Characterization of wheat-triticale doubled haploid lines by cytological and biochemical markers

Five wheat-triticale doubled haploid (DH) lines— M08, V209, DH220-14-2, DH696-3-4 and M16 —derived from anther culture of F₁s resulting from crosses involving hexaploid or octoploid triticale × hexaploid wheat, were characterized by cytological and biochemical markers. Cytological evidence from genomic in situ hybridization and C-banding indicated that DH lines M08 and V209 (2n= 42) each contained a pair of 1BL/1RS translocation chromosomes. DH220-14-2 (2n= 42) was also a translocated line with two pairs of chromosomes containing small fragments of rye. One of the translocation fragments carried the Sec-1R gene originating from the satellite region of 1RS; the origin of the other one remains unknown. DH696-3-4 (2n= 42) contained a 3D(3R) substitution. In M16 (2n= 44), three pairs of rye chromosomes, 3R, 4R and 6R, were present, 4R as an addition and 3D(3R) and 6D(6R) as substitutions. Biochemical, isozyme and storage protein markers confirmed the cytological conclusions. The advantages of transferring alien chromosomes or chromosome fragments into wheat and creating alien aneuploid lines by anther culture of hybrid F¹s are discussed.     

Doubled-haploid versus single-seed descent and S1-family variation for testcross performance in a maize population

Progress made in the in situ gynogenesis technique since 1990 now allows production of a high number of maize (Zea mays L.) doubled-haploid (DH) lines. The aim of the study was to compare DH lines versus selfing lines for testcross performance. DH and single-seed descent (SSD) lines were produced from random S₁ progenies of a broad-base population. For grain yield, kernel moisture, plant height, ear height and leaf length, the three population means were similar. Except for kernel moisture, the genetic variance of DH lines was nearly twice as high as the genetic variance of S₁ families, as expected. On the other hand, genetic variance among SSD lines was only 1.5 times higher than the genetic variance of S₁ families. This lower variance could be due to a selection bias in the method of production of SSD lines. However, for all traits, heritability of SSD or DH lines was higher than heritability of S₁ families. Epistasis effects in DH progenies were not significant. The consequence was a high correlation between S₁ testcross progenies and DH or SSD testcross progenies, meaning that the S₁ testcross value can be used to select the best families from which DH lines will be extracted. As a whole, the observed variation in DH lines appeared to be more in accordance with the observed variation among S₁ families than with the observed variation among SSD lines.     

Doubled haploids in tropical maize: <Emphasis Type="SmallCaps">II</Emphasis>. Quantitative genetic parameters for testcross performance

Single crosses (SC) of elite inbreds and open-pollinated populations (OP) are suitable source germplasm for doubled haploid (DH) line development in hybrid maize breeding, given that they combine a high population mean ([`(x)] \overline{x} ) for testcross performance with adequate response to selection ( \Updelta G \Updelta G ). This is the first study reporting testcross grain yield (TCGY) and dry matter content (TCDMC) evaluations of 131 DH lines developed from ten tropical source germplasm comprising five OP (OP1–OP5) and five SC (SC1–SC5). Gene diversity (d) and the average number of alleles (a <sub>r</sub> ) per locus was estimated for DH lines based on 24 simple sequence repeat markers. Analysis across three environments revealed no significant differences between [`(x)] \overline{x} of OP- and SC-derived DH lines for TCGY and TCDMC. Significant genetic variance for both traits was only detected among OP-derived DH lines which may be explained by a larger number of segregating quantitative trait loci (QTL) as suggested by higher d and a _r values than in SC-derived DH lines. The usefulness criterion ( U = [`(x)] + \Updelta G U = \overline{x} + \Updelta G ) was higher for OP-derived DH lines for TCDMC, but higher for SC-derived DH lines for TCGY. DH lines from OP1, OP2, and OP3 showed high TCGY, suggesting that they may be useful in tropical hybrid breeding. We conclude that tropical OP represent a valuable source of untapped genetic variation that can efficiently be exploited with DH technology for hybrid maize breeding.     

Segregation for isozymes and HMW glutenins in a doubled-haploid cross of winter wheat carrying 1BL.1RS and 5DL.5RS translocations from rye

The doubled haploid (DH) wheat line ‘dh 5841’ carrying two translocations from rye, 5DL.5RS and 1BL.1RS, has been crossed to the subline of wheat cultivar ‘Amadeus 7143’ with a 1BL.1RS translocation. The resulting F₁ hybrid IJ 98 with a heterozygous 5DL.5DS‐5DL.5RS chromosome pair has been used to produce doubled haploids. A total of 57 DH lines were obtained from plantlets regenerated in anther culture after successful colchicine treatment and seed set. These lines were identified regarding the constitution of chromosome 5D (5DL.5DS or 5DL.5RS) by means of isoenzyme marker analysis. Thirty DH lines possessed the 5DL.5DS chromosome, while the remaining 27 lines carried the 5DL.5RS translocation. For some of these lines, the 5DL.5RS chromosome was cytologically confirmed by C‐banding. Furthermore, the DH lines were evaluated for their high molecular weight glutenin subunit composition. All possible combinations for the four independent loci —Skdh, Glu‐Al, Glu‐B1 and Glu‐D1— were detected in only 57 DH lines and no segregation distortion was observed.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Field Performance of Androgenetic Doubled Haploid Spring Wheat Lines in Comparison with Lines Selected by the Pedigree System

A total of 316 doubled haploid lines (DH) of spring wheat were compared with 621 lines selected in a pedigree system (PS) under field conditions in a breeding nursery. The lines originated from 21 crosses and the samples tested represent mean values for variables comprising the main breeding goals such as disease resistance, baking quality, and agronomic traits. In general, the DH lines were later in days to heading and shorter than PS lines of different homozygosity levels. Except for leaf rust (Puccinia recondite), the DHs were more resistant to the artificially infected diseases such as powdery mildew (Erysiphe graminis), stripe rust (Puccinia striiformis), and septoria nodorum blotch (Septoria nodorum). These differences, which were relatively small for practical selection purposes, were probably due to easier and more precise disease assessment of the homozygous DH lines. For quality characters, the DHs had a higher protein level, while the rest of the parameters were similar for both origins. The data analyzed suggest so far that androgentic doubled haploids in spring wheat are very similar to lines selected in a pedigree system in respect to all the agronomic characters tested. However, the DH lines were produced in a much shorter period of time. It is suggested that androgenetic doubled haploids be produced from F₁ hybrids and that the well-established bulk method should continue to allow selection bulk method should continue to allow selection for rare recombinants as soon as homozygosity is reached.     

Studies of the Genetic Relationship between Anther Culture and Somatic Tissue Culture Abilities in Wheat

A population of thirty-eight doubled haploid lines, developed from the F₁ between two wheat parents differing in anther culture and somatic tissue culture responses, ‘was used to examine the genetical control of responses to these in vitro systems. During anther culture genetic variation between lines was exhibited for frequencies of callus induction., embryo production and embryo regeneration rates. In addition the relative frequencies of green and albino plants was shown to be genotype dependent. However, there was no correlation, between the frequencies of embryo production and the regeneration rate of those embryos suggesting an independent genetic control of these two components. Transgressive segregation for performance was observed for all components indicating that at least two genes are involved in the response of each, and lines for improved performance, combining high ernoryo production rates and good regeneration capacity were identified. No genetic variation for frequencies of callus induction from immature embryos was observed in this cross. However, genetic variation for the regeneration frequencies of plants was observed. Lines with an improved tissue culture response over the two parents were identified. There was no correlation between the performance of lines in anther culture and somatic tissue culture, indicating separate genetical control, and lines with alternative levels of response to the two systems were identified.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

Variation in Doubled Haploid Plants of Wheat Obtained through Wheat (Triticum aestivum) × Maize (Zea mays) Crosses

Variation was investigated in 110 doubled haploid (DH) lines of wheat derived from wheat × maize crosses. Field observation revealed visible variations in 15 DH2 lines such as extreme dwarfism, low seed fertility, alteration of spike type and stripes. Six agronomic traits, i.e., heading date, spike number/ plant, culm length, spike length, seed fertility and grain weight were statistically analyzed in the DH2 and DH3 generations. Out of the 88 DH2 lines/DH3 groups, 26 %/64 % showed significant differences from the parental variety in the means of one or more traits. Ranges of the DH3 lines were larger than those of the DH2 lines, except for spike number/plant. Furthermore, analyses of variance within and between DH lines showed the presence of heterogeneity/heterozygosity in the DH2 lines/plants. These results indicated the occurrence of gametoclonal variation in the DH lines. It is considered that most of the variations detected were due to the colchicine treatment rather than to the 2,4-D treatment or in vitro culture.     

Production of salt tolerant rice breeding line via doubled haploid

Summary A haploid breeding program was initiated to develop doubled haploid salt tolerant rice breeding line via anther culture. Two sensitive breeding lines BR4608-R₁-R₂ and BR4909-R₁-R₂ were crossed with a salt tolerant line IR13146-13-3-3 to transfer its salt tolerant character to the doubled haploids.Anther from confirmed F₁s of the two crosses were cultured in defined medium for callus induction and eventual plant regeneration. Fifteen doubled haploid (DH) lines were obtained from two crosses. Test for salt tolerance were done in vitro. Five out of 15 lines were found tolerant at the level of 8–10 decisiemens/m (ds/m) while the rests were sensitive to that level of salinity.Field experiment was conducted to evaluate the doubled haploids under saline and non saline soil. Five salt tolerant lines produced comparable yield with the resistant control (BR 23) under saline condition, whereas these lines yielded even higher in non saline soil under irrigated condition when evaluated with other 10 sensitive DH linesAbbreviations LSD Least Significant Difference - NAA Napthalene Acetic Acid     

Chromosomal regions associated with green plant regeneration in wheat (Triticum aestivum L.) anther culture

Genetic capacity for green plant regeneration in anther culture were mapped in a population comprising 50 doubled haploid lines from a cross between two wheat varieties ‘Ciano’ and ‘Walter’ with widely different capacity for green plant regeneration. Bulked segregant analysis with AFLP markers and composite interval mapping detected four QTLs for green plant percentage on chromosomes 2AL (QGpp.kvl-2A), 2BL (QGpp.kvl-2B.1 and QGpp.kvl-2B.2) and 5BL (QGpp.kvl-5B).The three QTLs detected on chromosome 2AL and 2BL all derived their alleles favouring green plant formation from the responsive parent ‘Ciano’.The remaining QTL on chromosome 5BL had the allele favouring green plants from the low responding parent ‘Walter’. In a multiple regression analysis the four QTLs could explain a total of 80% of the genotypic variation for green plant percentage. None of the chromosomal regions with QTLs for green plant percentage showed significant influence on either embryo formation or regeneration frequencies from the anther culture. The three major QTLs located on group two chromosomes were fixed in a second DH population derived from two parents ‘Ciano’ and ‘Benoist’,both with high capacity to produce green plants. A QTL explaining31.5% of the genetic variation for green plant formation were detected on chromosome 5BL in this cross as well. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distorted Segregation for Mildew Resistance in Doubled Haploid Lines of Spring Barley

The mildew reactions of the second generation of doubled haploid (DH) plants, derived from anther culture of crosses among three spring barley lines carrying different Mla mildew resistance alleles and the cv. ‘Pallas’, were analyzed by using a set of three European and one Israeli mildew isolate. The results indicated, (1) a significant level of distortion segregation in favour of resistant DH genotypes, which was possibly due to linkage of mildew resistance genes on chromosome 5 with genes for plant regeneration and (2) various degrees of dominance for the different resistance genes studied as well as the possible action of modifier genes.     

Effect of genetic background on the target fatty acids of acyl-ACP thioesterase transgenes in Brassica napus

Acyl‐acyl carrier protein (ACP) thioesterase (TE) is involved in the biosynthetic fatty acid pathway of plants. Conventional canola lines transformed individually with the bay‐TE (Uc FatB1), elm‐TE (Ua FatB1), nutmeg‐TE (Mf FatB1) or Cuphea‐TE transgene (Ch FatB1), produce seed oil with modified fatty acid compositions. This study assessed the effects of genetic background, cytoplasm, maternal parent, and interaction of different TE transgenes, on the target fatty acids using F1 seeds and double haploid (DH) lines. The F₁ seeds were produced by crossing four TE transgenic parental lines and three non‐transgenic cultivars with distinct fatty acid compositions. The DH lines were developed from microspores of F₁ plants. DH lines from different crosses showed that genetic background does not have an effect on the relative levels of the target fatty acids of the same TE, indicating the stability of the substrate specificity of the TE within canola. However, significant effects of genetic background on the content of the major target fatty acids, lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, were observed. Expression of the TE in low erucic acid (C22:1) genotypes resulted in higher target fatty acid levels than expression in high C22:1 genotypes. Reciprocal crosses showed maternal effects, but not cytoplasmic effects. In addition, co‐expression of two different TE transgenes in the same seeds was observed. These results indicate the importance of selection for appropriate genetic backgrounds in order to maximize the expression of the target fatty acids of TE transgenes, and also indicate potential uses of TE co‐expression in modifying canola seed oil.     

Quantitative trait loci for grain yield and yield components in a cross between a six-rowed and a two-rowed barley

Summary The positions of quantitative trait loci (QTL) for yield and yield components were estimated using a 85-point linkage map and phenotype data from a F₁-derived doubled haploid (DH) population of barley. Yield and its components were recorded in two growing seasons. Highly significant QTL effects were found for all traits at several sites in the genome. A major portion of the QTL was found on chromosome 2. The effect of the alleles in locus v on thousand grain weight and kernels per ear explained 70–80% of the genetic variation in the traits. QTL × year interaction was found for grain yield. Several different QTL were found within the two-rowed DH lines compared to those found in the six-rowed DH lines. Epistasis between locus v and several loci for yield and yield components indicates that genes are expressed differently in the two ear types. This may explain the difficulties of selecting high yielding lines from crosses between two-rowed and six-rowed barley.Abbreviations DH doubled haploid - QTL quantitative trait locus/loci - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - T. Prentice Tystofte Prentice - V. Gold Vogelsanger Gold     

RAPD markers linked to a clubroot-resistance locus in Brassica rapa L.

Linkage of random amplified polymorphic DNA (RAPD) markers with resistance genes to clubroot (Plasmodiophora brassicae Wor.) in Brassica rapa L. was studied in a doubled haploid (DH population obtained by microspore culture. Thirty-six DH lines were obtained from F₁ plants from a cross between susceptible ‘Homei P09’ and resistant ‘Siloga S2’ plants. ‘Homei P09’ was a DH line obtained by microspore culture of the Chinese cabbage variety ‘Homei’, which is highly responsive in microspore culture. The resistant line ‘Siloga S2’ was obtained by two rounds of selfing of the fodder turnip ‘Siloga’. Three RAPD markers, RA12-75A, WE22B and WE49B, were found to be linked to a clubroot-resistance locus. These three markers were linked in the DH lines and an F₂ population and should be useful for marker-assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Marker-assisted increase of genetic diversity in a double-low seed quality winter oilseed rape genetic background

Generation of novel genetic diversity for maximization of heterosis in hybrid production is a significant goal in winter oilseed rape breeding. Here, we demonstrate that doubled haploid (DH) production using microspore cultivation can simultaneously introgress favourable alleles for double‐low seed quality (low erucic acid and low‐glucosinolate content) into a genetically diverse Brassica napus genetic background. The DH lines were derived from a cross between a double‐low quality winter rapeseed variety and a genetically diverse semisynthetic B. napus line with high erucic acid and high glucosinolates (++ quality). Twenty‐three low‐glucosinolate lines were identified with a genome component of 50–67% derived from the ++ parent. Four of these lines, with a genome component of 50–55% derived from the ++ parent, also contained low erucic acid. Heterosis for seed yield was confirmed in test‐crosses using these genetically diverse lines as pollinator. The results demonstrate the potential of marker‐assisted identification of novel genetic pools for breeding of double‐low quality winter oilseed rape hybrids.     

Relationship between phenotypic and genetic diversity of parental genotypes and the frequency of transgression effects in barley (Hordeum vulgare L.)

The aim of the study was to evaluate the relationship between genetic and phenotypic distances of parents and the genetic potential of crosses as measured by the frequency of transgressive segregants in homozygous populations. Material for the study involved 17 barley cross‐combinations. In each cross, the parental genotypes, F₂ hybrids and doubled haploid (DH) lines were analysed. Yield and yield‐related traits were observed in the experiments. Phenotypic (univariate and multivariate) and genetic distances (GD) were investigated between pairs of parental genotypes. Genetic distance was evaluated by using random amplified polymorphic DNA markers. In F₂ generations, the genetic coefficient of variability (GCV) was evaluated. Within all the cross‐combinations studied, each DH line was compared with both parents to distinguish the positive and negative transgressive lines. In addition, the coefficient of gene distribution (r) along parental genomes was evaluated. Relationships between frequency of transgression and both phenotypic and GDs, GCV and r, were assessed by regression analysis. It was found that for all the traits studied the frequency of transgressive lines depended mainly on gene distribution (r). Genetic distance between parents appeared to be significant for the occurrence of transgression effects in plant height, ear length, grain weight per ear and grain yield per plot. Regression analysis has shown that phenotypic differences between parental genotypes were also important for the frequency of transgressive lines. A weak relationship was found between the variation of F₂ hybrids and the occurrence of transgressive lines. The results indicate that occurrence of transgressive segregants in a homozygous population should be considered as a phenomenon dependent simultaneously on several factors characterizing parental genotypes. Among them, the most important are: gene distribution, phenotypic diversity and GD.     

Genetic markers for doubled haploid response in barley

In order to analyse the genetic control of anther culture response in barley, a doubled-haploid (DH) population from the cross between a medium responsive cultivar ‘Dobla’ and the model cultivar ‘Igri’ was produced. A linkage map was constructed with 91 markers. A sub-population of 41 lines was characterised for different components of the anther culture response, and was used for quantitative trait loci (QTL) analysis. The vrs1 locus region on chromosome 2H, which determines inflorescence row type, was coincident with the largest putative QTL for number of embryos (nEMB) and albino plants. A region of chromosome 6H was associated with QTLs for nEMB and green plants. QTLs for number and percentage of green plants were located on the long arm of chromosome 5H. Therefore, new QTLs for main components of barley anther culture response were identified on chromosomes 2H, 5H and 6H, indicating that anther culture response in barley could be controlled by relative few genes of large effect. This work is a useful step towards the identification of new regions on the barley genome that could be associated with fundamental biological process implicated in the anther culture response.     

Comparison and analysis of main effects,epistatic effects,and QTL × environment interactions of QTLs for agronomic traits using DH and RILs populations in rice

Two genetic linkage maps based on doubled haploid (DH) and recombinant inbred lines (RILs) populations, derived from the same indica-japonica cross ‘Samgang × Nagdong’, were constructed to analyze the quantitative trait loci (QTLs) affecting agronomic traits in rice. The segregations of agronomic traits in RILs population showed larger variations than those in DH population. A total of 10 and 12 QTLs were identified on six chromosomes using DH population and seven chromosomes using RILs population, respectively. Three stable QTLs including pl9.1, ph1.1, and gwp11.1 were detected through different years. The percentages of phenotypic variation explained by individual QTLs ranged from 8 to 18% in the DH population and 9 to 33% in the RILs population. Twenty-three epistatic QTLs were identified in the DH population, while 21 epistatic QTLs were detected in the RILs population. Epistatic interactions played an important role in controlling the agronomic traits genetically. Four significant main-effect QTLs were involved in the digenic interactions. Significant interactions between QTLs and environments (QE) were identified in two populations. The QTLs affecting grain weight per panicle (GWP) were more sensitive to the environmental changes. The comparison and QTLs analysis between two populations across different years should help rice breeders to comprehend the genetic mechanisms of quantitative traits and improve breeding programs in marker-assisted selection (MAS).     

Use of barley seed vigour to discriminate drought and cold tolerance in crop years with high seed vigour and low trait variation

Seed vigour is a precondition for early and homogenous field emergence of barley, in addition to effective malting. This study aimed to assess the selection of barley varieties by using seed vigour as the indicator. Seed vigour of barley (quantified as the germination percentage) was evaluated under drought (?0.2 MPa) and temperature stress (10°C). At two locations over a 3‐year period, 1 population of 133 Derkado × B83‐12/21/5 doubled haploid (DH) lines (and parents) was evaluated for seed vigour, of which 108 DH lines were assessed for three malting parameters. The relatively high values of vigour during the 3‐year period (overall average 94–95%) probably impeded high variations in genetic potential. A total of 27 DH lines of the 133 evaluated showed transgression for vigour (up to 98%) in comparison with the parents (Derkado: 96%; B83: 92%). In conclusion, caution should be applied when selecting for seed vigour, even in good crop years with high levels of seed vigour and low trait variations. Such selection might improve vigour, particularly in crop years with unsuitable weather conditions.