酶H indⅢ和HK脱磷酶对其进行酶切和脱磷,将该载体自连并通过凝胶回收线性段DNA,获得了可用于构建BAC文库的线性载体。将HMW与载体进行连接,电击转化感受态DH 10β大肠杆菌,一次转化得到近2 000个克隆。插入片段大小为50~200 kb,基本满足建库的需要。     

Size Does Matter: Application-driven Approaches for Soil Metagenomics

Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification, and if necessary, cloning methods for the intended downstream analyses. The most important technical considerations in a metagenomic study include obtaining a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host. Size does matter in the context of the average insert size within a clone library or the sequence read length for a high-throughput sequencing approach. It is also imperative to select the appropriate metagenomic screening strategy to address the specific question(s) of interest, which should drive the selection of methods used in the earlier stages of a metagenomic project (e.g., DNA size, to clone or not to clone). Here, we present both the promising and problematic nature of soil metagenomics and discuss the factors that should be considered when selecting soil sampling, DNA extraction, purification, and cloning methods to implement based on the ultimate study objectives.     

Construction and characterization of a BAC library for sunflower (Helianthus annuus L.)

A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.     

Construction and characterization of a BAC library of soybean

We have constructed a soybean (Glycine max (L.) Merrill) bacterial artificial chromosome (BAC) library from green leaf protoplasts of the cultivar, Misuzudaizu. The library contains 53,760 clones with an average insert size of 116 kb. About 2.9% chloroplast DNA origin was revealed by PCR and colony hybridization. Apart from 2.8% clones having no insert, this library represents 5.2 genome equivalents. With this genome coverage, the probability of having any DNA sequence represented in the library is higher than 99.5%. Three-dimensional pools of the BAC library in combination with the use of a high efficiency genome scanning (HEGS) electrophoresis facilitate rapid and efficient PCR-based screening. An average of five positive clones were identified after screening the BAC library with SSR and STS markers. BAC-end walking was performed for three SSR associated BACs. This library will provide a good resource for positional cloning of agronomically and biologically important QTL genes that Misuzudaizu possesses.