棉花RAV基因家族的全基因组分析

在二倍体棉花D5基因组(Gossypium raimondii Ulb.)数据库中鉴定出10个RAV基因,分布于4、5、8、9、13号染色体;在二倍体棉花A2基因组(Gossypium arboreum L.)数据库中鉴定出10个RAV基因,与棉花D5基因组的R AV成员的数量和序列具有一一对应的同源关系,推测棉花A、D组的祖先种中可能存在10个RAV基因。对植物R AV蛋白序列做系统发育分析,将R AV成员分为4个组;发现棉花R AV基因可能参与了棉属所特有的基因组多倍化事件的证据。对N CBI中陆地棉(Gssypium hirsutum L.)EST、Unigene数据库做比对统计,得到陆地棉不同组织中R AV基因表达情况;对陆地棉受黄萎病菌胁迫后的荧光定量检测,发现棉花RAV基因与棉花响应黄萎病菌的胁迫相关。     

棉花单核苷酸多态性标记研究进展

单核苷酸多态性标记已在农作物研究中得到广泛应用并取得重大进展。为了便利棉花SNP(Single nucleotide polymorphism)标记的研究和应用,介绍了利用基因芯片、简化基因组测序、重测序等在棉花中开发SNP标记的方法 ,综述了SNP标记在棉花遗传图谱构建、数量位点的定位和分子标记辅助育种、基因组测序以及系统进化等研究中的应用。并对异源四倍体棉花中SNP标记开发时,同源序列位点和部分同源序列位点上的SNP标记辨别问题进行了系统探讨,对其快捷的开发、检测方式和在数量基因定位中的应用前景进行了展望。     

一个FIF1基因的SNP及定位研究

分子标记辅助选择逐步成为植物育种的重要方法。然而需要更多的实用的分子标记,尤其是与重要农艺性状紧密连锁的标记。目前,许许多多的方法已经应用到棉花结构基因组的标记开发,但是棉花SNP标记鲜见报道。FIF1基因是棉纤维优势表达的一个基因,研究证明它在棉纤维的发育过程中起着很重要的调控作用。根据已发表的亚洲棉FIF     

一个新的棉花MYB类基因(GhTF1)的克隆及染色体定位分析

MYB类转录因子是指含有MYB结构域的一类转录因子, 广泛参与植物发育和代谢调节。含2个MYB结构域的R2R3类MYB转录因子在植物体内主要参与次生代谢的调节和控制细胞的形态发生。从优质材料7235不同发育时期的棉纤维混合cDNA文库中克隆了一个棉花MYB转录因子基因GhTF1(GenBank登录号: EF651783)。该cDNA序列长1 115 bp, 其开放读码框长度为771 bp, 编码256个氨基酸。表达特征分析表明, 该基因在陆地棉7235不同组织中均表达, 但表达量不同, 特别在开花前1 d, 开花后8 d和11 d的纤维细胞中优势表达。该基因在二倍体棉种非洲棉和雷蒙德氏棉中开放读码框区的序列较保守, 但在非编码区差异较大, 在内含子区存在大片段插失和碱基替换现象。Southern杂交结果表明该基因在陆地棉基因组中存在2个拷贝, 推测A、D亚组中各有1个拷贝。利用海7124和TM-1两亲本配置的BC₁作图群体, 将GhTF1定位在染色体10上。     

草棉EST-SSRs的遗传评价

根据GenBank中公布的247条草棉EST序列,搜索SSR并进行引物设计。其中的25条序列含有27个SSR,1~6碱基重复类型都存在,二碱基和三碱基重复的频率较高。为了明确在A、D和AD基因组中的可转移性,依据25条序列共设计25对EST-SSR引物,其中22对引物扩增出清晰可辨的DNA条带,产生92个多态性片段,平均每对引物产生3.64个多态性片段。引物的多态性信息含量(PIC)在0.49~0.91之间,平均为0.81。6对引物在BC₁种间作图群体[(鄂棉22 × Pima3-79) ×鄂棉22]中表现多态性,产生7个多态性位点,其中5个为共显性,2个为显性。除HAU230b标记在BC₁分离群体中不符合孟德尔式分离比例,其余引物表现正常分离。6个位点被整合到陆地棉和海岛棉种间BC₁遗传连锁图谱上的6条染色体：有4个位于A亚基因组的4条染色体上(Chr.6、10、11和12),2个位于D亚基因组的2条染色体(Chr.19和20)。     

Essential oils and silver nanoparticles (SNP) as novel agents to extend vase-life of gerbera (Gerbera jamesonii cv. ‘Dune’) flowers

The aim of this study was to evaluate the efficacy of silver nanoparticles (SNP) and essential oils as novel antimicrobial agents in extending the vase-life of gerbera (Gerbera jamesonii cv. ‘Dune’) flowers. The vase-life of flowers held in a solution containing 5 mg L⁻¹ SNP plus 6% sucrose was found to be significantly higher than with 8-HQC (8-hydroxyquinoline citrate) or control treatments. However, the vase-life was not different to that of flowers held in similar concentrations of silver nitrate. All gerbera flowers held in SNP solutions showed significantly higher relative fresh weight than the control. Vase-life of gerbera flowers was extended by addition of either 50 or 100 mg L⁻¹ carvacrol and either 1 or 2 mg L⁻¹ SNP from 8.3 to 16 d. In addition, the relative fresh weight and solution uptake of gerbera flowers were increased by addition of 100 mg L⁻¹ essential oils and 1 or 2 mg L⁻¹ SNP as compared to that of control flowers. Our results suggest the potential application of essential oils or SNP as novel alternatives to common chemicals used in preservative solutions for gerbera flowers.     

陆地棉蔗糖磷酸合成酶基因家族的鉴定及表达分析

【目的】蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)是调控植物蔗糖代谢合成途径的关键酶,在植物光合产物的积累与分配方面发挥着重要作用,然而棉花中SPS基因的系统研究尚很少开展。本研究旨在对陆地棉SPS基因进行全基因组鉴定,并对它们的表达特性进行系统分析。【方法】基于已公布的陆地棉基因组序列,利用生物信息学和荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,qRT-PCR)等方法对陆地棉SPS家族基因的蛋白结构、进化关系、基因结构特征、染色体定位、基因复制和表达特性进行分析。【结果】(1)在陆地棉基因组中,共鉴定到10个Gh SPS基因(Gh SPS1-Gh SPS10);(2)Gh SPS蛋白具有植物SPS家族特有的两个保守的蛋白结构域和3个相对保守的蛋白磷酸化位点;(3)进化分析表明,Gh SPS蛋白可聚为A、B和C共3个亚族,其中A亚族成员最多,包含6个GhSPS蛋白;(4)位于同一亚族的GhSPS基因具有相似的外显子-内含子分布模式,但是外显子/内含子数目在不同亚族间差异很大;(5)GhSPS基因均匀地分布在陆地棉A亚组和D亚组的5条染色体上,片段复制可能导致了GhSPS基因在陆地棉基因组中的扩增;(6)转录组分析表明,不同亚族GhSPS基因具有不同的组织表达模式,A亚族Gh SPS基因在被检测的各个组织均有较高的表达,B亚族GhSPS基因主要在叶片中高表达,C亚族Gh SPS基因主要在纤维、叶片和花瓣中高表达;(7)进一步荧光定量PCR分析表明,GhSPS4在叶片中表达量很高,GhSPS1在叶片和花瓣中表达量较高,Gh SPS7和Gh SPS10在被检测的各个组织均有较高的表达,该结果与转录组分析结果相对一致。【结论】陆地棉SPS基因家族包含10个成员,分布在5条染色体上,可分为3个亚族,不同亚族成员呈现出不同的表达模式,为后续深入解析陆地棉SPS家族基因的功能奠定理论基础。     

A novel aphid resistance locus in cowpea identified by combining SSR and SNP markers

The utility of combining simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker genotyping was determined for genetically mapping a novel aphid (Aphis craccivora) resistance locus in cowpea breeding line SARC 1‐57‐2 and for introgressing the resistance into elite cultivars by marker‐assisted backcrossing (MABC). The locus was tagged with codominant SSR marker CP 171F/172R with a recombination fraction of 5.91% in an F₂ population from ‘Apagbaala’ x SARC 1‐57‐2. A SNP‐genotyped biparental recombinant inbred line population was genotyped for CP 171F/172R, which was mapped to position 11.5 cM on linkage group (LG) 10 (physical position 30.514 Mb on chromosome Vu10). Using CP 171F/172R for foreground selection and a KASP‐SNP‐based marker panel for background selection in MABC, the resistance from SARC 1‐57‐2 was introduced into elite susceptible cultivar ‘Zaayura’. Five BC₄F₃ lines of improved ‘Zaayura’ that were isogenic except for the resistance locus region had phenotypes similar to SARC 1‐57‐2. This study identified a novel aphid resistance locus and demonstrated the effectiveness of integrating SSR and SNP markers for trait mapping and marker‐assisted breeding.     

棉花茎端发育长链非编码RNA的鉴定及功能预测

长链非编码RNA(Long non-codingRNA,lncRNA)是一类长度在200 nt以上的非编码RNA。它不具备蛋白质编码功能,但在生物体中以RNA分子的形式参与众多生物过程。利用实验室前期获得的陆地棉茎尖转录组数据,鉴定了8044条lncRNA,其中3691条分布于At亚组,2852条分布于Dt亚组;通过基因组共定位、碱基互补配对等生物信息学方法对其中2227条lncRNA进行功能注释,其中1875条位于编码基因上下游,可能通过与编码基因的顺式作用元件或3’UTR区结合,在转录或者转录后水平调控基因表达;317条反义lncRNA与正义链的mRNA存在互作,通过碱基互补配对调控基因沉默、转录及mRNA的稳定性;20条lncRNA预测为microRNA的前体;5条lncRNA注释到4个lncRNA家族。功能注释表明,这些lncRNA主要参与转录调节、代谢、激素应答和信号转导等生物过程。本研究为充分利用高通量测序数据研究棉花lncRNA提供了新思路。     

陆地棉PPO基因全基因组鉴定及对黄萎病菌的响应分析

【目的】多酚氧化酶(Polyphenol oxidases,PPO)广泛参与植物抵抗病虫害等生物逆境胁迫的过程。本研究旨在研究棉花中的PPO基因及其表达模式,验证多酚氧化酶与棉花抗黄萎病的关系。【方法】分析了黄萎病菌V991侵染下异源四倍体陆地棉TM-1根系中PPO酶活力变化,并利用TM-1的基因组数据库,鉴定相关基因,并进行生物信息学和表达分析。【结果】共筛选到13个候选GhPPO基因。这些基因都不含内含子,所编码的蛋白包含2个铜离子结合位点和PPO的保守序列(酪氨酸酶、DWL和KFDV保守结构域)。进化分析显示,陆地棉PPO基因的种内相似性大于种间相似性,并且相互之间形成了多对重复基因。GhPPO的表达量差异较大,少数基因具有组织特异表达的特性,多数基因在棉花不同组织中表达量都很低。实时荧光定量聚合酶链式反应分析结果表明:GhPPO6D在棉花根系中表达量较高,且在黄萎病菌V991侵染后上调。【结论】GhPPO6D可能参与了棉花与黄萎病菌的互作过程,与V991侵染后棉花根系PPO酶活力上升相关。     

Identification of genome-wide SNPs associated with combining ability for grain quality traits in parents of hybrid japonica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Improving the combining ability for grain quality traits of the parents is the key factor to enhance averaged bulk grain quality of hybrid japonica rice. Eight quality traits including brown rice rate, milled rice rate (MRR), head rice rate (HRR), chalkiness degree (CD), percentage of chalky grain (PCG), alkali spreading value (ASV), gel consistency (GC) and amylose content (AC) of the bulked sample of rough rice harvested from the 81 F₁ hybrid plants were investigated in 2014 and 2015. By combining the phenotypic data of general combining ability (GCA) for the quality traits with genotypic data of single nucleotide polymorphism (SNP) obtained by genotyping by sequencing method, CAScreen1.0 program compiled by MATLAB language was conducted to identify the elite SNP genotype associated with combining ability of quality traits in parents. Totally 35 elite SNP genotypes involving 22 genes (genotypes) were detected associated with GCA of seven quality traits (P?<?0.05) in both 2014 and 2015. Seven and four SNPs were detected associated with combining ability for MRR and HRR, respectively. For PCG and CD, six and ten SNPs were detected associated with combining ability of parents, respectively. Six SNPs involving two genes were detected for combining ability of GC. Only two SNPs were detected which associated with combining ability for ASV. For combining ability of AC, six elite SNPs genotype were detected. Among them, elite SNP genotype of LOC_Os08g25220 (T/C) located on chromosome 8 could significantly reduce AC content in mixed rice samples of hybrid japonica rice.     

Identification and confirmation of quantitative trait loci for stachyose content in soybean seed

Stachyose is an unfavorable sugar in soybean meal that causes flatulence for non‐ruminant animals. Understanding the genetic control of stachyose in soybean will facilitate the modification of stachyose content at the molecular level. The objective of this study was to identify quantitative trait loci (QTL) associated with seed stachyose content using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. A normal stachyose cultivar, ‘Osage’, was crossed with a low stachyose line, V99‐5089, to develop a QTL mapping population. Two parents were screened with 33 SSR and 37 SNP markers randomly distributed on chromosome 10, and 20 SSR and 19 SNP markers surrounding a previously reported stachyose QTL region on chromosome 11. Of these, 5 SSR and 16 SNP markers were used to screen the F_3:4 lines derived from ‘Osage’ x V99‐5089. Seed samples from F_3:5 and F_3:6 lines were analyzed for stachyose content using high‐performance liquid chromatography (HPLC). Composite interval mapping analysis indicated that two stachyose QTL were mapped to chromosome 10 and 11, explaining 11% and 79% of phenotypic variation for stachyose content, respectively. The SSR/SNP markers linked to stachyose QTL could be used in breeding soybean lines with desired stachyose contents. Chi‐square tests further indicated that these two QTL probably represent two independent genes for stachyose content. Therefore, a major QTL was confirmed on chromosome 11 and a novel QTL was found on chromosome 10 for stachyose content.     

Mapping resistance gene analogs (RGAs) in cultivated tetraploid cotton using RGA-AFLP analysis

Diseases cause significant losses in cotton production throughout the US Cotton Belt. Growing resistant cultivars can significantly improve cotton yields and effectively reduce production inputs. Disease resistance (R) genes have been isolated in numerous plant species and the R genes with domains of nucleotide binding sites (NB) and leucine rich repeats (LRR) represent the largest R gene family. Degenerate primers designed based on conserved motifs of plant disease resistance genes were used alone or in combination with AFLP primers to analyze disease resistance gene analogs (RGAs) in a recombinant inbred line (RIL) population of Pima (Gossypium barbadense) 3–79 and Upland cotton (G. hirsutum) line NM 24016. Eighty-eight polymorphic RGA markers were amplified by 8 pairs of RGA degenerate primers, while 131 polymorphic RGA-AFLP markers were produced from six pairs of RGA-AFLP primer combinations. Of the 219 polymorphic RGA and RGA-AFLP markers that were identified, 212 were assigned to 18 chromosomes and linkage groups based on existing SSR markers that are on known chromosomes. However, the RGA and RGA-AFLP markers are not evenly distributed among chromosomes in that 189 RGA and RGA-AFLP markers (88%) are assigned onto three “giant” chromosomes, i.e., C6, C12, and C15, suggesting RGA clusters in the cotton genome. Several RGA and RGA-AFLP markers were mapped to the same linkage group carrying a root-knot nematode resistance gene. The identification and mapping of RGA and RGA-AFLP markers provide a framework to facilitate marker-assisted selection of disease resistance in cotton breeding and to understand the physical relationship of cotton resistance genes.     

Investigations on F1 and F2 hybrids between Brassica oleraceae Var. Acephala and Raphanus sativus

A cross was made between B. oleraceae var. acephala and R. sativus. The use of a piece of cotton soaked with the growth regulator, N-m-tolyphthalamic acid at 100 ppm and attached to the pedicel at the time of pollination resulted in set of several pods with one viable seed. The hybrid kale x radish appeared vegetatively intermediate between the parents except for the flower color which resembled the radish. The F₁ plant propagated asexually and maintained under screen isolation cages with pollinating insects for a period of four years yielded only one viable F₂ seed. The single F₂ plant obtained, an allotetraploid, failed to yield any fertile seeds through selfing. Backcrossing of this F₂ plant to the radish has yielded two seeds.Department of Horticulture, Michigan State UniversityWith 7 figuresJournal article No. 2923. From the Michigan Agricultural Expreriment Station.     

<Emphasis Type="Italic">LKF</Emphasis>, the locus regulating large grains in the rice cultivar ‘Fusayoshi’, is identical to the loci encoding a serine/threonine protein phosphatase with Kelch motif

The LKF locus, which regulates grain size in the rice cultivar ‘Fusayoshi’ showing large grain, has been mapped to the proximal part of the long arm of chromosome 3. An incomplete dominant allele, Lkf, caused large grain size of Fusayoshi. The structure and function of this locus, however, have not yet been determined. In a similar position to LKF on chromosome 3, two loci, Os03g0407400 (GS3) and LOC_Os03g44500, have been already reported as loci also regulating rice grain size. The objective of the present study was to determine the nucleotide sequences of both Os03g0407400 and LOC_Os03g44500 for different alleles at the LKF locus. Results showed that only one known single nucleotide polymorphism (SNP) in exon 10 of LOC_Os03g44500 was detected between a large-grain allele (Lkf) and a small-grain allele at the LKF locus, whereas no polymorphisms in Os03g0407400. This SNP, visualized using a dCAPS marker, clearly demonstrated nearly complete co-segregation with grain length in an F₂ population segregating the Lkf at LKF. Other large-grain mutant lines with large-grain alleles at the LKF locus, which originated from another cultivar ‘Gimbozu’, also showed the same SNP in exon 10 of LOC_Os03g44500. It was concluded from these results that LKF is identical to LOC_Os03g44500, and the detected SNP in exon 10, at least, which is included in Kelch-like repeat motif, could be essential for expression of the large-grain phenotype.     

Hybridization between cotton and Malvaceae species as a tool for production of partial interspecific aneuploid cotton plants

Cotton although is an autogamous species could be cross pollinated under favorable climate conditions and/or in the presence of pollinators. The coexistence of cotton with Malvaceae species raises questions on the possibility pollen to be exchanged among Malvaceae species and on the resulted consequences. The present work was undertaken to evaluate the in situ response of cotton flowers (G.hirsutum L, G. barbadense L.) and their F₁ interspecific hybrids when are artificially pollinated with Malva sylvestris L., Hibiscus syriacus L. and Abelmoschus esculentus Moench. Furthermore, an in vitro protocol was attempted to support embryos’ growth in order to produce viable progenies originating from crosses between cotton and the aforementioned Malvaceae species. The obtained results gave evidence that pollen from the above Malvaceae species stimulated cotton ovaries without successful hybridization. The interaction between pollen and cotton’s stigmas was higher at early stages when M. sylvestris was used as pollinator but in crosses with H. syriacus and A. esculentus more ovules were activated as revealed by the percentage of carpodesis and life-time of bolls onto maternal plants. Only crosses between cotton with okra produced cotton seeds under in situ conditions. The in vitro embryo-ovule culture protocol, used, increased the number of regenerated cotton plants, especially in crosses among F₁ interspecific cotton hybrids and A. esculentus. In this case, regenerated plants were recombinant aneuploids, combining traits from both cotton species. This novel cotton germplasm possessing unique chromosome rearrangements, at aneuploid level could be proved useful after cytogenetic, molecular or QTL genetic analysis referring to important agronomic traits.     

Construction of a high-density genetic map using genotyping by sequencing (GBS) for quantitative trait loci (QTL) analysis of three plant morphological traits in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

In recent years, the production costs of cotton (Gossypium hirsutum L.) in China have continued to rise, and this has been accompanied by relatively low productivity, diminished enthusiasm of Chinese farmers for planting cotton, and the difficulty caused by high subsidies as well as the high degree of mechanized harvesting for competing crops like grains. Therefore, it is urgent to improve the level of mechanization and the scale of cotton production in China. Morphological traits play an important role in the mechanized harvesting of cotton. Plant height (PH), height of the first fruiting branch node (HFFBN), and the number of vegetative shoot (NOVS) are key cotton morphological traits that influence mechanical harvesting. The genetic basis of PH, HFFBN, and NOVS were examined in the Z571 and CCRI 49 parents as well as 188 individuals comprising the F₂ mapping population. This F₂ population was examined using genotyping by sequencing (GBS) with 5571 high-density polymorphism single nucleotide polymorphism (SNP) markers to construct a genetic linkage map comprised of 3187 polymorphic markers. The genetic map spanned 3828.551 cM, with an average distance of 0.687 cM between markers. The complete interval mapping method identified 17 quantitative trait loci (QTL) for PH, HFFBN, and NOVS located on chromosomes 3, 4, 5, 7, 9, 17, 19, 23, and 25. Our study provides an efficient approach for fast detection of QTL underlying complex trait variation with high accuracy, thus providing preliminary information that can improve the efficiency of subsequent machine cotton picking through breeding and molecular marker-assisted selection methods.     

Identification of resistance gene analogs in cotton (Gossypium hirsutum L.)

Sequence analyses of numerous plant disease resistance genes have revealed the presence of conserved motifs common to this class of genes, namely a nucleotide binding site (NBS) and leucine rich repeat region. In this study, thirty-three resistance gene analogs (RGAs) were cloned and sequenced from cotton (Gossypium hirsutum L.) following PCR with degenerate primers designed from the conserved NBS motif of plant resistance (R) genes. Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into four distinct classes from which several subgroups were delineated based on nucleic acid sequences. Gene database searches with the consensus protein sequences of each of the four classes and respective subgroups of cotton RGAs revealed their conserved NBS domains and homology to RGAs and known resistance genes from a variety of plant genera. Given the complete lack of knowledge regarding molecular organization of R genes in cotton, the cloned RGAs described here may be useful as probes to map, characterize, and manipulate R genes of the cotton genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymorphisms of the <Emphasis Type="Italic">FT</Emphasis> gene as a tool to identify underground rhizome types of bamboos

The Flowering Locus T (FT)-like genes of angiosperms are highly conserved. The FT-encoded proteins include a phosphatidylethanolamine-binding domain that is involved in the control of the shoot apical meristem identity and flowering time. In the present study, FT genes were investigated in 20 bamboo species that are grouped into sympodial, mixed and scattered bamboos based on their morphology. All examined orthologous FT genes consisted of four exons and three introns. Their encoded protein sequences contained the critical amino acid residues Tyr85, Glu109, Leu128, Tyr134, Trp138, Arg139, Gln140 and Asn152, of which each possesses a biological function. The DNA sequences were rich in single nucleotide polymorphism (SNP) sites. The SNP frequency was 1 SNP/16.8 bp, and the nucleotide diversity (π) equaled 0.265. Some SNPs altered restriction enzyme sites or resulted in changes in amino acid contents. The correlation analysis showed that several SNPs were informative in relation to the underground rhizome types of bamboos. Therefore, FT polymorphisms could be used as a tool to identify the underground rhizome types of bamboos. The phylogenetic tree constructed based on the FT gene sequences showed that the obtained clustering was consistent with the underground rhizome types. The SNP markers developed in the present study will provide information on the genetic diversity of bamboos and they can aid taxonomic study as well.     

Introgression genetics and breeding between Upland and Pima cotton: a review

The narrow genetic base of elite Upland cotton (Gossypium hirsutum L.) germplasm has been a significant impediment to sustained progress in the development of cotton cultivars to meet the needs of growers and industry in recent years. The prospect of widening the genetic base of Upland cotton by accessing the genetic diversity and fiber quality of Pima cotton (Gossypium barbadense L.) has encouraged interspecific hybridization and introgression efforts for the past century. However, success is limited due mainly to genetic barriers between the two species in the forms of divergent gene regulatory systems, accumulated gene mutations, gene order rearrangements and cryptic chromosomal structure differences that have resulted in hybrid breakdown, hybrid sterility and selective elimination of genes. The objective of this paper is to provide a mini-review in interspecific hybridization between Upland and Pima cotton relevant to breeding under the following sections: (1) qualitative genetics; (2) cytogenetic stocks; (3) quantitative genetics; (4) heterosis, and (5) introgression breeding. Case studies of successful examples are provided.