Development of a core RFLP map in maize using an immortalized F2 population.

A map derived from restriction fragment length polymorphisms (RFLPs) in maize (Zea mays L.) is presented. The map was constructed in an immortalized Tx303 x CO159 F2 mapping population that allowed for an unlimited number of markers to be mapped and pooled F3 seed to be distributed to other laboratories. A total of 215 markers consisting of 159 genomic clones, 16 isozymes and 35 cloned genes of defined function have been placed on 10 chromosomes. An examination of segregation data has revealed several genomic regions with aberrant segregation ratios favoring either parent or the heterozygote. Mapping of cloned genes and isozymes that have been previously mapped by functional criteria has provided 29 points of alignment with the classical maize genetic map. Screening of all mapped RFLP probes against a collection of U.S. Corn Belt germplasm using EcoRI, HindIII and EcoRV has resulted in a set of 97 core markers being defined. The designation of a set of core markers allows the maize genome to be subdivided into a series of bins which serve as the backbone for maize genetic information and database boundaries. The merits and applications of core markers and bins are discussed.

Novel sources of resistance to Striga hermonthica in Tripsacum dactyloides, a wild relative of maize.

• The parasitic weed Striga hermonthica lowers cereal yield in small-holder farms in Africa. Complete resistance in maize to S. hermonthica infection has not been identified. A valuable source of resistance to S. hermonthica may lie in the genetic potential of wild germplasm. • The susceptibility of a wild relative of maize, Tripsacum dactyloides and a Zea mays-T. dactyloides hybrid to S. hermonthica infection was determined. Striga hermonthica development was arrested after attachment to T. dactyloides. Vascular continuity was established between parasite and host but there was poor primary haustorial tissue differentiation on T. dactyloides compared with Z. mays. Partial resistance was inherited in the hybrid. • Striga hermonthica attached to Z. mays was manipulated such that different secondary haustoria could attach to different hosts. Secondary haustoria formation was inhibited on T. dactyloides, moreover, subsequent haustoria formation on Z. mays was also impaired. • Results suggest that T. dactyloides produces a signal that inhibits haustorial development: this signal may be mobile within the parasite haustorial root system.

Molecular marker mapping of leaf rust resistance gene lr46 and its association with stripe rust resistance gene yr29 in wheat.

ABSTRACT Leaf and stripe rusts, caused by Puccinia triticina and P. striiformis, respectively, are globally important fungal diseases of wheat that cause significant annual yield losses. A gene that confers slow rusting resistance to leaf rust, designated as Lr46, has recently been located on wheat chromosome 1B. The objectives of our study were to establish the precise genomic location of gene Lr46 using molecular approaches and to determine if there was an association of this locus with adult plant resistance to stripe rust. A population of 146 F(5) and F(6) lines produced from the cross of susceptible 'Avocet S' with resistant 'Pavon 76' was developed and classified for leaf rust and stripe rust severity for three seasons. Using patterns of segregation for the two diseases, we estimated that at least two genes with additive effects conferred resistance to leaf rust and three to four genes conferred resistance to stripe rust. Bulked segregant analysis and linkage mapping using amplified fragment length polymorphisms with the 'Avocet' x 'Pavon 76' population, F(3) progeny lines of a single chromosome recombinant line population from the cross 'Lalbahadur' x 'Lalbahadur (Pavon 1B)', and the International Triticeae Mapping Initiative population established the genomic location of Lr46 at the distal end of the long arm of wheat chromosome 1B. A gene that is closely linked to Lr46 and confers moderate levels of adult plant resistance to stripe rust is identified and designated as Yr29.

Quantitative trait locus analysis and construction of consensus genetic map for drought tolerance traits based on three recombinant inbred line populations in cultivated groundnut (Arachis hypogaea L.).

Groundnut (Arachis hypogaea L.) is an important food and cash crop grown mainly in semi-arid tropics (SAT) regions of the world where drought is the major constraint on productivity. With the aim of understanding the genetic basis and identification of quantitative trait loci (QTL) for drought tolerance, two new recombinant inbred line (RIL) mapping populations, namely ICGS 76 × CSMG 84-1 (RIL-2) and ICGS 44 × ICGS 76 (RIL-3), were used. After screening of 3,215 simple sequence repeat (SSR) markers on the parental genotypes of these populations, two new genetic maps were developed with 119 (RIL-2) and 82 (RIL-3) SSR loci. Together with these maps and the reference map with 191 SSR loci based on TAG 24 × ICGV 86031 (RIL-1), a consensus map was constructed with 293 SSR loci distributed over 20 linkage groups, spanning 2,840.8 cM. As all these three populations segregate for drought-tolerance-related traits, a comprehensive QTL analysis identified 153 main effect QTL (M-QTL) and 25 epistatic QTL (E-QTL) for drought-tolerance-related traits. Localization of these QTL on the consensus map provided 16 genomic regions that contained 125 QTL. A few key genomic regions were selected on the basis of the QTL identified in each region, and their expected role in drought adaptation is also discussed. Given that no major QTL for drought adaptation were identified, novel breeding approaches such as marker-assisted recurrent selection (MARS) and genomic selection (GS) approaches are likely to be the preferred approaches for introgression of a larger number of QTL in order to breed drought-tolerant groundnut genotypes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9660-0) contains supplementary material, which is available to authorized users.

The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.).

Molecular markers and genetic linkage maps are pre-requisites for molecular breeding in any crop species. In case of peanut or groundnut (Arachis hypogaea L.), an amphidiploid (4X) species, not a single genetic map is, however, available based on a mapping population derived from cultivated genotypes. In order to develop a genetic linkage map for tetraploid cultivated groundnut, a total of 1,145 microsatellite or simple sequence repeat (SSR) markers available in public domain as well as unpublished markers from several sources were screened on two genotypes, TAG 24 and ICGV 86031 that are parents of a recombinant inbred line mapping population. As a result, 144 (12.6%) polymorphic markers were identified and these amplified a total of 150 loci. A total of 135 SSR loci could be mapped into 22 linkage groups (LGs). While six LGs had only two SSR loci, the other LGs contained 3 (LG_AhXV) to 15 (LG_AhVIII) loci. As the mapping population used for developing the genetic map segregates for drought tolerance traits, phenotyping data obtained for transpiration, transpiration efficiency, specific leaf area and SPAD chlorophyll meter reading (SCMR) for 2 years were analyzed together with genotyping data. Although, 2-5 QTLs for each trait mentioned above were identified, the phenotypic variation explained by these QTLs was in the range of 3.5-14.1%. In addition, alignment of two linkage groups (LGs) (LG_AhIII and LG_AhVI) of the developed genetic map was shown with available genetic maps of AA diploid genome of groundnut and Lotus and Medicago. The present study reports the construction of the first genetic map for cultivated groundnut and demonstrates its utility for molecular mapping of QTLs controlling drought tolerance related traits as well as establishing relationships with diploid AA genome of groundnut and model legume genome species. Therefore, the map should be useful for the community for a variety of applications.

cisprimertool: software to implement a comparative genomics strategy for the development of conserved intron scanning (CIS) markers.

The availability of complete, annotated genomic sequence information in model organisms is a rich resource that can be extended to understudied orphan crops through comparative genomic approaches. We report here a software tool (cisprimertool) for the identification of conserved intron scanning regions using expressed sequence tag alignments to a completely sequenced model crop genome. The method used is based on earlier studies reporting the assessment of conserved intron scanning primers (called CISP) within relatively conserved exons located near exon-intron boundaries from onion, banana, sorghum and pearl millet alignments with rice. The tool is freely available to academic users at http://www.icrisat.org/gt-bt/CISPTool.htm.

WebStruct and VisualStruct: Web interfaces and visualization for Structure software implemented in a cluster environment.

Structure, is a widely used software tool to investigate population genetic structure with multi-locus genotyping data. The software uses an iterative algorithm to group individuals into "K" clusters, representing possibly K genetically distinct subpopulations. The serial implementation of this programme is processor-intensive even with small datasets. We describe an implementation of the program within a parallel framework. Speedup was achieved by running different replicates and values of K on each node of the cluster. A web-based user-oriented GUI has been implemented in PHP, through which the user can specify input parameters for the programme. The number of processors to be used can be specified in the background command. A web-based visualization tool "Visualstruct", written in PHP (HTML and Java script embedded), allows for the graphical display of population clusters output from Structure, where each individual may be visualized as a line segment with K colors defining its possible genomic composition with respect to the K genetic sub-populations. The advantage over available programs is in the increased number of individuals that can be visualized. The analyses of real datasets indicate a speedup of up to four, when comparing the speed of execution on clusters of eight processors with the speed of execution on one desktop. The software package is freely available to interested users upon request.

Grouping of accessions of Mexican races of maize revisited with SSR markers.

Mexican races of maize (Zea mays L.) represent a valuable genetic resource for breeding and genetic surveys. We applied simple sequence repeat (SSR) markers to characterize 25 accessions of races of maize from Mexico. Our objectives were to (1) study the molecular genetic diversity within and among these accessions and (2) examine their relationships as assumed previously on the basis of morphological data. A total of 497 individuals were fingerprinted with 25 SSR markers. We observed a high total number of alleles (7.84 alleles per locus) and total gene diversity (0.61), confirming the broad genetic base of the maize races from Mexico. In addition, the accessions were grouped into distinct racial complexes on the basis of a model-based clustering approach. The principal coordinate analyses of the four Modern Incipient hybrids corroborated the proposed parental races of Chalqueño, Cónico Norteño, Celaya, and Bolita on the basis of the morphological data. Consequently, for some of the accessions, hybridizations provide a clue that can further be used to explain the associations among the Mexican races of maize.

Wheat genetic diversity trends during domestication and breeding.

It has been claimed that plant breeding reduces genetic diversity in elite germplasm which could seriously jeopardize the continued ability to improve crops. The main objective of this study was to examine the loss of genetic diversity in spring bread wheat during (1) its domestication, (2) the change from traditional landrace cultivars (LCs) to modern breeding varieties, and (3) 50 years of international breeding. We studied 253 CIMMYT or CIMMYT-related modern wheat cultivars, LCs, and Triticum tauschii accessions, the D-genome donor of wheat, with 90 simple sequence repeat (SSR) markers dispersed across the wheat genome. A loss of genetic diversity was observed from T. tauschii to the LCs, and from the LCs to the elite breeding germplasm. Wheat's genetic diversity was narrowed from 1950 to 1989, but was enhanced from 1990 to 1997. Our results indicate that breeders averted the narrowing of the wheat germplasm base and subsequently increased the genetic diversity through the introgression of novel materials. The LCs and T. tauschii contain numerous unique alleles that were absent in modern spring bread wheat cultivars. Consequently, both the LCs and T. tauschii represent useful sources for broadening the genetic base of elite wheat breeding germplasm.

Molecular markers for plant breeding: comparisons of RFLP and RAPD genotyping costs.

Three molecular marker protocols, chemiluminescent restriction fragment length polymorphisms (c-RFLPs), radioactivity-based restriction fragment length polymorphisms (r-RFLPs), and randomly amplified DNA polymorphisms (RAPDs) were compared in terms of cost and time efficiency. Estimates of cost of supplies and time requirements were obtained from simulations of maize (Zea mays L.) genotyping experiments utilizing protocols currently in use. The increase in total cost with increasing numbers of individuals genotyped and markers analyzed is higher for RAPDs than for RFLPs. RAPDs were generally found to be more cost and time efficient for studies involving small sample sizes, while RFLPs have the advantage for larger sample sizes. Because of the shorter exposure times involved, c-RFLPs require less time than r-RFLPs to obtain a given amount of information. Variations in the protocols, such as number of re-uses of Southern blots or cost of Taq DNA polymerase per reaction of amplification, also affect the relative merits of RAPDs and RFLPs. Two examples were analyzed where molecular markers are used: a germ plasm survey and quantitative trait loci (QTL) mapping in a segregating population. No protocol was found to be the most cost and time efficient over the entire range of sample sizes and number of marker loci studied.

Use of AA and NA in the treatment of chemical dependencies of traumatic brain injury survivors.

Alcoholics Anonymous and Narcotics Anonymous have been under-utilized in the treatment of chemical dependency in traumatic brain injury survivors. Both offer a social support network and a self-help recovery programme. The fellowships can be viewed as three progressive levels focusing on behavioural, cognitive and gestalt issues. Finally observations and suggestions are offered which may help in incorporating this therapeutic modality which is the most widespread treatment of individuals with chemical dependencies.

Plant regeneration from immature embryos of 48 elite CIMMYT bread wheats.

Forty-eight bread wheat (Triticum aestivum L.) released cultivars and elite advanced lines were evaluated for their ability to produce embryogenic callus using three different media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,4-D (E3) or MS medium containing 2,4-D plus different amino acids (E5) were used for callus initiation and maintenance. Plant regeneration was achieved on basal MS medium with indole-3-acetic acid (IAA) and 6-benzylamino purine (BAP) and rooting on MS with 1-naphthaleneacetic acid (NAA). Percentage regeneration varied widely with both genotype and initiation medium, with values ranging from 2% to 94%. The number of plantlets produced per embryo ranged from 6 to 42. Thirteen genotypes showed at least 50% regeneration after culture on E5 medium; 3 genotypes after culture on E3 initiation medium and 1 after initiation on E1. After four subcultures, over a 16-week period, 41 genotypes (85%) lost their ability to regenerate plants while the remaining 7 lines (15%) retained plant regeneration potential but at reduced levels. E3 medium was found to be the best for maintaining regeneration potential after four subcultures.

Detection of quantitative trait loci associated with leaf rust resistance in bread wheat.

Leaf rust, caused by Puccinia recondita Rob. ex Desm., is a common disease in wheat. The objective of this study was to develop molecular markers associated with the quantitative trait loci (QTLs) putatively conferring durable leaf rust resistance in Triticum aestivum L. em. Thell. A population of 77 recombinant inbred lines (RILs) developed from 'Parula' (resistant) and 'Siete Cerros' (moderately susceptible) was used. Bulked segregant analysis was done using random amplified polymorphic DNAs (RAPDs) with DNA enriched for low-copy sequences using hydroxyapatite chromatography. Out of 400 decamer primers screened, 3 RAPD markers were identified between the bulk of the most resistant and the bulk of the most susceptible lines. These were cloned and used as probes on the RILs in Southern hybridizations. Two probes revealed two tightly linked loci. One-way analysis of variance showed that these two loci, and another revealed by the third probe, were linked to QTLs controlling leaf rust resistance based on data taken from 2 years of replicated field trials. Cytogenetic analysis placed the two tightly linked loci on the long arm of chromosome 7B. The third probe detected loci located on the short arms of chromosomes 1B and 1D. It is suggested that the QTL detected on 7BL may well be homoeoallelic to Lr34.

Identification of quantitative trait loci under drought conditions in tropical maize. 1. Flowering parameters and the anthesis-silking interval.

Drought is an important climatic phenomenon which, after soil infertility, ranks as the second most severe limitation to maize production in developing countries. When drought stress occurs just before or during the flowering period, a delay in silking is observed, resulting in an increase in the length of the anthesis-silking interval (ASI) and in a decrease in grain yield. Selection for reduced ASI in tropical open-pollinated varieties has been shown to be correlated with improved yields under drought stress. Since efficient selection for drought tolerance requires carefully managed experimental conditions, molecular markers were used to identify the genomic segments responsible for the expression of ASI, with the final aim of developing marker-assisted selection (MAS) strategies. An F2population of 234 individuals was genotyped at 142 loci and F3 families were evaluated in the field under several water regimes for male flowering (MFLW), male sterility (STER), female flowering (FFLW) and ASI. The genetic variance of ASI increased as a function of the stress intensity, and the broad-sense heritabilites of MFLW, FFLW and ASI were high under stress conditions, being 86%, 82% and 78%, respectively. Putative quantitative trait loci (QTLs) involved in the expression of MFLW and/or FFLW under drought were detected on chromosomes 1, 2, 4, 5, 8, 9 and 10, accounting for around 48% of the phenotypic variance for both traits. For ASI, six putative QTLs were identified under drought on chromosomes 1, 2, 5, 6, 8 and 10, and together accounted for approximately 47% of the phenotypic variance. Under water stress conditions, four QTLs were common for the expression of MFLW and FFLW, one for the expression of ASI and MFLW, and four for the expression of ASI and FFLW. The number of common QTLs for two traits was related to the level of linear correlation between these two traits. Segregation for ASI was found to be transgressive with the drought-susceptible parent contributing alleles for reduced ASI (4 days) at two QTL positions. Alleles contributed by the resistant line at the other four QTLs were responsible for a 7-day reduction of ASI. These four QTLs represented around 9% of the linkage map, and were stable over years and stress levels. It is argued that MAS based on ASI QTLs should be a powerful tool for improving drought tolerance of tropical maize inbred lines.

Comparative map and trait viewer (CMTV): an integrated bioinformatic tool to construct consensus maps and compare QTL and functional genomics data across genomes and experiments.

In the past few decades, a wealth of genomic data has been produced in a wide variety of species using a diverse array of functional and molecular marker approaches. In order to unlock the full potential of the information contained in these independent experiments, researchers need efficient and intuitive means to identify common genomic regions and genes involved in the expression of target phenotypic traits across diverse conditions. To address this need, we have developed a Comparative Map and Trait Viewer (CMTV) tool that can be used to construct dynamic aggregations of a variety of types of genomic datasets. By algorithmically determining correspondences between sets of objects on multiple genomic maps, the CMTV can display syntenic regions across taxa, combine maps from separate experiments into a consensus map, or project data from different maps into a common coordinate framework using dynamic coordinate translations between source and target maps. We present a case study that illustrates the utility of the tool for managing large and varied datasets by integrating data collected by CIMMYT in maize drought tolerance research with data from public sources. This example will focus on one of the visualization features for Quantitative Trait Locus (QTL) data, using likelihood ratio (LR) files produced by generic QTL analysis software and displaying the data in a unique visual manner across different combinations of traits, environments and crosses. Once a genomic region of interest has been identified, the CMTV can search and display additional QTLs meeting a particular threshold for that region, or other functional data such as sets of differentially expressed genes located in the region; it thus provides an easily used means for organizing and manipulating data sets that have been dynamically integrated under the focus of the researcher's specific hypothesis.

Plant genetic resources: what can they contribute toward increased crop productivity?

To feed a world population growing by up to 160 people per minute, with >90% of them in developing countries, will require an astonishing increase in food production. Forecasts call for wheat to become the most important cereal in the world, with maize close behind; together, these crops will account for approximately 80% of developing countries' cereal import requirements. Access to a range of genetic diversity is critical to the success of breeding programs. The global effort to assemble, document, and utilize these resources is enormous, and the genetic diversity in the collections is critical to the world's fight against hunger. The introgression of genes that reduced plant height and increased disease and viral resistance in wheat provided the foundation for the "Green Revolution" and demonstrated the tremendous impact that genetic resources can have on production. Wheat hybrids and synthetics may provide the yield increases needed in the future. A wild relative of maize, Tripsacum, represents an untapped genetic resource for abiotic and biotic stress resistance and for apomixis, a trait that could provide developing world farmers access to hybrid technology. Ownership of genetic resources and genes must be resolved to ensure global access to these critical resources. The application of molecular and genetic engineering technologies enhances the use of genetic resources. The effective and complementary use of all of our technological tools and resources will be required for meeting the challenge posed by the world's expanding demand for food.

Interpreting genotype†×†environment interaction in tropical maize using linked molecular markers and environmental covariables.

An understanding of the genetic and environmental basis of genotype×environment interaction (GEI) is of fundamental importance in plant breeding. In mapping quantitative trait loci (QTLs), suitable genetic populations are grown in different environments causing QTLs×environment interaction (QEI). The main objective of the present study is to show how Partial Least Squares (PLS) regression and Factorial Regression (FR) models using genetic markers and environmental covariables can be used for studying QEI related to GEI. Biomass data were analyzed from a multi-environment trial consisting of 161 lines from a F(3:4) maize segregating population originally created with the purpose of mapping QTLs loci and investigating adaptation differences between highland and lowland tropical maize. PLS and FR methods detected 30 genetic markers (out of 86) that explained a sizeable proportion of the interaction of maize lines over four contrasting environments involving two low-altitude sites, one intermediate-altitude site, and one high-altitude site for biomass production. Based on a previous study, most of the 30 markers were associated with QTLs for biomass and exhibited significant QEI. It was found that marker loci in lines with positive GEI for the highland environments contained more highland alleles, whereas marker loci in lines with positive GEI for intermediate and lowland environments contained more lowland alleles. In addition, PLS and FR models identified maximum temperature as the most-important environmental covariable for GEI. Using a stepwise variable selection procedure, a FR model was constructed for GEI and QEI that exclusively included cross products between genetic markers and environmental covariables. Higher maximum temperature in low- and intermediate-altitude sites affected the expression of some QTLs, while minimum temperature affected the expression of other QTLs.

Molecular characterization of Asian maize inbred lines by multiple laboratories.

This study focuses on the standardization of techniques across laboratories to enable multiple datasets to be compared and combined in order to obtain reliable and robust wide-scale patterns of diversity. A set of protocols using a core collection of simple sequence repeat (SSR) markers, reference lines and standard alleles, plus a common system of allele nomenclature, was adopted in the study of maize genetic diversity in a network of laboratories in Asia. Pair-wise allele comparisons of the reference lines, done to assess the general agreement between datasets from four laboratories, showed error rates (raw) ranging from 5.8% to 9.7%, which were reduced to less than 8% after adjustments of correctable errors, and further reduced to less than 6% after the exclusion of all markers with greater than 10% individual error rates. Overall, 45% of the total mismatches were due to frameshift errors, 39% to wrong allele size, 15% to failed amplification and 1% to "extra" alleles. Higher genetic similarity values of the reference lines were achieved using fewer markers with data of higher quality rather than with more markers of questionable quality. Cluster analysis of the merged datasets showed the lines from southern China to be highly diverse, falling into six of the seven clusters observed and all well represented by tester lines. The lines from Indonesia fell into five of six groups, with two main groups represented by tester lines. The CIMMYT lines developed for the Asian region showed a relatively narrow genetic base, falling in two out of seven and in three out of six clusters in China and Indonesia, respectively. In contrast to the case in southern China where 95% of the lines clustered separately from the CIMMYT lines, lines in the Indonesian breeding program show a closer relationship with the CIMMYT lines, reflecting a long history of germplasm exchange.

Use of SSRs for establishing heterotic groups in subtropical maize.

Heterotic groups and patterns are of fundamental importance in hybrid breeding. The objectives of our research were to: (1) investigate the relationship of simple sequence repeats (SSR) based genetic distances between populations and panmictic midparent heterosis (PMPH) in a broad range of CIMMYT maize germplasm, (2) evaluate the usefulness of SSR markers for defining heterotic groups and patterns in subtropical germplasm, and (3) examine applications of SSR markers for broadening heterotic groups by systematic introgression of other germplasm. Published data of two diallels and one factorial evaluated for grain yield were re-analyzed to calculate the PMPH in population hybrids. Additionally, 20 pools and populations widely used in CIMMYT's breeding program were assayed with 83 SSR markers covering the entire maize genome. Correlations of squared modified Roger's distance (MRD(2)) and PMPH were mostly positive and significant, but adaption problems caused deviations in some cases. For intermediate- and early-maturity subtropical germplasm, two heterotic groups could be suggested consisting of a flint and dent composite. We concluded that the relationships between the populations obtained by SSR analyses are in excellent agreement with pedigree information. SSR markers are a valuable complementation to field trials for identifying heterotic groups and can be used to introgress exotic germplasm systematically.

QTL for insect resistance and drought tolerance in tropical maize: prospects for marker assisted selection.

Insects and drought cause severe losses in the production of maize in many developing countries. Conventional breeding efforts to enhance the level of resistance to a number of insect pests and tolerance to drought have been successful, although only through large efforts of many breeders and over a large period of time. Continued improvements will only be possible through substantial investment of resources. Recently, success in identifying quantitative trait loci (QTL) in several plant species using various molecular marker systems offers alternative methods for accelerating conventional breeding programs. As the first step towards using molecular markers in CIMMYT's maize breeding program, restriction fragment length polymorphisms (RFLPs) have been used to understand the genetic basis of resistance to two corn borer species, southwestern corn borer and sugarcane borer, and to one major component of drought tolerance, anthesis-silking interval. A number of QTL with effects large enough to be regarded as significant in breeding were detected for each of these traits and many of them presented stable effects over environments. While variability in the number and location of QTL has been found when compared across populations, several loci were found to be quite consistent. Simple calculations can be made which estimate that the total genetic potential in maize for these traits is high. It is argued that to ultimately access and manipulate this potential, the use of linked molecular markers as indirect selectable markers is both feasible and necessary.