Molecular markers and their applications in wheat breeding

In recent years, considerable emphasis has been placed on the development of molecular markers to be used for a variety of objectives. This review attempts to give an account of different molecular markers—restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), sequence-tagged sites (STS), DNA amplification fingerprinting (DAF), amplified fragment length polymorphisms (AFLPs) and microsatellites (STMS)—currently available for genome mapping and for tagging different traits in wheat. Other markers, including microsatellite-primed polymerase chain reaction (MP-PCR), expressed sequence tags (ESTs) and single nucleotide polymorphisms (SNPs) are also discussed. Recent information on synteny in cereal genomes, marker-assisted selection, marker validation and their relevance to cereal breeding in general and wheat breeding in particular are also examined.     

Wild emmer: genetic resources,gene mapping and potential for wheat improvement

Wild emmer, Triticum dicoccoides, the progenitor of cultivated wheat, harbors rich genetic resources for wheat improvement. They include many agronomic traits such as abiotic stress tolerances (salt, drought and heat), biotic stress tolerances (powdery mildew, rusts, and Fusarium head blight), grain protein quality and quantity, and micronutrient concentrations (Zn, Fe, and Mn). In this review, we summarize (1) traits and controlling genes identified and mapped in T. dicoccoides; and (2) the genes transferred to cultivated wheat from T. dicoccoides. These genes, controlling important agronomic traits such as disease resistance, high protein and micronutrient content, should contribute to wheat production and food nutrition. However, most of the rich genetic reservoir in wild emmer remains untapped, highlighting the need for further exploration and utilization for long-term wheat breeding programs.     

Wheat breeding assisted by markers: CIMMYT’s experience

Significant progress has been made in the characterization of loci controlling traits of importance using molecular markers. A number of markers are currently available in wheat for genes of interest to the breeders. Markers can be used to better characterize parental material, thereby improving the efficiency and effectiveness of parental selection for crossing and to track genes in segregating progenies through the selection process. Although a number of breeding programs are using molecular markers at modest levels, the costs associated with marker assisted selection (MAS) are frequently cited as the main constraint to their wide-spread use by plant breeders. However, this is likely to change when user-friendly, high-throughput, automated marker technologies based on single nucleotide polymorphisms become available. These evolving technologies will increase the number of available markers, and will improve the efficiency, throughput, and cost effectiveness of MAS, thereby making it more attractive and affordable to many breeding programs. This article examines the extent to which molecular markers have been used at the International Maize and Wheat Improvement Center (CIMMYT) in applied wheat breeding and reviews the limited publicly available information on MAS from other wheat breeding programs. As markers are currently available for relatively few traits, we believe that MAS must be integrated with ongoing conventional breeding to maximize its impact. When used in tandem with phenotypic selection, MAS will improve response to selection for certain traits, thereby increasing rates of genetic progress.     

基于AFLP分子图谱的蝴蝶兰3个叶片性状QTL分析

本研究以一个蝴蝶兰杂交组合Phal.462×Phal.20后代为材料,测定其后代群体中88个单株的叶长、叶宽和株幅,并基于已构建的AFLP分子连锁图谱,应用区间作图法对调控这3个性状的基因进行了QTL分析。统计分析结果表明,3个性状在群体中呈连续分布,具有数量性状的典型特征,叶长与株幅之间存在极显著正相关。QTL分析结果表明,在父本中共检测到QTL60个,分布于8个连锁群上,包括控制叶长性状的QTL20个,控制叶宽性状19个,控制株幅性状21个;各QTL的LOD值在3.05～14.78之间,可解释44.1%～89.4%的表型变异。在母本中共检测到QTL28个,分布于五个连锁群上,包括控制叶长性状10个,控制叶宽性状8个,控制株幅性状10个;各QTL的LOD值在3.1~9.24之间,可解释57.7%~76.3%的表型变异。这是蝴蝶兰上首个QTL分析研究,可为今后蝴蝶兰的基因克隆与分子标记辅助育种等提供基础。     

Phenotypic and marker-assisted evaluation of spring and winter wheat germplasm for resistance to fusarium head blight

Fusarium head blight (FHB) caused by Fusarium species, is among the most devastating wheat diseases, causing losses in numerous sectors of the grain industry through yield and quality reduction, and the accumulation of poisonous mycotoxins. A germplasm collection of spring and winter wheat, including nine reference cultivars, was tested for Type II FHB resistance and deoxynivalenol (DON) content. Genetic diversity was evaluated on the basis of Simple Sequence Repeat (SSR) markers linked to FHB resistance quantitative trait loci (QTLs) and Diversity Arrays Technology (DArT) markers. The allele size of the SSR markers linked to FHB resistance QTLs from known resistance sources was compared to a germplasm collection to determine the presence of these QTLs and to identify potentially novel sources of resistance. Forty-two accessions were identified as resistant or moderately resistant to Fusarium spread, and two also had very low DON concentrations. Genetic relationships among wheat accessions were generally consistent with their geographic distribution and pedigree. SSR analysis revealed that several resistant accessions carried up to four of the tested QTLs. Resistant and moderately resistant lines without any known QTLs are considered to be novel sources of resistance that could be used for further genetic studies.     

Molecular markers in Brassica oilseed breeding: current status and future possibilities

As PCR techniques have developed over the last 15 years, a wealth of new DNA marker technologies have arisen which have enabled the generation of high‐density molecular maps for all the major Brassica crop species. Molecular markers have also been heavily used in analyses of genetic diversity in Brassica crops. The majority of the work utilizing molecular markers in Brassica oilseed breeding has to date been based on genetic mapping using various DNA marker systems in segregating populations generated for specific investigations of particular traits of interest. For numerous qualitative traits, traditional mapping approaches have led to the development of marker‐assisted selection strategies in oilseed Brassica breeding, and in some cases to map‐based cloning of the responsible genes. For quantitative traits, however, it has become apparent that traditional mapping of quantitative trait loci (QTL) is often not sufficient to develop effective markers for trait introgression or for identification of the genes responsible. In this case, allele‐trait association studies in non‐structured genetic populations represent an interesting new approach, provided the degree of gametic phase disequilibrium between the QTL and the marker loci is sufficient. Because Brassica species represent the closest crop plant relatives to the model plant Arabidopsis thaliana, significant progress will be achieved in the coming years through integration of candidate gene approaches in crop brassicas, using the detailed information now available for the Arabidopsis genome. Integration of information from the model plant with the increasing supply of data from physical mapping and sequencing of the diploid Brassica genomes will undoubtedly give great insight into the genetics underlying both simple and complex traits in oilseed rape. This review describes the current use of available genetic marker technologies in oilseed rape breeding and provides an outlook for use of new technologies, including single‐nucleotide polymorphism markers, candidate gene approaches and allele‐trait association studies.     

The Power of Integrative Approaches to Develop Markers for Maize Molecular Breeding

Recent advances in genomics and bioinformatics now offer real opportunities for dissecting complex Waits into their component sub-traits, which will simplify the process of developing the tools necessary to manipulate the underlying genes （Varshney et al., 2005）. The value of molecular markers as a complement to phenotyping under several breeding scenarios is largely unquestioned, as demonstrated by the increasing number of successful studies published （Varshney et al., 2006）. However most experiments have targeted crop improvement for disease resistance, morphological waits or quality waits （Franca et al., 2005） and there is still some way to go before markers can be used routinely and ubiquitously to breed for complex waits, such as tolerance to abiotic stress （Ribaut and Ragot, 2007）. The combination of genetic mapping and association studies has considerable potential to generate a catalogue of genetic variation, and thereby present novel opportunities for selection based on genome-wide scans （Biswas and Akey, 2006）. Nevertheless, it remains to be seen whether the outcome of gene interactions, particularly where significant gene networks are involved,     

Characterization of dinucleotide and trinucleotide EST-derived microsatellites in the wheat genome

Over the past decade microsatellites or simple sequence repeats (SSRs) have attracted a considerable amount of attention from researchers. The aim of the present paper was to analyse expressed sequence tag-derived SSR (EST-SSR) marker variability in wheat and to investigate the relationships between the number and type of repeat units and the level of microsatellite polymorphism. Two hundred and forty-one new EST-SSR markers available in a public database () were characterized in eight durum wheat cultivars (Svevo, Ciccio, Primadur, Duilio, Meridiano, Claudio, Latino, Messapia), two accessions of Triticum turgidum var. dicoccoides (MG4343, MG29896), one accession of T. turgidum var. dicoccum (MG5323) and in the common wheat cv. Chinese Spring. Of these, 201 primer pairs (83.4%) amplified PCR products successfully, while the remaining 40 (16.6%) failed to amplify any product. Of the EST-SSRs analysed, 45.2% of the primer pairs amplified one or two PCR products. Multiple discrete PCR products were observed among both di- and trinucleotide EST-SSR markers (31.2 and 40.5%, respectively). Markers based on dinucleotide microsatellites were more polymorphic than those based on trinucleotide SSRs in the 12 wheat genotypes tested (68.9 and 52.7%, respectively). An average of 2.5 alleles for dinucleotide and 2.0 alleles for trinucleotide SSRs was observed. The data reported in the present work indicate the presence of a significant relationship between motif sequence types and polymorphism. The primer set based on the AG repeat motif showed the lowest percentage of polymorphism (55.0%), while the primer set based on the AC repeat motif showed t he highest percentage (85.0%). Among trinucleotide SSRs, the AGG microsatellite markers showed the highest percentage of polymorphism (70.0%), and the ACG motif the lowest value (25.0%). The characterization of these new EST-SSR markers and the results of our studyon the effect of repeat number and type of motifs could have important applications in the genetic analysis of agronomically important traits, quantitative trait locus discovery and marker-assisted selection.     

Molecular genetic analysis of flour color using a doubled haploid population in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Flour color is an important trait in the assessment of flour quality for the production of many end products. In this study, quantitative trait loci (QTLs) with additive effects, epistatic effects, and QTL × environment (QE) interactions for flour color in bread wheat (Triticum aestivum L.) were studied, using a set of 168 doubled haploid (DH) lines derived from a Huapei 3 × Yumai 57 cross. A genetic map was constructed using 283 simple sequence repeats (SSR) and 22 expressed sequence tags (EST)-SSR markers. The DH and parents were evaluated for flour color in three environments. QTL analyses were performed using QTLNetwork 2.0 software based on a mixed linear model approach. A total of 18 additive QTLs and 24 pairs of epistatic QTLs were detected for flour color, which were distributed on 19 of the 21 chromosomes. One major QTL, qa1B, closely linked to barc372 0.1 cM, could account for 25.64% of the phenotypic variation of a* without any influence from the environments. So qa1B could be used in the molecular marker-assisted selection (MAS) in wheat breeding programs. The results showed that both additive and epistatic effects were important genetic basis for flour color, and were also sometimes subject to environmental modifications. The information obtained in this study should be useful for manipulating the QTLs for flour color by MAS in wheat breeding programs. Kun-Pu Zhang and Guang-Feng Chen contributed equally to this study.     

Molecular markers and doubled haploids in European plant breeding programmes

The breeding companies and laboratories involved in this article cover a wide range of crops grown in the temperate climate zone: small grain cereals, oilseed crops, forage crops, turf, vegetables and potato. Speed and efficiency are becoming increasingly important in variety breeding and doubled haploids (DH) and genetic markers are important biotechnological tools to accelerate materials to market. Collaborative research between universities, research institutions and breeding companies has resulted in the routine use of DH technology and molecular markers in practical breeding of barley, wheat and rapeseed. DH populations have been established not only for barley, wheat and rapeseed, but for rye, oat and triticale, where DH technology is less developed. A driver here is the value of the crop e.g. although wheat is less responsive to DH production the value of the end product makes the effort worthwhile. Simple and rapid DNA extraction methods used in high-throughput marker assisted selection (MAS) systems are essential for routine use of markers. MAS is used both to monitor the presence of genes of interest and also to monitor the genetic background. DH technology in forage, turf and vegetables is still in progress and the practical use of markers in all crops is limited by access to trait linked markers. Collaboration and technology transfer with universities, research institutions and breeding companies is essential for the improvement of both DH protocols in recalcitrant crops and marker technology in all crops.     

分子标记辅助选择小麦抗白粉病基因Pm2、Pm4a、Pm21 的聚合体

利用与小麦抗白粉病基因Pm2、Pm4a和Pm21紧密连锁的PCR标记,对含有Pm2、Pm4a和Pm21的小麦品系复合杂交后代经3轮分子标记选择,得到了一批聚合有Pm2+Pm4a+Pm21 3个基因的抗病植株,以及若干株Pm2+Pm21、Pm4a+Pm21和Pm2+Pm4a 2个基因聚合的植株。同时,还对中选植株进行抗病性人工接种鉴定。结果表明,含有Pm21的聚合体与Pm21基因单独存在时抗性相当,均对白粉病免疫,聚合体Pm2+Pm4a的抗性好于Pm2或Pm4a单独存在时的抗性。为降低分子标记选择成本,将检测Pm4a和Pm21的2种PCR放在一个反应体系中进行,扩增产物经1次电泳,可同时检测出Pm4a和Pm21,不同引物之间没有明显交叉扩增现象。     

Applications of pedigree-based genome mapping in wheat and barley breeding programs

The aim of the pedigree-based genome mapping project is to investigate and develop systems for implementing marker assisted selection to improve the efficiency of selection and increase the rate of genetic gain in breeding programs. Pedigree-based whole genome marker application provides a vehicle for incorporating marker technologies into applied breeding programs by bridging the gap between marker–trait association and marker implementation. We report on the development of protocols for implementation of pedigree-based whole genome marker analysis in breeding programs within the Australian northern winter cereals region. Examples of applications from the Queensland DPI&F wheat and barley breeding programs are provided, commenting on the use of microsatellites and other types of molecular markers for routine genomic analysis, the integration of genotypic, phenotypic and pedigree information for targeted wheat and barley lines, the genomic impacts of strong selection pressure in case study pedigrees, and directions for future pedigree-based marker development and analysis.     

Quantitative trait loci for early maturity and their potential in breeding for earliness in Brassica juncea

A doubled haploid population of Brassica juncea, developed from a cross between two parental lines differing for days to maturity, was used to study the efficiency of indirect selection for a primary trait through selection of secondary trait(s) over direct selection for the primary trait when quantitative trait loci information is available for both primary and secondary traits, and applied. Days to maturity was considered as primary trait, while days to first flowering, days to end of flowering, flowering period and plant height were considered as secondary traits. An RFLP linkage map was employed for QTL analysis of maturity and maturity-determinant traits, and a stable QTL B6 simultaneously affecting these two types of traits was identified. This linked QTL explained 11.7% phenotypic variation for days to maturity, 20.7% variation for days to first flowering, 24.3% variation for days to end of flowering and 14.4% variation for plant height. Phenotypic evaluation of maturity and/or maturity-determinant traits, viz. days to first flowering, days to end of flowering and plant height revealed that limited genetic advance for early maturity can be achieved through phenotypic selection of the primary and/or the secondary trait(s). However, the estimates of genetic advance for early maturity based on combined phenotypic evaluation and linked QTL data was found to be, at least, three times higher compared to genetic advance based on phenotypic evaluation only, demonstrating the potential of marker-assisted selection in breeding for early maturity in B. juncea.     

Application of new knowledge,technologies, and strategies to wheat improvement

There have been many changes impacting wheat improvement since the 1996 International Maize and Wheat Improvement Center Wheat Yield Symposium. This review highlights a few of the technological advances and impacts of new knowledge on wheat improvement that have occurred in the past 10 years as well as on-going challenges. One of the most dramatic discoveries has been the revelation that the genomes of graminaceous crops are complex, rapidly evolving, and heterogeneous, even within species. The use of marker-assisted selection for improving complex traits is one of the challenges facing wheat breeders. Integration of association analysis into conventional breeding programs is proposed as a crop improvement strategy that has the potential to improve the efficiency of molecular breeding.     

An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts

Recognizing the enormous potential of DNA markers in plant breeding, many agricultural research centers and plant breeding institutes have adopted the capacity for marker development and marker-assisted selection (MAS). However, due to rapid developments in marker technology, statistical methodology for identifying quantitative trait loci (QTLs) and the jargon used by molecular biologists, the utility of DNA markers in plant breeding may not be clearly understood by non-molecular biologists. This review provides an introduction to DNA markers and the concept of polymorphism, linkage analysis and map construction, the principles of QTL analysis and how markers may be applied in breeding programs using MAS. This review has been specifically written for readers who have only a basic knowledge of molecular biology and/or plant genetics. Its format is therefore ideal for conventional plant breeders, physiologists, pathologists, other plant scientists and students.     

Molecular marker analysis of kernel size and shape in bread wheat

The economic value of wheat grain is determined by the kernel morphology which is an important parameter for manufacturing different food products requiring specific grain characteristics. Although kernel size and shape have emerged as important breeding objectives, not much information is available about the number or location of associated gene(s)/quantitative trait loci. In the present study, a recombinant inbred line population of 106 plants (F₇) was phenotyped for four traits, namely kernel length, width, weight and factor form density (FFD) and genotyped with different polymerase chain reaction‐based markers. Transgressive segregants were observed for all the traits and genetic correlation studies showed positive correlations between the majority of the traits. The number of markers associated with each trait ranged from two to nine and the phenotypic contribution by an individual marker ranged from 3.3 to 16.6%. Many of the markers showed linkage to more than one trait. Strategies for improving the wheat grain quality traits and the utility of such markers in marker‐assisted selection (MAS) efforts are discussed.     

QTL mapping for flour and noodle colour components and yellow pigment content in common wheat

Improvement of flour colour is an important breeding objective for various wheat-based end-products. The objectives of this study were to identify quantitative trait loci (QTL) for flour colour components and yellow pigment content (YPC), using 240 recombinant inbred lines (RILs) derived from a cross between the Chinese wheat cultivars PH82-2 and Neixiang 188. Field trials were performed in a Latinized α-lattice design in Anyang and Jiaozuo, Henan Province and Taian, Shandong, in the 2005–2006 and 2006–2007 cropping seasons providing data for six environments. One hundred and eighty-eight polymorphic SSR markers, rye secalin marker Sec1, STS markers YP7A for a phytoene synthase gene (Psy-A1), and four glutenin subunit markers, were used to genotype the population and construct the linkage map for subsequent QTL analysis. Two major QTL were detected for YPC, associated with 1RS (1B.1R translocation) and the Psy-A1 (7A) gene, explaining 31.9% and 33.9% of the phenotypic variances, respectively. 1RS also had large influences on Fa*, Fb*, KJ, NL*and Nb*, and Psy-A1 genes showed large effects on Fa*, Fb*, Kj, Fci, NL*, Na* and Nb*, explaining from 4.5 to 26.1% and 4.3 to 35.9% of the phenotypic variances, respectively. In addition, QTL for flour colour parameters and yellow pigment content were also detected on chromosomes 1A and 4A, accounting for 1.5–4.1% of the phenotypic variance. The genetic effect of the 1B.1R translocation on flour colour parameters was also discussed.     

SSR and STS markers for wheat stripe rust resistance gene <Emphasis Type="Italic">Yr26</Emphasis>

Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide. Triticum aestivum-Haynaldia villosa 6VS/6AL translocation lines carrying the Yr26 gene on chromosome 1B, are resistant to most races of Pst used in virulence tests. In order to better utilize Yr26 for wheat improvement, we attempted to screen SSR and EST-based STS markers closely linked with Yr26. A total of 500 F₂ plants and the F_2:3 progenies derived from a cross between 92R137 and susceptible cultivar Yangmai 5 were inoculated with race CYR32. The analysis confirmed that stripe rust resistance was controlled by a single dominant gene, Yr26. Among 35 pairs of genomic SSR markers and 81 pairs of STS markers derived from EST sequences located on chromosome 1B, Yr26 was flanked by 5 SSR and 7 STS markers. The markers were mapped in deletion bins using CS aneuploid and deletion lines. The closest flanking marker loci, Xwe173 and Xbarc181, mapped in 1BL and the genetic distances from Yr26 were 1.4 cM and 6.7 cM, respectively. Some of these markers were previously reported on 1BS. Eight common wheat cultivars and lines developed from the T. aestivum-H. villosa 6VS/6AL translocation lines by different research groups were tested for presence of the markers. Five lines with Yr26 carried the flanking markers whereas three lines without Yr26 did not. The results indicated that the flanking markers should be useful in marker-assisted selection for incorporating Yr26 into wheat cultivars.     

Development and validation of class I SSR markers targeting (GATA) n repeat motifs in rice

SSR markers targeting (GATA) _n motifs are known to be highly polymorphic and useful in many organisms. (GATA) _n motif specific SSR markers covering the whole rice genome are not available. The present study was carried out with an objective to identify class I rice microsatellites in the rice genome with (GATA)_n motifs, in-silico, and validate their potential as molecular markers. A total of 243 such motifs were identified; 65 of these were present in the genic region, 59 were in the upstream region and the remaining motifs were found in the intergenic regions. Many of the (GATA) _n motifs were found within and/or upstream of genes associated with biotic or abiotic stress tolerance. A total of 230 PCR-based markers targeting all the class I (GATA) _n microsatellites were developed and 35 of these markers spread across the rice genome were validated in a set of 24 representative rice varieties belonging to five distinct cultivar groups. All the markers were polymorphic, with average polymorphism information content (PIC) value of 0.61, and the rice cultivars could be uniquely distinguished into different cultivar groups based on marker analysis. These informative markers targeting (GATA) _n motifs representing a new set of markers in rice will be highly useful for genetic studies and marker-assisted selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Passoupathy Rajendrakumar, Akshaya Kumar Biswal and Kannabiran Sakthivel contributed equally.     

The potential of genomic‐assisted breeding to improve Fusarium head blight resistance in winter durum wheat

Durum wheat is the most important tetraploid wheat mainly used for semolina and pasta production, but is notorious for its high susceptibility to Fusarium head blight (FHB). Our objectives were to identify and characterize quantitative trait loci (QTL) in winter durum and to evaluate the potential of genomic approaches for the improvement of FHB resistance. Here, we employed an international panel of 170 winter and 14 spring durum lines, phenotyped for Fusarium culmorum resistance at five environments. Heading date, plant height and mean FHB severity showed significant genotypic variation with high heritabilities and FHB resistance was negatively correlated with both heading date and plant height. The dwarfing gene Rht‐B1 significantly affected FHB resistance and the genome‐wide association scan identified eight additional QTL affecting FHB resistance, explaining between 1% and 14% of the genotypic variation. A genome‐wide prediction approach yielded only a slightly improved predictive ability compared to marker‐assisted selection based on the four strongest QTL. In conclusion, FHB resistance in durum wheat is a highly quantitative trait and in breeding programmes may best be tackled by classical high‐throughput recurrent phenotypic selection that can be assisted by genomic prediction if marker profiles are available.