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     

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.     

Segregation of genetic markers among wheat doubled haploid lines derived from wheat x maize crosses

Summary Wheat doubled haploid (DH) lines were produced from the F₁ hybrid, Fukudo-komugi x Oligo Culm, through intergeneric crosses between wheat and maize. F₂ plants and 203 DH lines were analyzed for the segregation of the eight genetic markers, namely, grain proteins, grain esterases, GA-insensitivity and glume traits. The segregation in the F₂ plants fitted to the expected ratios. No deviation was observed among the DH lines, either, except for the glume pubescence. The result indicates the absence of correlation between the markers investigated and the efficiency of embryo formation in the DH lines.     

Potential use of doubled haploid lines for the screening of resistance to yellow rust (Puccinia striiformis) in hexaploid wheat

Doubled haploid lines derived from anther culture of two Iranian spring wheat genotypes‘Ghods’susceptible and‘9106’resistant to yellow rust in Iranian field conditions, and their F₁ hybrids were used in this study. Seedlings of 36 doubled haploid lines, selected out of 96 according to their agronomic traits and the two parental genotypes were inoculated with eight races of yellow rust. The parental genotypes (‘Ghods’and‘9106’) were segregating for some of the races but their doubled haploid lines were either resistant or susceptible to them.‘Ghods’was susceptible to three of the races studied but three doubled haploid lines derived from it were resistant to them. Five selected doubled haploids from the‘9106’genotype and six from F₁ hybrid plants were resistant to all eight races tested. After further investigations in Iranian field conditions it was found that some of these lines can be used as donor genotypes for resistance to yellow rust in wheat breeding programmes. Use of these genotypes should be possible if the French yellow rust races used for selection also represent the dominant races in Iran. It can be concluded that anther culture provides an efficient method for fixing genes of resistance to yellow rust and desirable doubled haploids from F₁ plants can be derived.     

Haploidy breeding and mutagenesis for drought tolerance in wheat

Several intraspecific crosses between known drought tolerant wheat varieties and stable high yielding recombinants were made with the objective to develop improved cultivars for the moisture stressed rainfed areas of Pakistan. Five of these crosses were selected for further creation of useful mutations through the application of low doses of gamma rays and development of doubled haploids through anther culture. Anther culture response of the selected irradiated F₁ generations was studied on liquid and solid induction media. The highest number of calli among almost all crosses was produced on Potato-2induction medium. All the crosses varied greatly in response to callus induction and maximum calli (75%) were obtained from Lyl-73/vee’s’ cross. Similarly, genotypic differences were found for green vs. albino regenerants. The highest number of green plantlets (12.1%) was recorded for Lu-26/3062. From the developed doubled haploid population 25 DH-mutants were initially selected and nine lines were finally included in multi-locational field tests. Two DH-mutants (i.e. DHML-50 and DHML-9) have potential for better grain yield, earliness, disease resistance and moisture stress tolerance. This revised version was published online in August 2006 with corrections to the Cover Date.     

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     

Discrimination of haploid and diploid maize kernels via multispectral imaging

The use of doubled haploids (DHs) in maize has become ubiquitous in maize breeding programmes as it allows breeders to go from cross to evaluation in as little as 2 years. Two important aspects of the in vivo DH system used in maize are as follows: (i) the identification of haploid progeny and (ii) doubling of the haploid genome to produce fertile inbred lines. This study is focused on the first step. Currently, identification of maize haploid progeny is performed manually using the R1‐nj seed colour marker. This is a labour‐intensive and time‐consuming process; a method for automated sorting of haploids would increase the efficiency of DH line development. In this study, six inbred lines were crossed with the maternal haploid inducer ‘RWS/RWK‐76’ and a sample of seed was sorted manually for each line. Using the VideometerLab 3 system, spectral imaging techniques were applied to discriminate between haploids and hybrids. Using DNA markers to confirm the haploid/diploid state of the tested seed, for the majority of genotypes haploid identification was possible with over 50% accuracy.     

Early spontaneous diploidization of maternal maize haploids generated by in vivo haploid induction

The production of doubled haploid (DH) lines has become a key technology in maize (Zea mays L.) research and breeding. However, most of the haploid plants are sterile and in many cases artificial chromosome doubling involves the use of costly and toxic chemicals. Here, we report a special kind of doubled haploid named the early doubled haploid (EH) that was generated directly by in vivo haploid induction. We found 83 EH plants induced from the hybrid Zhengdan958, 55 families of its F_2:3 population and the parental lines, all of which were confirmed to be homozygous diploids via flow cytometry and 104 SSR markers. The progeny of EH₀ (EH₁) behaved in the same manner and showed the same potentialities as the parents of Zheng58 and Chang7-2. EH plants were also detected in other genetic backgrounds at a frequency of 1–3.5 % based on the total number of haploid plants. Because the EH lines exhibited completely fertility and were obtained from induction directly in one step, they could be used in DH breeding as a new breeding strategy. According to our observations, it is likely that spontaneous doubling in EH occurred during embryo development when haploid induction. The possible mechanism of EH is also discussed.     

A Comparison of Genetic Segregation in Traditional and Microspore-Derived Populations of Brassica juncea L. Czern and Coss.

An isolated microspore culture procedure was used to produce doubled haploid lines of Brassica juncea from F¹ plants of reciprocal crosses between the cultivar‘RLM514’and a canola quality breeding line. The inheritance of two qualitative markers, seed color and leaf hairiness, was compared using traditional and microspore-derived populations from these crosses. Chi-square tests indicated that each trait is controlled by different sets of duplicate pairs of genes. Brown seeds or hairy leaves can result from the presence of either of two dominant alleles, whereas yellow seed or glabrous leaves are produced when alleles at both loci are recessive. The segregation of genes controlling seed color and leaf hairs in doubled haploid progeny did not differ significantly from that expected under random assortment, indicating that doubled haploids can be used in this species for genetic studies, and probably cultivar development as well.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

Conditions favouring doubled haploid breeding over conventional breeding of self-fertilizing crops

Summary Monte Carlo computer simulation was used to investigate the conditions favouring doubled haploid breeding over conventional breeding of self-fertilizing crops. Two different systems of doubled haploid breeding and three systems of conventional breeding were compared for two criterion parameters, i.e., the probability of obtaining desirable genotypes and the expected genetic advance of selected lines. It was inferred that the efficiency of production of haploid and doubled haploid plants primarily determines the success of the doubled haploid breeding method. In doubled haploid breeding, about 1/5, hopefully 1/2 as many test plants need to be raised as in conventional breeding to achieve the same level of success. With this condition begin satisfied, the doubled haploid breeding method can efficiently be used when one or more of the following conditions are met: (i) a relatively small number of loci, presumably ten of less, is involved with the breeding objective concerned, (ii) desirable alleles are recessive to undesirable ones at most, if not all, of the segregating loci, and (iii) the genes are not strongly linked. It was confirmed that the doubling of haploids can better be applied to selected F₂ plants rather than to F₁ plants.     

Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SSR markers

The traditional process of obtaining maize hybrids involves the generation of inbred lines through successive generations of selfing and subsequent testcrosses in order to identify the best combining ability by allelic complementation. A fast alternative to obtain inbred lines is to induce the formation of haploids followed by chromosome doubling. However, even with the aid of haploid-inducing genetic sources, this strategy has not been widely used in maize breeding programs, partly due to difficulties inherent to haploid generation and identification. In order to evaluate the possibility of using dihaploids to generate homozygous maize tropical lines, we used the androgenetic haploid inducer line W23 as a female parent in crosses with the tropical single-cross hybrid BRS1010. Within the progeny of these crosses, 462 seeds were phenotypically selected as putative haploids by the purple-colored endosperm and colorless embryo conditioned by the R1-nj gene. Among these, only four individuals were confirmed as being haploids using SSR markers, chromosome counting and flow cytometry, showing that the phenotypic marker was not efficient in detecting haploids in the tropical maize genotype used. All four haploids as well as some diploid plants presented reduced size, corroborating the difficulties for haploid identification by phenotypic evaluation. Genetic diversity analysis revealed by SSR markers divided the haploids in two groups represented by flint and dent maize inbred lines, which could be helpful in identifying complementary dihaploid lines. The present article demonstrates that a combination of haploid production and SSR fingerprinting is a feasible strategy for maize hybrid development in tropical germplasm.     

Haploid plantlet screening in the development of blackleg resistant DH lines of Brassica napus

This study was conducted to expedite disease-resistance breeding in canola by screening haploid plants against blackleg disease. Microspore-derived haploid plantlets from 14 unrelated crosses were inoculated with Leptosphaeria maculans pycnidiospores and were rated as resistant, intermediate or susceptible. Blackleg-inoculated and control haploid plants were then colchicine-treated to produce doubled haploid (DH) lines. The DH lines thus produced were again screened against blackleg using the cotyledon bioassay. In general, the proportion of the resistant DH lines derived from selected resistant haploid plants was much higher than the proportion of resistant DH lines from control haploid populations. There was no detrimental effect on the survival of haploid plants after the combined treatment of blackleg inoculation and colchicine. The importance of the micro-environment on the final outcome of plant-pathogen interaction in case of non-obligate pathogens is also discussed.     

Transfer of cytoplasmic male sterility by spontaneous androgenesis in tobacco (Nicotiana tabacum L.)

Summary Five cytoplasmic male sterile lines of Nicotiana tabacum homozygous for a dominant rootless mutation [Rac^-] were used as female parents in crosses with three male fertile varieties. Androgenetic haploids were selected by their ability to form plantlets with a normal root system, whereas hybrid plantlets failed to grow. High frequencies of androgenetic haploid plants were obtained with N. debneyi cytoplasm, and with one paternal genotype. Chromosome doubling was performed using three different methods (pollination, in vitro culture, acenaphthen) and cytoplasmic male sterile doubled haploid plants have been obtained. The results demonstrate the efficiency of this method for a rapid cytoplasm transfer in Nicotiana tabacum; the integrity of the cytoplasm is conserved. For the first time, we possess a counter selectable marker universally usable for Nicotiana tabacum and closely related species.     

Inheritance of salt tolerance in rice

Summary The genetic behavior of salt tolerance was studied in artificially salinized conditions at the International Rice Research Institute.Divergent selection, carried out at a salinity level where the ECe was 15.2 mmhos/cm at 25 C in F₃ lines from two crosses confirmed the effects of salt tolerance on F₄ progeny with realized heritability values of 0.39 and 0.62, respectively.In a cross between two tolerant cultivars there was clear over-dominance for tolerance, despite the high environmental fluctuation which resulted in a low genetic response as indicated by a low but significant repeatability of 0.20–0.25, and many progeny lines more tolerant than the parents were recovered. The superior tolerance of these progenies compared to the parents was confirmed subsequently at 3 different salt levels. In the same experiment a cross between tolerant and susceptible cultivars produced some progeny of comparable tolerance with tolerant sources.In a 6×6 diallel cross experiment with two tolerant, moderate, and susceptible varieties each, both general and specific combining ability were significant.The findings indicate the possibility of breeding rices more tolerant than existing tolerant cultivars through cumulative crosses of tolerant cultivars. Further improvement can be attained by crossing highly tolerant lines with donors of good agronomic traits and pest and disease resistance.     

Yield of doubled haploid lines of Nordic spring barley infected with net blotch, Pyrenophora teres

Five Nordic spring barley lines (‘Rolfi’, ‘Arve’, ‘Botnia’, ‘Pohto’ and WW7977) and doubled haploid (DH) populations from a half diallel of crosses between them, were sown in the field in Finland over 2 years and were artificially infected with Pyrenophora teres, the causal agent of net blotch. The purpose of the experiments was to determine the extent of yield loss under net blotch infection in a range of parent barleys and DH populations differing in symptom expression. Analysis of foliar damage symptoms, yield and aerial biomass data indicated that, in both years, there were statistically significant differences among parents and crosses, but the relationships between symptom expression and yield maintenance and between symptom expression and aerial biomass maintenance were stronger in 1997, when yields were higher and net blotch was less severe.     

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.     

Double haploids,markers and QTL analysis in vegetable brassicas

Double haploid (DH) plants of Brassica spp. can be produced via anther culture or culture of microspores. This paper reviews the uses of double haploids in crop improvement research in vegetable brassicas (B. oleracea). Applications of DH lines are described for breeding; construction of linkage maps; genetic analysis of quantitative traits and capturing genetic variation. The advantages and disadvantages of DH lines are discussed     

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.     

Classification approaches for sorting maize (Zea mays subsp. mays) haploids using single-kernel near-infrared spectroscopy

Doubled haploids (DHs) are an important breeding tool for creating maize inbred lines. One bottleneck in the DH process is the manual separation of haploids from among the much larger pool of hybrid siblings in a haploid induction cross. Here, we demonstrate the ability of single-kernel near-infrared reflectance spectroscopy (skNIR) to identify haploid kernels. The skNIR is a high-throughput device that acquires an NIR spectrum to predict individual kernel traits. We collected skNIR data from haploid and hybrid kernels in 15 haploid induction crosses and found significant differences in multiple traits such as percent oil, seed weight, or volume, within each cross. The two kernel classes were separated by their NIR profile using Partial Least Squares Linear Discriminant Analysis (PLS-LDA). A general classification model, in which all induction crosses were used in the discrimination model, and a specific model, in which only kernels within a specific induction cross, were compared. Specific models outperformed the general model and were able to enrich a haploid selection pool to above 50% haploids. Applications for the instrument are discussed.