The agronomic performance of wheat doubled haploid lines derived from wheat x maize crosses

Summary The agronomic performance of 9 doubled haploid (DH) lines of Chinese Spring, 6 DH lines of Hope, 14 DH lines of the single chromosome substitution line Chinese Spring (Hope 5 A) and their respective parents was analyzed under field conditions. Seventeen Chinese Spring DH lines derived from wheat x Hordeum bulbosum crosses were also included for comparison. No significant variation was detected in either population of Chinese Spring DH lines and neither DH population differed from its parent. The Hope DH lines differed significantly for tiller biomass, spikelet number per ear, ear grain weight and 50-grain weight. However, all the variation could be attributed to the poor performance of only one line. Chinese Spring (Hope 5 A) DH lines showed significant variation for ear emergence time, but this was probably due to genetic heterogeneity in the parental stock. Overall, the results suggest that most DH lines produced by the wheat x maize method resemble their wheat parent, and that the variation induced in DH production is likely to be similar to that found in DHs from wheat x Hordeum bulbosum crosses.     

Comparison of frequency distributions of doubled haploid and single seed descent lines in barley

Summary Both doubled haploid (DH) and single seed descent (SSD) methods were used to derive homozygous lines from two crosses of barley. The frequency distributions of grain yield, heading date, and plant height of the DH and SSD lines were compared by the Mann-Whitney U test, Kolmogorov-Smirnov twosample test and Wald-Wolfowitz runs test. It was found that the DH lines distributed in the same manner as the SSD lines with respect to the three characters. The results indicated that although the SSD method had more opportunity for recombination than the DH method, it did not produce a sample of recombinants which differed significantly from the DH sample; thus both methods were equally efficient for use in deriving homozygous lines from F₁ hybrids in a relatively short time.Contribution no. 455 Charlottetown Research Station, Agriculture Canada, P.E.I. (Canada)     

Hybrid maize breeding with doubled haploids: III. Efficiency of early testing prior to doubled haploid production in two-stage selection for testcross performance

Early testing prior to doubled haploid (DH) production is a promising approach in hybrid maize breeding. We (1) determined the optimum allocation of the number of S₁ families, DH lines, and test locations for two different breeding schemes, (2) compared the maximum selection gain achievable under both breeding schemes, and (3) investigated limitations in the current method of DH production. Selection gain was calculated by numerical integration in two-stage breeding schemes with evaluation of testcross progenies of (1) DH lines in both stages (DHTC), or (2) S₁ families in the first and DH lines within S₁ families in the second stage (S₁TC-DHTC). Different assumptions were made regarding the budget, variance components, and time of DH production within S₁ families. Maximum selection gain in S₁TC-DHTC was about 10% larger than in DHTC, indicating the large potential of early testing prior to DH production. The optimum allocation of test resources in S₁TC-DHTC involved similar numbers of test locations and test candidates in both stages resulting in a large optimum number of S₁ families in the first stage and DH lines within the best two S₁ families in the second stage. The longer cycle length of S₁TC-DHTC can be compensated by haploid induction of individual S₁ plants instead of S₁ families. However, this reduces selection gain largely due to the current limitations in the DH technique. Substantial increases in haploid induction and chromosome doubling rates as well as reduction in costs of DH production would allow early testing of S₁ lines and subsequent production and testing of DH lines in a breeding scheme that combines high selection gain with a short cycle length. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of haploid and doubled haploid plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) for use in breeding for multiple virus resistance

We have developed improved procedures for recovery of haploid and doubled haploid (DH) melon plants, using hybrids derived from crosses of lines with multiple virus resistance. Seeds formed after pollination with irradiated pollen were cultured in liquid medium for 10 days before excision of the embryos for further culture. This made it easier to identify the seeds containing parthenogenetic embryos, thereby reducing the effort required and increasing the percentage of plants recovered. The plants obtained (approximately 175) were transferred to a greenhouse for evaluation. Three fertile lines were identified, and selfed seeds were obtained for evaluating virus resistance. Flow cytometry of leaf tissues showed that two of these lines were spontaneous DH and the third was a mixoploid containing haploid and diploid cells. The other plants remained sterile through the flowering stage. Flow cytometry of 20 sterile plants showed that all were haploid. Attempts to induce chromosome doubling by applying colchicine to greenhouse-grown plants were unsuccessful. Shoot tips from the haploid plants were used to establish new in vitro cultures. In vitro treatment of 167 micropropagated haploid shoots with colchicine produced 10 diploid plants as well as 100 mixoploid plants. Pollen from male flowers that formed in vitro on the colchicine-treated plants was examined. High percentages of viable pollen that stained with acetocarmine were found not only in the diploids but also in >60% of the plants scored as mixoploid or haploid by flow cytometry. Efficient recovery of DH from hybrid melon lines carrying combinations of important horticultural traits will be a valuable tool for melon breeders.     

Comparison of bread wheat lines selected by doubled haploid, single-seed descent and pedigree selection methods

 Yield performance of each group of ten spring bread wheat lines selected by doubled haploid (DH), single-seed descent (SSD) and pedigree selection (PS) methods from three F₁ crosses was compared with the aim of evaluating the DH method in breeding programs. Populations of 65–97 DH lines and 110 SSD lines per cross were used for selection. PS lines were developed by repeated selections from 1500 F₂ plants. Yield evaluation was performed at the F₆ generation of SSD and PS lines along with DH lines in a 2-year field experiment. It took only 2 years from the planting of wheat materials for DH production to the planting of selected DH lines for yield evaluation. There was no significant difference in grain yield between DH lines and PS lines selected from an F₁ cross whose parental varieties were closely related in their pedigrees. In two crosses with low coefficients of parentage and a large variation in their progenies, grain yield of selected DH lines was significantly lower than those of selected SSD and PS lines. These results confirm that the DH method can save time in obtaining recombinant inbred lines ready for yield evaluation. However, a larger DH population is required to achieve the same level of genetic advance with the PS method in crosses containing greater genetic variation. Received: 23 December 1997 / Accepted: 12 March 1998     

Plants from haploid tissue culture of Lavix decidua

Haploid embryogenic tissue was initiated on 1/2 LM medium supplemented with 500 mg/l glutamine, 1000 mg/l casein hydrolysate, 100 mg/l inositol, 30000 mg/l sucrose, and 0.1 mg/l 2,4-D. The embryoids matured to produce plantlets. One plant from one of the two lines survived. The chromosome complement of tissue cultures, of the needle bases from the source plant, and of the plant produced in vitro were established by squashes. DNA content was assessed by DNA microdensiometry. In vitro tissues were haploid (n = 12). The plant produced was mixoploid, with a predominance of diploid cells (2n = 24).     

The agronomic performance of anther culture derived plants of barley produced via pollen embryogenesis

Anther culture was used to generate microspore-derived doubled haploid (DH) plants from four spring barley crosses. The culture medium used contained maltose as the sole carbohydrate source and the mode of plantlet regeneration was mainly via pollen embryogenesis. Both haploid and spontaneously doubled regenerants were produced and the doubled haploids were compared to recom-binant inbred lines generated by several rounds of selfing (single seed descent). Parental, DH and single seed descent (SSD) lines were grown in randomised, replicated field trials and the samples were scored for a range of agronomic traits. The mean performance and phenotypic distribution of the DH and SSD samples were similar and there was little evidence to support the conclusion that anther culture derived lines exhibit a reduction in vigour. Where significant differences were detected between groups these were mainly confined to crosses which were segregating for the denso dwarfing gene. The differential transmission of particular regions of the barley genome may therefore influence and confound the expression of agronomic traits in DH populations. This is the first report of the agronomic performance of anther culture lines produced via pollen embryogenesis and the results are discussed in relation to the exploitation of anther culture technology in barley breeding.     

Novel technologies in doubled haploid line development

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.     

Whole genome mapping in a wheat doubled haploid population using SSRs and TRAPs and the identification of QTL for agronomic traits

Genetic maps are useful for detecting quantitative trait loci (QTL) associated with quantitative traits and for marker-assisted selection (MAS) in breeding. In this research, we used the wheat × maize method to develop a doubled haploid (DH) population derived from the synthetic hexaploid wheat (SHW) line TA4152-60 and the North Dakota hard red spring wheat line ND495. The population consisted of 213 lines, of which a subset of 120 lines was randomly selected and used to construct linkage maps of all 21 chromosomes and for QTL detection. The whole genome maps consisted of 632 markers including 410 SSRs, 218 TRAPs, 1 RFLP, and 3 phenotypic markers, and spanned 3,811.5 cM with an average density of one marker per 6.03 cM. Telomere sequence-based TRAPs allowed us to define the ends of seven linkage groups. Analysis revealed major QTLs associated with the traits of days to heading on chromosomes 5A and 5B, plant height on chromosomes 4D and 5A, and spike characteristics on chromosomes 3D, 4A, 4D, 5A and 5B. The DH population and genetic map will be a useful tool for the identification of disease resistance QTL and agronomically important loci, and will aid in the identification and development of markers for MAS. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Agronomic performance of lines derived from anther culture, maize pollination and single-seed descent in a spring wheat cross

Anther culture and maize hybridization are two frequently used techniques for doubled haploid production in wheat (Triticum aestivum L.). Information on the field performance of lines derived from these techniques is limited. This study was conducted to compare the performance of F_4:6 lines obtained by single-seed descent with lines obtained by anther culture and maize (Zea mays L.) pollination from the same cross of spring wheat, ’Chris’/MN 7529. Thirty-three lines derived from each of those techniques were evaluated in six environments for grain yield, protein content, test weight, heading date, kernel weight and plant height. Mean performance of the single-seed descent lines exceeded performance of the anther culture lines for grain yield, kernel weight and plant height with no apparent differences for grain protein content, test weight and heading date. No differences between trait means for the single-seed descent and maize pollination lines were found except for plant height. The best 5 lines from each method for grain yield, protein content and test weight were similar in performance except that the protein content was higher for the maize pollination lines than for the single-seed descent lines. Acceptable levels of agronomic performance could be found among lines from each method. Wide acceptance of the doubled haploid technique for pure line production in breeding programs may, however, be limited by the often poor efficiency of doubled haploid line production, resulting in smaller population sizes for selection of desirable traits in comparison to the single-seed descent method. Received: 31 July 1998 / Accepted: 28 November 1998     

RFLP mapping of Brassica napus using doubled haploid lines

The combined use of doubled haploid lines and molecular markers can provide new genetic information for use in breeding programs. An F₁-derived doubled haploid (DH) population of Brassica napus obtained from a cross between an annual canola cultivar (Stellar) and a biennial rapeseed (Major) was used to construct a linkage map of 132 restriction fragment length polymorphism loci. The marker loci were arranged into 22 linkage groups and six pairs of linked loci covering 1016 cM. The DH map was compared to a partial map constructed with a common set of markers for an F₂ population derived from the same F₁ plant, and the overall maps were not significantly different. Comparisons of maps in Brassica species suggest that less recombination occurs in B. napus (n = 19) than expected from the combined map distances of the two hypothesized diploid progenitors, B. oleracea (n = 9) and B. rapa (n=10). A high percentage (32%) of segregating marker loci were duplicated in the DH map, and conserved linkage arrangements of some duplicated loci indicated possible intergenome homoeology in the amphidiploid or intragenome duplications from the diploid progenitors. Deviation from Mendelian segregation ratios (P < 0.05) was observed for 30% of the marker loci in the DH population and for 24% in the F₂ population. Deviation towards each parent occurred at equal frequencies in both populations and marker loci that showed deviation clustered in specific linkage groups. The DH lines and molecular marker map generated for this study can be used to map loci for agronomic traits segregating in this population. Present address Embrapa/Cenargen, C.P. 0.2372, CEP 70.770, Brasilia DF, Brazil     

Magnitude of hybrid vigor retained in double haploid lines of some heterotic rice hybrids

Sixty one double haploid (DH) lines were evaluated for yield components and yield and compared with the three corresponding hybrids from which they were derived through anther culture. Analysis of a 6×6 diallel experiment led to the selection of these hybrids based on their high heterosis and revealed the nature of gene action of the characters under evaluation. The DH lines along with the hybrids and parents were planted following a simple lattice design with two replications. The results show that in DH lines the values of the characters expressing predominantly additive genetic effects could reach the heterotic level of the hybrids. For characters including yield showing predominantly dominance effects, values in the DH lines were significantly lower than those of the corresponding hybrids. The promising DH lines, however, possess a higher yield potential than the better parents.     

Assessment of androclonal variation in an indica rice PTB 28

Anther culture (AC) derived 160 doubled haploid (DH) families of an indica rice PTB 28 were field evaluated at Port Blair (lat 11 degrees.41'13.04"N; long 92 degrees 43'30.16"E), to assess the extent and range of induced variability among the androclones at segregating A2 generation and identify promising selectants for genetic improvement. Though the coefficient of variation (CV) for plant height was least (2.65%), majority of the androclones were shorter (81.7%). CV was high in case of panicle number per plant (24.59%) and tiller number per plant (28.28%), while it was maximum in respect of yield per plant (37.0%). Tiller and panicle number per plant registered maximum reduction compared to parent, however, displayed larger variations in double haploid (DH) lines. Out of 160 DH families, only 10 (6.3%) lines out-yielded the parent. Extreme types with wide variations were encountered which reaffirms the possibility of employing anther culture in rice improvement by restoring yield advantage in advanced generations or by tailoring them through recombination breeding.     

Mapping QTLs with epistatic effects and QTL×environment interactions for plant height using a doubled haploid population in cultivated wheat

Quantitative trait loci (QTLs) for plant height in wheat (Triticum aestivum L.) were studied using a set of 168 doubled haploid (DH) lines, which were derived from the cross Huapei 3/Yumai 57. A genetic linkage map was constructed using 283 SSR and 22 EST-SSR markers. The DH population and the parents were evaluated for wheat plant height in 2005 and 2006 in Tai’an and 2006 in Suzhou. QTL analyses were performed using the software of QTLNetwork version 2.0 based on the mixed linear model. Four additive QTLs and five pairs of epistatic effects were detected, which were distributed on chromosomes 3A, 4B, 4D, 5A, 6A, 7B, and 7D. Among them, three additive QTLs and three pairs of epistatic QTLs showed QTL×environment interactions (QEs). Two major QTLs, Qph4B and Qph4D, which accounted for 14.51% and 20.22% of the phenotypic variation, were located similar to the reported locations of the dwarfing genes Rht1 and Rht2, respectively. The Qph3A-2 with additive effect was not reported in previous linkage mapping studies. The total QTL effects detected for the plant height explained 85.04% of the phenotypic variation, with additive effects 46.07%, epistatic effects 19.89%, and QEs 19.09%. The results showed that both additive effects and epistatic effects were important genetic bases of wheat plant height, which were subjected to environmental modifications, and caused dramatic changes in phenotypic effects. The information obtained in this study will be useful for manipulating the QTLs for wheat plant height by molecular marker-assisted selection (MAS).     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Excessive homozygosity in doubled haploids — advantages and disadvantages for plant breeding and fundamental research

The utility of haploids and DH lines for breeding programmes and basic research is discussed here. High effectiveness of haploid induction from random responding gametes and from valuable donor plants is not sufficient to ensure success in breeding. DH lines often appeared inferior to conventionally obtained inbred lines. One of the causes may be a very high level of homozygosity, in this paper referred to as excessive homozygosity. Besides, colchicine treatment as well as gametoclonal and somaclonal variation could have a negative effect on the agronomic performance of DH plants. Lack of natural selection in the first stages of haploid development is another important factor, negatively influencing haploid utility. On the other hand, there is a wide range of possibilities of using haploids as model plants for basic research. Among different applications, including mapping, genetic analysis, mutations, transformation, somatic hybridisation, biochemical and physiological studies, artificial seed production and germplasm storage, the first seems to be explored most effectively and give promising results.     

Tests for the presence of gametoclonal variation in barley and wheat doubled haploids produced using the Hordeum bulbosum system

Summary To investigate whether the Hordeum bulbosum system of doubled haploid production generates gametoclonal variation, populations of second generation doubled haploid lines were developed from first generation doubled haploid lines of two barley varieties and three wheat genotypes. In barley, no variation between doubled haploids from doubled haploids was detected for a range of quantitative characters, suggesting the absence of any gametoclonal effects. However, the original selfed-seed stocks were shown to contain cryptic allelic variation for some of the characters investigated. In wheat, gametoclonal variation was detected for ear emergence time, plant height and yield, and its components for two out of the three genotypes investigated. The type and range of variation was similar to that reported from studies of somaclonal variation from immature embryos and gametoclonal variation from anther culture. Generally, the effects appeared to reduce the yield performance of individual lines. The difference in response between the two species and the consequences for the use of the doubled haploid system in breeding programmes are discussed.     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.     

Genotypic and phenotypic characterization of isogenic doubled haploid exotic introgression lines in maize

We characterized the genotypic and phenotypic variation in cell wall digestibility (CWD) and other agronomic traits of 50 backcross 1 generation doubled haploid (BC1DH) lines developed from the Germplasm Enhancement of Maize project. These lines were generated by introgressing 31 exotic unadapted maize races into PHZ51 and PHB47, temperate inbred lines with expired Plant Variety Protection. The 50 BC1DH lines and five check lines were genotyped with 199 single nucleotide polymorphism markers distributed across the genome. We identified, on average, 11.8% of markers with exotic donor parent alleles. This likely underestimates the proportion of donor introgressions, since we cannot discriminate monomorphic alleles from donor and recurrent parents. The potential roles of natural selection and the doubled haploid process in favouring selection of the recurrent genome are discussed. Although the proportion of donor parent genome was underestimated, donor fragments evaluated across the 50 BC1DH lines covered 92.9% of the recurrent parent genome. The evaluation of BC1DH lines for CWD revealed promising lines with CWD not differing significantly (???=?0.05) from forage quality lines used as checks. The introgression of exotic genome segments, however, was generally associated with higher ears, lodging, and late flowering. Even with limited power, our association analysis revealed quantitative trait polymorphisms associated with CWD, flowering date, and lodging.