OBJECTIVES:
The objective of this presentation is to briefly describe 1. methods of double-haploid (DH) production, and 2. uses of DH production in a) alien introduction and germplasm development, b) quantitative genetics and inheritance studies, c) trait loci mapping and marker production, and d) small grain breeding. The proceedings document is in the form of a literature review, the slide presentation will highlight portions of this paper with examples from Nairn program.
1. METHODS OF DOUBLE-HAPLOID PRODUCTION:
Haploid plants are sporophytes containing the gametic chromosome number (Kasha and Reinbergs 1981). They can be produced spontaneously at low levels (corn - Gowen 1952) or efficiently by interspecific hybridization (barley - bulbosum, Kasha and Kao 1970). Hayes (person. comm.) has increased the efficiency of the bulbosum method by rescuing florets instead of embryos. Intergeneric hybridization has been utilized (wheat - maize, Laurie and O'Donoughue 1988, improved technique (Suenaga and Nakajima 1989) and much improved (Howes pers. comm.). Both bulbosum and maize chromosomes are eliminated from the embryos after fertilization.
Anther culture techniques (in Aegilops, Clapham 1973, in barley, Kao 1983 and wheat, Baenziger et al. 1989) and microspore culture in barley and wheat (Kasha 1998) can efficiently produce haploids. There have been reports of ploidy variations in anther culture of barley (Sunderland 1980) and unexpected variability among progeny plants in wheat, due to gametoclonal and somoclonal variations (Baenziger et al.1989). In contrast, Kertesz et al (1998) found that DH lines of wheat using anther culture on cultivars, were comparable agronomically to their original versions. Ovule (San Noeum, 1976) and hap initiator gene (Hagberg and Hagberg 1980) methods have been reported but have not been put to common use.
Therefore each DH system has its own drawbacks:
Methods 2. through 6. above, have two important thing in common: 1. Completely homozygous lines at all loci and 2. They are expensive, need indoor facilities and expertise and wise parent selection.
2a) ALIEN INTRODUCTION AND GERMPLASM DEVELOPMENT:
Homozygosity of all loci may be obtained in the shortest possible time by doubling the haploid chromosome number using colchicine (anther culture in barley can produce a significant number of diploids without the use of colchicine). Using DH methodologies, development of aneuploid stocks is easier (Finch 1980, Islam and Shephard, 1981), maintenance of aneupoids can be improved (Comeau and Plourde 1987, Fedak, pers. comm.) and exotic germplasm can be selected and stabilized quickly (Darvey 1998).
Fedak suggested that DH in wheat was relatively new but could be of great use in wide crosses. Many researchers have noticed instability even at advanced stages due to linkage drag. Each wide hybrid has to be taken on a case by case basis depending on how much recombination has taken place or been induced. He suggested that a) recombination should take place, maybe for two cycles, b) use DH to purify the material then c) proceed with integration (i.e. translocations of the smallest possible units). Transformation techniques has taken this subject into new heights but the intent is the same; the right genes still have to find the right combinations in the right place.
2b) QUANTITATIVE INHERITANCE AND GENETIC STUDIES:
The use of DH has provided considerable information in genetic studies. A small sample are outlined in this section: gene action and interaction, number of genes, heritability, estimation of genetic variances (additive, additive x additive epistasis), general and specific combining abilities, detection of gene linkages, pleiotropy, chromosomal locations. In some cases DH alone is utilized in small grains (Choo 1981a), Powell et al 1983, Choo et al 1986, Choo and Reinbergs 1987 and Jui et al 1997). In other quantitative genetic studies, comparisons are made using parents, F1 and DH lines in barley (Choo 1981b) and in wheat (Suenaga 1998).
DH, F1 and F2 are both utilized and compared in barley (Ho et al 1996) and DH lines with F6, F7 lines developed using SSD, in wheat Fusarium studies (Ban and Suenaga 1998). Comparisons of means, variances and skewnesses have been made in barley with DH lines developed from F1 and F2 plants to detect linkage (Snape and Simpson 1881,). F1 equaled F2 derived DH lines when linkage was not detected but in the presence of linkage, F2 benefited from increased recombination. F2 and F3 were similar in the frequency of the best recombinant. Some tips from above studies: The skewness of a biparental doubled haploid population is equal to zero in the absence of gene interaction but greater or less than zero in the presence of complementary and duplicate interactions, respectively and kurtosis is positive only where gene interaction is present. This type of analysis can detect additive gene interactions and multiple gene action (Kasha and Reinbergs 1981).
The number of genes affecting a quantitative trait can be estimated by dividing the square of the deviation of the most extreme double-haploid from the sample mean by the genotypic (or additive) variance of the double-haploids (Choo 1981c). Careful choice of parents with known resistances and physical markers when using DH lines can yield i. number of genes responsible for a trait comparing observed vs. expected under chi2 scrutiny, ii. gene linkage for disease and chromosome locations using chi2 and the Fisher exact probability test (Ho et al. 1996). A review by Choo et al.1986 on use of DH lines reminded us that h2 is the slope of the regression of the offspring phenotype divided by the average parental phenotype so that:
i. Covariance between DH progeny and one parent and also covariance between DH progeny and mid-parent can be calculated to determine h2, ii. Response to selection was predicted and variance of response to selection was estimated, iii. three methods were used to detect linkage disequilibrium in the DH lines based on the facts that the overall parental mean would be significantly different from the mean of all DH's when linkage disequilibrium and additive x additive epistasis are present, using a t-test and compared the parental variance with the variance of all DH lines using an F'-test; the two variances should be homogenous in the absence of linkage disequilibrium. The measurement of the covariance between DH progeny and one parent compared to the covariance between DH progeny and mid-parent should be the same in the absence of linkage and linkage disequilibrium.
2c) TRAIT LOCI MAPPING AND MARKER PRODUCTION:
Double-haploid lines are ideal for genetic mapping (i.e. the use of DH in the NABGMP) because of their homozygosity, uniformity and each line can be replicated indefinitely over several locations (Tinker et al 1996).
A MQTL software package (RFLP's) has been developed for interval mapping using QTL (quantitative trait loci) analysis in multi-locations for completely homozygous lines (Tinker and Mather 1995). Using QTL analysis on DH lines, regions of the genome that affect agronomic performance in two-rowed barley (Tinker et al 1996), regions that affect grain and malting quality of barley (Mather et al 1997) and the mapping of disease resistant loci in barley (Spaner et al 1998) and rice (Ando et al. 1997) have been reported.
The detection of polymorphism using RFLP's ( Liu et al. 1990) and microsatellites ( Ma et al 1996) has been lower in hexaploid wheat than in diploids but comparative molecular mapping of agronomically important genes in the Triciceae genomes indicate that in most cases the orders of molecular markers on the linkage maps are identical, although distances may differ (Borner 1998). Thus placement of genes on particular chromosomes in hexaploids have been realized by utilizing mapping information from other related species.
Although much information has been directly resolved using deficiency stocks (Sears and Sears 1978) and deletion stocks (Endo and Gill 1996, Gill and Gill 1998) in wheat, RFLP mapping in diploid species of Triticae have been successful (Pagnotta et al 1998). Ma and Lapitan (1998) reported that AFLP analysis in hexaploid wheat can detect up to 8 times more polymorphism than RFLP analysis, thus may detect 16 times more loci. They suggested that AFLP's might be useful for high density mapping in regions containing genes of interest. The Wheat Genetics Resource Center web page (http://www.ksu.edu/wgrc/Probe/probes.html) contains markers used for mapping and their location on AE. tauschii, T. aestivum, H. vulgare and T. monococcum.
Buerstmayr et al. 1998 reported an ongoing study using 250 and 180 each, DH lines from crosses between two parents resistant to Fuarium (Frontana and Sumai 3 based) and a susceptible line of winter wheat. Different types of DNA markers are being tested for polymorphism in these populations. Armstrong (1998) stated that microsatellites (SSR'S) are his markers of choice, because they are PCR based, are locus specific and are the most highly polymorphic of the markers per locus. From his study in progress, 70 % of the SSR's used (50 were screened on a wheat population) were polymorphic. He estimated that were now 350 + available markers and that 245 could be eventually mapped on the same population of DH lines. The use of DH lines in QTL mapping, may provide benefit, by the ability to score in a completely homozygous background over several environments but they only generate two genotypes at each marker locus compared to three for F2 analysis which has the estimation of the degree of dominance associated with detected QTL's (Lynch and Walsh 1997).
To come back to earth, Wolfe et al 1996, reported the creation of multiple recessive and dominant genetic physical marker stocks in barley. Over 110 DH lines were produced at that time and under increase, between the master recessive and dominant lines (the parents were doubled first). Thus genetic purity and maintenance of important traits have been satisfied to a greater extent using double-haploidy.
2d) SMALL GRAINS BREEDING:
It should be obvious that the above sections are also part of plant breeding; under the categories of tools, finding needed traits, producing variability, measurement of gene action and control and placement of genes for access. We will present some examples on the knowledge gained in section 2 b) above and the actual breeding improvement gained in a few of their studies, in Idaho Falls. Examples of our use of DH in our breeding programs at Nairn in barley and wheat, will also be reported. The presentation will include i. a brief history of DH production and success (20 + spring barley and 2 winter barley varieties released since 1998 at Nairn), ii. examples of particular crosses (very narrow and very wide) that have worked and possibly why, iii. a brief review of some particular breeding projects at Nairn - all utilizing DH (bulbosum in barley; maize in wheat as follows: (see Appendix 1 for DH scheme at Nairn)
- Fusarium resistance in soft wheat using winter wheat resistant donors from Frontana
- lower pentosans content in soft red winter wheat with emphasis on 1B1R vs non-1B1R populations
- reduce pre-harvest sprouting in soft white winter wheat using Mark Sorrell's donor parent material ( Mark is collaborating on the pentosans and sprouting projects)
- increase protein quantity and quality of hard red and white winter wheat for eastern Canada using Rollie Sears' Karl-92 and KS2W as donors ( multi-location study and breeding effort with parents, DH lines to correlate mixographs - farinographs, estimate genetic/environmental interaction with protein quantity and quality, produce genetic markers, determine agronomic adaptability of these DH lines from the Maritimes to Ontario to Saskatoon to Kansas and of course; produce marketable varieties.
There are three other projects underway that are particularly related to transformation and the use of haploids - marker assisted selection at the haploid stage - doubling and then testing of phenotypes: i. moving to microspore culture at Nairn, ii. producing markers for Fusarium, pre-harvest sprouting resistance and iii. transforming winter barley and wheat to increase winter survival and storage capacity.
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