Doubled haploidy
Encyclopedia
A doubled haploid is a genotype formed when haploid cells undergo chromosome doubling. Artificial production of doubled haploids is important in plant breeding
.
Haploid cells are produced from pollen
or egg
cells or from other cells of the gametophyte
, then by induced or spontaneous chromosome doubling, a doubled haploid cell is produced, which can be grown into a doubled haploid plant. If the original plant was diploid, the haploid cells are monoploid
, and the term doubled monoploid may be used for the doubled haploids. Haploid organisms derived from tetraploids are sometimes called dihaploids (and the doubled dihaploids are tetraploid).
Conventional inbreeding
procedures take six generations to achieve approximately complete homozygosity, whereas doubled haploidy achieves it in one generation. Dihaploid plants derived from tetraploid crop plants may be important for breeding programs that involve diploid wild relatives of the crops.
(Kasha and Kao, 1970) and tobacco
(Burk et al., 1979). Tobacco, rapeseed
, and barley are the most responsive species for doubled haploid production. Doubled haploid methodologies have now been applied to over 250 species.
or in vitro
. Haploid embryos are produced in vivo by parthenogenesis
, pseudogamy
, or chromosome elimination after wide crossing. The haploid embryo is rescued, cultured, and chromosome-doubling produces doubled haploids. The in vitro methods include gynogenesis (ovary
and flower culture) and androgenesis (anther and microspore culture).Androgenesis is preferred method. Another method of producing the haploids is wide crossing. In barley, haploids can be produced by wide crossing with the related species Hordeum
bulbosum, fertilization is effected, but during the early stages of seed development the H. bulbosum chromosomes are eliminated leaving a haploid embryo. In tobacco (Nicotiana tabacum
), wide crossing with Nicotiana africana
is widely used. When N. africana is used to pollinate N. tabacum, 0.25 to 1.42 percent of the progeny
survive and can readily be identified as either F1 hybrid
s or maternal haploids. Although, these percentages appear small, the vast yield of tiny seeds and the early death of most seedlings provide significant numbers of viable hybrids and haploids in relatively small soil containers. This method of interspecific pollination serves as a practical way of producing seed-derived haploids of N. tabacum, either as an alternative method or complementary method to anther culture.
Studies were conducted comparing DH method and other conventional breeding methods and it was concluded that adoption of doubled haploidy does not lead to any bias of genotypes in populations, and random DHs were even found to be compatible to selected line produced by conventional pedigree method.
or related species in to a recipient elite line through repeated backcrossing. A problem in this procedure is being able to identify the lines carrying the trait of interest at each generation. The problem is particularly acute if the trait of interest is recessive, as it will be present only in a heterozygous condition after each backcross. The development of molecular markers provides an easier method of selection based on the genotype (marker) rather than the phenotype. Combined with doubled haploidy it becomes more effective. In marker assisted backcross conversion, a recipient parent is crossed with a donor line and the hybrid (F1) backcrossed to the recipient. The resulting generation (BC1) is backcrossed and the process repeated until the desired genotypes are produced. The combination of doubled haploidy and molecular marker provides the short cut. In the back cross generation one itself a genotype with the character of interest can be selected and converted into homozygous doubled haploid genotype. Chen et al. (1994) used marker assisted backcross conversion with doubled haploidy of BC1 individuals to select stripe rust resistant lines in barley.
These two disadvantages can be over come by DHs, and more elite crosses can be evaluated and selected within less time.
The relevance of DHs to plant breeding has increased markedly in recent years owing to the development of protocols for 25 species. Doubled haploidy already plays an important role in hybrid cultivar production of vegetables, and the potential for ornamental production is being vigorously examined. DHs are also being developed in the medicinal herb Valeriana officinalis to select lines with high pharmacological activity. Another interesting development is that fertile homozygous DH lines can be produced in species that have self-incompatibility systems.
In haploids produced from anther culture, it is observed that some plants are aneuploids and some are mixed haploid-diploid types. Another disadvantage associated with the double haploidy is the cost involved in establishing tissue culture and growth facilities. The over-usage of doubled haploidy may reduce genetic variation in breeding germplasm. Hence one has to take several factors into consideration before deploying doubled haploidy in breeding programmes.
Plant breeding
Plant breeding is the art and science of changing the genetics of plants in order to produce desired characteristics. Plant breeding can be accomplished through many different techniques ranging from simply selecting plants with desirable characteristics for propagation, to more complex molecular...
.
Haploid cells are produced from pollen
Pollen
Pollen is a fine to coarse powder containing the microgametophytes of seed plants, which produce the male gametes . Pollen grains have a hard coat that protects the sperm cells during the process of their movement from the stamens to the pistil of flowering plants or from the male cone to the...
or egg
Egg (biology)
An egg is an organic vessel in which an embryo first begins to develop. In most birds, reptiles, insects, molluscs, fish, and monotremes, an egg is the zygote, resulting from fertilization of the ovum, which is expelled from the body and permitted to develop outside the body until the developing...
cells or from other cells of the gametophyte
Gametophyte
A gametophyte is the haploid, multicellular phase of plants and algae that undergo alternation of generations, with each of its cells containing only a single set of chromosomes....
, then by induced or spontaneous chromosome doubling, a doubled haploid cell is produced, which can be grown into a doubled haploid plant. If the original plant was diploid, the haploid cells are monoploid
Ploidy
Ploidy is the number of sets of chromosomes in a biological cell.Human sex cells have one complete set of chromosomes from the male or female parent. Sex cells, also called gametes, combine to produce somatic cells. Somatic cells, therefore, have twice as many chromosomes. The haploid number is...
, and the term doubled monoploid may be used for the doubled haploids. Haploid organisms derived from tetraploids are sometimes called dihaploids (and the doubled dihaploids are tetraploid).
Conventional inbreeding
Inbreeding
Inbreeding is the reproduction from the mating of two genetically related parents. Inbreeding results in increased homozygosity, which can increase the chances of offspring being affected by recessive or deleterious traits. This generally leads to a decreased fitness of a population, which is...
procedures take six generations to achieve approximately complete homozygosity, whereas doubled haploidy achieves it in one generation. Dihaploid plants derived from tetraploid crop plants may be important for breeding programs that involve diploid wild relatives of the crops.
History
The first report of the haploid plant was published by Blakeslee et al. (1922) in Datura stramonium. Subsequently, haploids were reported in many other species. Guha and Maheshwari (1964) developed an anther culture technique for the production of haploids in the laboratory. Haploid production by wide crossing was reported in barleyBarley
Barley is a major cereal grain, a member of the grass family. It serves as a major animal fodder, as a base malt for beer and certain distilled beverages, and as a component of various health foods...
(Kasha and Kao, 1970) and tobacco
Tobacco
Tobacco is an agricultural product processed from the leaves of plants in the genus Nicotiana. It can be consumed, used as a pesticide and, in the form of nicotine tartrate, used in some medicines...
(Burk et al., 1979). Tobacco, rapeseed
Rapeseed
Rapeseed , also known as rape, oilseed rape, rapa, rappi, rapaseed is a bright yellow flowering member of the family Brassicaceae...
, and barley are the most responsive species for doubled haploid production. Doubled haploid methodologies have now been applied to over 250 species.
Production of Doubled Haploids
Doubled haploids can be produced in vivoIn vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
or in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
. Haploid embryos are produced in vivo by parthenogenesis
Parthenogenesis
Parthenogenesis is a form of asexual reproduction found in females, where growth and development of embryos occur without fertilization by a male...
, pseudogamy
Pseudogamy
Pseudogamy refers to aspects of reproduction. It has come to have different meanings in zoology and in botany.- In zoology :In zoology it is used for a type of parthenogenesis in which the sperm stimulates the egg cell to develop into an embryo, but no male inheritance is transmitted...
, or chromosome elimination after wide crossing. The haploid embryo is rescued, cultured, and chromosome-doubling produces doubled haploids. The in vitro methods include gynogenesis (ovary
Ovary
The ovary is an ovum-producing reproductive organ, often found in pairs as part of the vertebrate female reproductive system. Ovaries in anatomically female individuals are analogous to testes in anatomically male individuals, in that they are both gonads and endocrine glands.-Human anatomy:Ovaries...
and flower culture) and androgenesis (anther and microspore culture).Androgenesis is preferred method. Another method of producing the haploids is wide crossing. In barley, haploids can be produced by wide crossing with the related species Hordeum
Hordeum
Hordeum is a genus of about 30 species of annual and perennial grasses, native throughout the temperate Northern Hemisphere, temperate South America, and also South Africa....
bulbosum, fertilization is effected, but during the early stages of seed development the H. bulbosum chromosomes are eliminated leaving a haploid embryo. In tobacco (Nicotiana tabacum
Nicotiana tabacum
Nicotiana tabacum, or cultivated tobacco, is a perennial herbaceous plant. It is found only in cultivation, where it is the most commonly grown of all plants in the Nicotiana genus, and its leaves are commercially grown in many countries to be processed into tobacco. It grows to heights between 1...
), wide crossing with Nicotiana africana
Nicotiana africana
Nicotiana africana is a species of plant in the Solanaceae family. It is endemic to Namibia. Its natural habitats are subtropical or tropical dry shrubland and rocky areas.-References:* Craven, P. 2004. . Downloaded on 22 August 2007....
is widely used. When N. africana is used to pollinate N. tabacum, 0.25 to 1.42 percent of the progeny
Progeny
Progeny can refer to:*A genetic descendant or offspring*An academic progeny Other uses*Progeny Linux Systems*Progeny - an episode of the television series Stargate Atlantis...
survive and can readily be identified as either F1 hybrid
F1 hybrid
F1 hybrid is a term used in genetics and selective breeding. F1 stands for Filial 1, the first filial generation seeds/plants or animal offspring resulting from a cross mating of distinctly different parental types....
s or maternal haploids. Although, these percentages appear small, the vast yield of tiny seeds and the early death of most seedlings provide significant numbers of viable hybrids and haploids in relatively small soil containers. This method of interspecific pollination serves as a practical way of producing seed-derived haploids of N. tabacum, either as an alternative method or complementary method to anther culture.
Genetics of DH population
In DH method only two types of genotypes occur for a pair of alleles, A and a, with the frequency of ½ AA and ½ aa, while in diploid method three genotypes occur with the frequency of ¼ AA, ½ Aa, ¼ aa. Thus , if AA is desirable genotype, the probability of obtaining this genotype is higher in haploid method than in diploid method. If n loci are segregating, the probability of getting the desirable genotype is (1/2)n by the haploid method and (1/4)n by the diploid method. Hence the efficiency of haploid method is obviously high when the number of genes concerned is large.Studies were conducted comparing DH method and other conventional breeding methods and it was concluded that adoption of doubled haploidy does not lead to any bias of genotypes in populations, and random DHs were even found to be compatible to selected line produced by conventional pedigree method.
Mapping Quantitative Trait Loci
Most of the economic traits are controlled by genes with small but cumulative effects. Although the potential of DH populations in quantitative genetics has been understood for some time, it was the advent of molecular marker maps that provided the impetus for their use in identifying loci controlling quantitative traits. As the quantitative trait loci (QTL) effects are small and highly influenced by environmental factors, accurate phenotyping with replicated trials is needed. This is possible with doubled haploidy organisms because of their true breeding nature and because they can conveniently be produced in large numbers. Using DH populations, 130 quantitative traits have been mapped in nine crop species. In total, 56 DH populations were used for QTL detection.Backcross breeding
In backcross conversion, genes are introgressed from a donor cultivarCultivar
A cultivar'Cultivar has two meanings as explained under Formal definition. When used in reference to a taxon, the word does not apply to an individual plant but to all those plants sharing the unique characteristics that define the cultivar. is a plant or group of plants selected for desirable...
or related species in to a recipient elite line through repeated backcrossing. A problem in this procedure is being able to identify the lines carrying the trait of interest at each generation. The problem is particularly acute if the trait of interest is recessive, as it will be present only in a heterozygous condition after each backcross. The development of molecular markers provides an easier method of selection based on the genotype (marker) rather than the phenotype. Combined with doubled haploidy it becomes more effective. In marker assisted backcross conversion, a recipient parent is crossed with a donor line and the hybrid (F1) backcrossed to the recipient. The resulting generation (BC1) is backcrossed and the process repeated until the desired genotypes are produced. The combination of doubled haploidy and molecular marker provides the short cut. In the back cross generation one itself a genotype with the character of interest can be selected and converted into homozygous doubled haploid genotype. Chen et al. (1994) used marker assisted backcross conversion with doubled haploidy of BC1 individuals to select stripe rust resistant lines in barley.
Bulked segregant analysis (BSA)
In bulked segregant analysis, a population is screened for a character of interest and the genotypes at the two extreme ends form two bulks. Then the two bulks were tested for the presence or absence of molecular markers. Since the bulks are supposed to contrast for alleles contributing positive and negative effects, any marker polymorphism between the two bulks indicates the linkage between the marker and character of interest. BSA is dependent on accurate phenotyping and the DH population has particular advantage in that they are true breeding and can be tested repeatedly. DH populations are commonly used in bulked segregant analysis, which is a popular method in marker assisted breeding. This method has been applied mostly to rapeseed and barley.Genetic maps
Genetic maps are very important to understand the structure and organization of genomes from which evolution patterns and syntenic relationships between species can be deduced. Genetic maps also provide a framework for the mapping of genes of interest and estimating the magnitude of their effects and aid our understanding of genotype/phenotype associations. DH populations have become standard resources in genetic mapping for species in which DHs are readily available. Doubled haploid populations are ideal for genetic mapping. It is possible to produce a genetic map within two years of the initial cross regardless of the species. Map construction is relatively easy using a DH population derived from a hybrid of two homozygous parents as the expected segregation ratio is simple, i.e. 1:1. DH populations have now been used to produce genetic maps of barley, rapeseed, rice, wheat, and pepper. DH populations played a major role in facilitating the generation of the molecular marker maps in eight crop species.Genetic studies
Genetic ratios and mutation rates can be read directly from haploid populations. A small doubled haploid (DH) population was used to demonstrate that a dwarfing gene in barley is located chromosome 5H. In another study the segregation of a range of markers has been analyzed in barley.Genomics
Although QTL analysis has generated a vast amount of information on gene locations and the magnitude of effects on many traits, the identification of the genes involved has remained elusive. This is due to poor resolution of QTL analysis. The solution for this problem would be production of recombinant chromosome substitution line, or stepped aligned recombinant inbred lines. Here, backcrossing is carried out until a desired level of recombination has occurred and genetic markers are used to detect desired recombinant chromosome substitution lines in the target region, which can be fixed by doubled haploidy. In rice, molecular markers have been found to be linked with major genes and QTLs for resistance to rice blast, bacterial blight, and sheath blight in a map produced from DH population.Elite crossing
Traditional breeding methods are slow and take 10-15 years for cultivar development. Another disadvantage is inefficiency of selection in early generations because of heterozygosity.These two disadvantages can be over come by DHs, and more elite crosses can be evaluated and selected within less time.
Cultivar development
Uniformity is a general requirement of cultivated line in most species, which can be easily obtained through DH production. There are various ways in which DHs can be used in cultivar production. The DH lines themselves can be released as cultivars, they may be used as parents in hybrid cultivar production or more indirectly in the creation of breeders lines and in germplasm conservation. Barley has over 100 direct DH cultivars. According to published information there are currently around 300 DH derived cultivars in 12 species world wide.The relevance of DHs to plant breeding has increased markedly in recent years owing to the development of protocols for 25 species. Doubled haploidy already plays an important role in hybrid cultivar production of vegetables, and the potential for ornamental production is being vigorously examined. DHs are also being developed in the medicinal herb Valeriana officinalis to select lines with high pharmacological activity. Another interesting development is that fertile homozygous DH lines can be produced in species that have self-incompatibility systems.
Advantages of DHs
The ability to produce homozygous lines after a single round recombination saves a lot of time for the plant breeders. Studies conclude that random DH’s are comparable to the selected lines in pedigree inbreeding. The other advantages include development of large number of homozygous lines, efficient genetic analysis and development of markers for useful traits in much less time. More specific benefits include the possibility of seed propagation as an alternative to vegetative multiplication in ornamentals, and in species such as trees in which long life cycles and inbreeding depression preclude traditional breeding methods, doubled haploidy provides new alternatives.Disadvantages of DHs
The main disadvantage with the DH population is that selection cannot be imposed on the population. But in conventional breeding selection can be practised for several generations: thereby desirable characters can be improved in the population.In haploids produced from anther culture, it is observed that some plants are aneuploids and some are mixed haploid-diploid types. Another disadvantage associated with the double haploidy is the cost involved in establishing tissue culture and growth facilities. The over-usage of doubled haploidy may reduce genetic variation in breeding germplasm. Hence one has to take several factors into consideration before deploying doubled haploidy in breeding programmes.