Marker-assisted Plant Breeding Research Articles

Metabolic profiles of peanut (Arachis hypogaea L.) in response to Puccinia arachidis fungal infection

Background Puccinia arachidis fungus causes rust disease in the peanut plants (Arachis hypogaea L.), which leads to high yield loss. Metabolomic profiling of Arachis hypogaea was performed to identify the pathogen-induced production of metabolites involved in the defense mechanism of peanut plants. In this study, two peanut genotypes, one susceptible (JL-24) and one resistant (GPBD-4) were inoculated with Puccinia arachidis fungal pathogen. The metabolic response was assessed at the control stage (0 day without inoculation), 2 DAI (Day after inoculation), 4 DAI and 6 DAI by Gas Chromatography-Mass Spectrometry (GC-MS). Results About 61 metabolites were identified by NIST library, comprising sugars, phenols, fatty acids, carboxylic acids and sugar alcohols. Sugars and fatty acids were predominant in leaf extracts compared to other metabolites. Concentration of different metabolites such as salicylic acid, mannitol, flavonoid, 9,12-octadecadienoic acid, linolenic acid and glucopyranoside were higher in resistant genotype than in susceptible genotype during infection. Systemic acquired resistance (SAR) and hypersensitive reaction (HR) components such as oxalic acid was elevated in resistant genotype during pathogen infection. Partial least square-discriminant analysis (PLS-DA) was applied to GC-MS data for revealing metabolites profile between resistant and susceptible genotype during infection. Conclusion The phenol content and oxidative enzyme activity i.e. catalase, peroxidase and polyphenol oxidase were found to be very high at 4 DAI in resistant genotype (p-value < 0.01). This metabolic approach provides information about bioactive plant metabolites and their application in crop protection and marker-assisted plant breeding.

Mining and validation of novel simple sequence repeat (SSR) markers derived from coconut (Cocos nucifera L.) genome assembly

BackgroundIn the past, simple sequence repeat (SSR) marker development in coconut is achieved through microsatellite probing in bacterial artificial chromosome (BAC) clones or using previously developed SSR markers from closely related genomes. These coconut SSRs are publicly available in published literatures and online databases; however, the number is quite limited. Here, we used a locally established, coconut genome-wide SSR prediction bioinformatics pipeline to generate a vast amount of coconut SSR markers. ResultsA total of 7139 novel SSR markers were derived from the genome assembly of coconut ‘Catigan Green Dwarf’ (CATD). A subset of the markers, amounting to 131, were selected for synthesis based on motif filtering, contig distribution, product size exclusion, and success of in silico PCR in the CATD genome assembly. The OligoAnalyzer tool was also employed using the following desired parameters: %GC, 40–60%; minimum ΔG value for hairpin loop, −0.3 kcal/mol; minimum ΔG value for self-dimer, −0.9 kcal/mol; and minimum ΔG value for heterodimer, −0.9 kcal/mol. We have successfully synthesized, optimized, and amplified 131 novel SSR markers in coconut using ‘Catigan Green Dwarf’ (CATD), ‘Laguna Tall’ (LAGT), ‘West African Tall’ (WAT), and SYNVAR (LAGT × WAT) genotypes. Of the 131 SSR markers, 113 were polymorphic among the analyzed coconut genotypes. ConclusionThe development of novel SSR markers for coconut will serve as a valuable resource for mapping of quantitative trait loci (QTLs), assessment of genetic diversity and population structure, hybridity testing, and other marker-assisted plant breeding applications.

Food Security and the Role of Biotechnology

Biotechnological approaches are given a greater significance as they continue to show promising potential to facilitate food security. These biotechnologies may consist of manipulation and interference in plant breeding (Marker-assisted plant breeding, mutational breeding) genetically manipulating the crop themselves (GM foods), or even just influencing the environmental factors to bring about the desired changes in the crop production (use of bio-fertilizers and bio-pesticides).Some of the greater benefits such as, increased crop yield, enhanced nutritional quality, upgraded ability to adapt and survive, and lengthened shelf-life brought about by these techniques are making developing countries including Bangladesh lean heavily towards the increased commercialization of biotech crops. Even amidst strong oppositions and a few limitations, the popularity of Bt brinjal and the continual development of many biotech rice varieties in Bangladesh alone, further substantiate the favourability of biotech crops and their potential to better the situation favouring both the environment and the economy. Bangladesh J. Nutr. Vol. 34, December 2020, P: 01-13

English

The extraction of high-quality DNA from okra (Abelmoschus esculentus L. Moench) is notoriously troublesome due to the high contents of polysaccharides, polyphenols, and different secondary metabolites. We have tested seven extraction buffers on silica dried okra leaves. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from okra, which is amenable for downstream analyses. In contrast to Cetyl-trimethyl-ammonium bromide (CTAB) methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, PVP-10, and chloroform. It also uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. The procedure employed a high concentration of Sodium dodecyl sulphate (SDS) to rid the problems associated with polysaccharides and polyphenols. The average yield was between 36 and 45 &mu;g of total DNA from 90 mg of dried leaf weight. The DNA is adequate for molecular analysis of okra, such as genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation. &nbsp; Key words: PVP-10, polyvinylpyrrolidone, non cetyltrimethylammonium bromide, okra, genomic DNA, Sodium dodecyl sulphate (SDS).

A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

Extraction of high-quality DNA from Gossypium (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10–15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation.•Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively.•Ribonuclease A was added before chloroform extraction.•A simple, rapid and inexpensive DNA extraction method is described.

Development and application of marker-assisted reverse breeding using hybrid maize germplasm

Humankind has been through different periods of agricultural improvement aiming at enhancing our food supply and the performance of food crops. In recent years, whole genome sequencing and deep understanding of genetic and epigenetic mechanisms have facilitated new plant breeding approaches to meet the challenge of growing population, dwindling resources, and changing climate. Here we proposed a simple and fast molecular breeding method, marker-assisted reverse breeding (MARB), which will revert any maize hybrid into inbred lines with any level of required similarity to its original parent lines. Since all the pericarp DNA of a hybrid is from the maternal parent, whereas one half of the embryo DNA is from the maternal parent and the other half from the paternal parent, so we firstly extract DNA from seed embryo and pericarp of a selected elite hybrid separately and then we derived the genotypes of the two parents with high-density single nucleotide polymorphism (SNP) chips. The following marker-assisted selection was performed based on an Illumina low-density SNP chip designed with 192 SNPs polymorphic between the two parental genotypes, which were uniformly distributed on 10 maize chromosomes. This method has the advantages of fast speed, fixed heterotic mode, and quick recovery of beneficial parental genotypes compared to traditional pedigree breeding using elite hybrids. Meanwhile, MARB has the advantage of not requiring sophisticated transformation and double haploid (DH) technologies over RNA interference (RNAi)-mediated reverse breeding. In addition, MARB can also be used with feed corn harvested from big farms, which is often similar to F2 populations, and the relevant transgenes in the population can be eliminated by marker-assisted selection. As a result, the whole global commercial maize hybrids can be utilized as germplasm for breeding with MARB technology. Starting with an F2 population derived from an elite hybrid, our experiment indicates that with three cycles of marker-assisted selection, selected lines could recover over 80% of the parental genotypes and associated beneficial genes in a fixed heterotic mode. The success application of MARB in maize suggests that this technology is applicable to any hybrid crop to breed new inbreds with improved hybrid performance but the same heterotic mode. As chip technology becomes cheap, it would be expected that polymorphism screening and following marker-assisted selection could be done with one all-purpose high density chip. Several issues associated with MARB were discussed, including its rationale, efficiency and advantages, along with food/feed and environmental safety issues and applications of MARB in variety protection and marker-assisted plant breeding.

Development and Utilization of InDel Markers to Identify Peanut (Arachis hypogaea) Disease Resistance.

Peanut diseases, such as leaf spot and spotted wilt caused by Tomato Spotted Wilt Virus, can significantly reduce yield and quality. Application of marker assisted plant breeding requires the development and validation of different types of DNA molecular markers. Nearly 10,000 SSR-based molecular markers have been identified by various research groups around the world, but less than 14.5% showed polymorphism in peanut and only 6.4% have been mapped. Low levels of polymorphism limit the application of marker assisted selection (MAS) in peanut breeding programs. Insertion/deletion (InDel) markers have been reported to be more polymorphic than SSRs in some crops. The goals of this study were to identify novel InDel markers and to evaluate the potential use in peanut breeding. Forty-eight InDel markers were developed from conserved sequences of functional genes and tested in a diverse panel of 118 accessions covering six botanical types of cultivated peanut, of which 104 were from the U.S. mini-core. Results showed that 16 InDel markers were polymorphic with polymorphic information content (PIC) among InDels ranged from 0.017 to 0.660. With respect to botanical types, PICs varied from 0.176 for fastigiata var., 0.181 for hypogaea var., 0.306 for vulgaris var., 0.534 for aequatoriana var., 0.556 for peruviana var., to 0.660 for hirsuta var., implying that aequatoriana var., peruviana var., and hirsuta var. have higher genetic diversity than the other types and provide a basis for gene functional studies. Single marker analysis was conducted to associate specific marker to disease resistant traits. Five InDels from functional genes were identified to be significantly correlated to tomato spotted wilt virus (TSWV) infection and leaf spot, and these novel markers will be utilized to identify disease resistant genotype in breeding populations.

Leaf Rubisco turnover in a perennial ryegrass (Lolium perenne L.) mapping population: genetic variation, identification of associated QTL, and correlation with plant morphology and yield

This study tested the hypotheses that: (i) genetic variation in Rubisco turnover may exist in perennial ryegrass (Lolium perenne L.); (ii) such variation might affect nitrogen use efficiency and plant yield; and (iii) genetic control of Rubisco turnover might be amenable to identification by quantitative trait loci (QTL) mapping. A set of 135 full-sib F1 perennial ryegrass plants derived from a pair cross between genotypes from the cultivars 'Grasslands Impact' and 'Grasslands Samson' was studied to test these hypotheses. Leaf Rubisco concentration at different leaf ages was measured and modelled as a log-normal curve described by three mathematical parameters: D (peak Rubisco concentration), G (time of D), and F (curve standard deviation). Herbage dry matter (DM) yield and morphological traits (tiller weight (TW), tiller number (TN), leaf lamina length (LL), and an index of competitive ability (PI)) were also measured. The progeny exhibited continuous variation for all traits. Simple correlation and principal component analyses indicated that plant productivity was associated with peak Rubisco concentration and not Rubisco turnover. Lower DM was associated with higher leaf Rubisco concentration indicating that Rubisco turnover effects on plant productivity may relate to energy cost of Rubisco synthesis rather than photosynthetic capacity. QTL detection by a multiple QTL model identified seven significant QTL for Rubisco turnover and nine QTL for DM and morphological traits. An indication of the genetic interdependence of DM and the measures of Rubisco turnover was the support interval overlap involving QTL for D and QTL for TN on linkage group 5 in a cluster involving QTL for DM and PI. In this region, alleles associated with increased TN, DM, and PI were associated with decreased D, indicating that this region may regulate Rubisco concentration and plant productivity via increased tillering. A second cluster involving QTL for LL, TN, PI and DM was found on linkage group 2. The two clusters represent marker-trait associations that might be useful for marker-assisted plant breeding applications. In silico comparative analysis indicated conservation of the genetic loci controlling Rubisco concentration in perennial ryegrass and rice.

Recycling of superfine resolution agarose gel

Genetic markers are now routinely used in a wide range of applications, from forensic DNA analysis to marker-assisted plant and animal breeding. The usual practice in such work is to extract the DNA, prime the markers of interest, and sift them out by electrically driving them through an appropriate matrix, usually a gel. The gels, made from polyacrylamide or agarose, are of high cost, limiting their greater applications in molecular marker work, especially in developing countries where such technology has great potential. Trials using superfine resolution (SFR) agarose for SSR marker screening showed that it is capable of resolving SSR loci and can be reused up to 14 times, thus greatly reducing the cost of each gel run. Furthermore, for certain applications, low concentrations of agarose sufficed and switching to lithium borate buffer, instead of the conventional Tris-borate-ethylenediaminetetraacetic acid buffer, will further save time and cost. The 2.5% gel was prepared following the Agarose SFR(TM)manual by adding 2.5 g agarose powder into 100 mL 1X lithium borate buffer in a 250-mL flask with rapid stirring. Two midigels (105 x 83 mm, 17 wells) or 4 minigels (50 x 83 mm, 8 wells), 4 mm thickness can be prepared from 100 mL gel solution. A total of 1680 PCR products amplified using 140 SSR markers from oil palm DNA samples were tested in this study using SFR recycled gel. As average, the gel can be recycled 8 times with good resolution, but can be recycled up to 14 times before the resolutions get blurred.

Erratum to: Whole-genome strategies for marker-assisted plant breeding

Erratum to: Whole-genome strategies for marker-assisted plant breeding

Whole-genome strategies for marker-assisted plant breeding

Molecular breeding for complex traits in crop plants requires understanding and manipulation of many factors influencing plant growth, development and responses to an array of biotic and abiotic stresses. Molecular marker-assisted breeding procedures can be facilitated and revolutionized through whole-genome strategies, which utilize full genome sequencing and genome-wide molecular markers to effectively address various genomic and environmental factors through a representative or complete set of genetic resources and breeding materials. These strategies are now increasingly based on understanding of specific genomic regions, genes/alleles, haplotypes, linkage disequilibrium (LD) block(s), gene networks and their contribution to specific phenotypes. Large-scale and high-density genotyping and genome-wide selection are two important components of these strategies. As components of whole-genome strategies, molecular breeding platforms and methodologies should be backed up by high throughput and precision phenotyping and e-typing (environmental assay) with strong support systems such as breeding informatics and decision support tools. Some basic strategies are discussed in this article, including (1) seed DNA-based genotyping for simplifying marker-assisted selection (MAS), reducing breeding cost and increasing scale and efficiency, (2) selective genotyping and phenotyping, combined with pooled DNA analysis, for capturing the most important contributing factors, (3) flexible genotyping systems, such as genotyping by sequencing and arraying, refined for different selection methods including MAS, marker-assisted recurrent selection and genomic selection (GS), (4) marker-trait association analysis using joint linkage and LD mapping, and (5) sequence-based strategies for marker development, allele mining, gene discovery and molecular breeding.

Recombination frequency variation in maize as revealed by genomewide single‐nucleotide polymorphisms

With 2 tablesAbstractRecombination frequency greatly affects selection efficiency in plant breeding. A high‐density single‐nucleotide polymorphism (SNP) map integrated with physical map and other molecular maps is very useful for characterizing genetic recombination variation. In this study, recombination frequency in maize was investigated through SNP linkage maps constructed with three recombinant inbred line populations. The integrated map consisted of 1443 molecular markers, including 1155 SNPs, spanning 1346 cM. A 100‐fold difference in recombination frequency was observed between different chromosomal regions, ranging from an average of 0.09 cM/Mb for pericentromeric regions to 7.08 cM/Mb for telomeric regions. Recombination suppression in non‐centromeric regions identified nine recombination‐suppressed regions, one of which likely contained condensed heterochromatin (knobs). Recombination variation along chromosomes was highly predictable for pericentromeric and telomeric regions, but population‐specific with 4.5‐fold difference for the same marker interval across the three populations or specific chromosome regions because of the presence of knobs. As recombination variation can be identified and well characterized as shown in this study, the related information will facilitate future genetic studies, gene cloning and marker‐assisted plant breeding.

Improvement of High-throughput Genotype Analysis After Implementation of a Dual-curve Sybr Green I-based Quantification and Normalization Procedure

The ability to rapidly genotype a large number of individuals is the key to any successful marker-assisted plant breeding program. One of the primary bottlenecks in high-throughput screening is the preparation of DNA samples, particularly the quantification and normalization of samples for downstream processing. A rapid and simple Sybr Green I-based quantification procedure that can be performed in a 96-well format is outlined. In this procedure, a dual standard curve method is used to allow better resolution of dilute samples and to reduce fluorescence value variation between samplings. A method to quickly normalize samples, and the importance of normalization, is also explored. We demonstrate that successful fragment amplification of a Theobroma grandiflorum (Willd ex Spreng) Schum. population is increased from 70% to 98% when each DNA extract is quantified and normalized as opposed to quantifying only a subset and normalizing all the samples based on the average of that subset. Improved microsatellite amplification was also observed among individuals in the monocot genus Phaedranassa Herb. ssp. Additionally, when our normalization method is applied to a Persea americana Mill. population, 97% of the samples normalized to 4 ng·μL−1 amplify at least three of six microsatellite regions, whereas only 30% of the samples below 4 ng·μL−1 (i.e., samples that could not be normalized) amplify at least three regions. We describe an undemanding method to quantify and normalize a large number of samples, which can be done manually or can be automated.

Single nucleotide polymorphism genotyping and its application on mapping and marker-assisted plant breeding

The nucleotide diversity across a genome is the source of most phenotypic variation. Such DNA polymorphism is the basis for the development of molecular markers, an indispensable tool in genetic mapping studies. In general, the high resolution fine mapping of genes is often limited by lack of sufficient number of polymorphic molecular markers. This problem is compounded with traits controlled by multi-genes because in several such studies, QTL cannot be resolved to a workable resolution that could be feasible for predicting the candidate gene(s) associated with traits of interests. The availability of abundant, high-throughput sequence-based markers is the key for detailed genomewide trait analysis. Single-nucleotide polymorphisms (SNP) are the most common sequence variation and a significant amount of effort has been invested in re-sequencing alleles to discover SNPs. In fully sequenced small-genome model organisms, SNP discovery is relatively straight forward, although highthroughput SNP discovery in natural populations remains both expensive and time-consuming. Here five central biochemical reaction principles that underlie SNP-genotyping methods specifically for large panel sizes and an intermediate number of SNPs are reviewed.

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F2(I.4RxI.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.

Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes

Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.

Molecular Markers in Plant Breeding-II. Some Pre-requisites for Use

This paper describes some of the pre-requisites for applying molecular markers in plant breeding or crop improvement programmes. This includes, possible answers to some of the very pertinent questions regarding marker-assisted plant breeding. For example, I) how an effective marker system is to be selected, ii) how, when and where these expensive technologies can be used efficiently and iii) what particular problem would be solved just by using a particular marker system. The most commonly used marker systems including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), micro-satellite or simple sequence repeat (SSR) and random amplified polymorphic DNAs (RAPDs) have been described in detail. The advantages and disadvantages (if any) of these marker systems and their practical utilization in different areas of crop improvement programmes have been discussed. Different methods of integrating molecular markers in conventional plant breeding programmes have also been described in greater detail with special reference to varieta l identification and germplasm characterization, marker-assisted selection for qualitative and quantitative traits and abiotic stress tolerance. It is suggested that in all these cases, cost-effective application of markers can best be achieved through collaboration with those who excel in this technology. This collaboration is also necessary in order to integrate the systems and technologies to deliver the product in minimum possible time and with minimum extra input/investment in terms of operational expenses.

Molecular Markers in Plant Breeding-I: Concepts and Characterization

Plant breeding is a combination of principles and methods of changing the genetic constitution of a plant to make it more suitable for human needs. Conventional plant breeding has evolved with the passage of time from simple seed saving of the best harvest to the selection of seeds according to the laws of Mendel. With the advent of morphological and biochemical markers, the selection process has hastened and the scope of conventional plant breeding increased many folds during the recent years. However, the process that actually revolutionized the plant breeding in the 20 century was the realization that th there exist a widespread polymorphism in natural populations, the degree of which can be assessed by sequencing or making restriction maps: an application of new tools of molecular biology. The first and the foremost molecular markers system i.e., restriction fragment length polymorphism (RFLP) was developed in early 1980. These are co-dominant markers and are available in unlimited number. Another breakthrough was the emergence of polymerase chain reaction (PCR) in 1990. With this technology, a new generation of DNA markers such as randomly amplified polymorphic DNA (RAPDs), sequence characterized amplified regions (SCARs), sequence tagged sites (STS), single polymorphic amplification test (SPLAT), variable number of tendom repeats (VNTRs), amplified fragment length polymorphism (AFLP), DNA amplification fingerprinting (DFA), single strand conformational polymorphism (SSCP), single nucleotide polymorphism (SNPs), micro-satellites or short tandem repeats (STRs), cDNA, DNA micro arrays and rDNA-ITS were introduced into the modern plant breeding systems. The concept of DNA based markers has increased our ability many folds, to follow minute regions of chromosome through opportunities such as map based cloning and Marker-assisted Plant (MAP) Breeding. In MAP breeding, the new ideas and concepts have been introduced which need to be understood thoroughly, before applying these ideas in practical breeding programmes particular in country like Pakistan where application of molecular biological approaches are still in its infancy. In order to achieve this objective, efforts were made to write a series of review articles in which concept of MAP breeding is being described thoroughly yet in a simple way so that student and researchers can build their understanding of plant molecular breeding and application of DNA based markers for the genetic dissection of qualitative and quantitative traits. In the present paper, concepts of MAP breeding and the type and characteristics of DNA markers is presented so that choice of the marker(s) can be made rational and for the defined purposes.

PCR‐genotyping of barley seedlings using DNA samples from tissue prints

AbstractScreening of large numbers of plant samples with polymerase chain reaction (PCR)‐based techniques is a central task in marker‐assisted plant breeding and molecular genetics. While PCR and electrophoresis have been streamlined as a result of high‐throughput analysis methods, the collection of plant material and its processing to make it accessible for reliable amplification reactions remain labour‐intensive, costly and not amenable to automation. As an alternative to traditional extraction protocols, DNA can be bound to specially treated paper (FTA‐paper). For PCR analysis, instead of being added as a liquid suspension, DNA is then released from small paper disks that are immersed into the reaction mixture. In the present study, several parameters were investigated for the successful amplification of various single‐copy sequence characterized amplified region markers from barley tissue prints. The results show that the FTA‐paper approach offers a quick, reliable and low‐cost alternative for PCR screening even in a crop plant with a large genome.

Genetic and physical characterization of chromosome 4DL in wheat

The long arm of chromosome 4D in wheat (Triticum aestivum L.) has been shown in previous studies to harbor genes of agronomic importance. A major dominant gene conferring Aluminum (Al) tolerance (Alt2 in 'Chinese Spring' and AltBH in 'BH 1146'), and the Knal locus controlling the K+/Na+ discrimination in saline environments have been mapped to this chromosome arm. However, accurate information on the genetic and physical location of markers related to any of these genes is not available and would be useful for map-based cloning and marker-assisted plant breeding. In the present study, using a population of 91 recombinant inbred lines segregating for Al tolerance, we provide a more extensive genetic linkage map of the chromosome arm 4DL based on RFLP, SSR, and AFLP markers, delimiting the AltBH gene to a 5.9-cM interval between markers Xgdm125 and Xpsr914. In addition, utilizing a set of wheat deletion lines for chromosome arm 4DL, the AltBH gene was physically mapped to the distal region of the chromosome, between deletion breakpoints 0.70 and 0.86, where the kilobase/centimorgan ratio is assumed to be low, making the map-based cloning of the gene a more realistic goal. The polymorphism rates in chromosome arm 4DL for the different types of markers used were extremely low, as confirmed by the physical mapping of AFLPs. Finally, analysis of 1 Mb of contiguous sequence of Arabidopsis chromosome 5 flanking the gene homologous to the BCD1230 clone (a cosegregating marker in our population coding for a ribulose-5-phosphate-3-epimerase gene), revealed a previously identified region of stress-related and disease-resistance genes. This could explain the collinearity observed in comparative mapping studies among different species and the low level of polymorphism detected in the chromosome arm 4DL in hexaploid wheat.