Doubled haploids of Coffea canephora: development,fertility and agronomic characteristics

Summary Doubled haploids (DH) of Coffea canephora Pierre were developed using haploid embryos which occur spontaneously in association with polyembryony. The frequencies of polyembryonic seeds and haploid embryos varied according to the parental genotypes. However, production of a large number of DH seemed possible from all genotypes. More than 750 DHs produced from various genotypes were grown under field conditions and evaluated for different characters of agronomic importance. Approximately half of DH genotypes did not survive, suggesting a strong, negative effect of homozygosity. Inbreeding depression is particularly severe on general vigor and reproductive aspects. For several characters studied such as leaf shape, leaf rust resistance and hundred bean weight, considerable genetic variations were observed within and between groups of DHs constituted by the DHs produced from the same clone. Despite their low vigor and reduced fertility, the DHs of C. canephora offer new possibilities in genetic research and coffee breeding.     

Breeding of cabbage (Brassica oleracea L. var. capitata) with fusarium wilt resistance based on microspore culture and marker-assisted selection

Cabbage Fusarium wilt (CFW), caused by Fusarium oxysporum f. sp. conglutinans, is one of the most devastating diseases of cabbage worldwide. In recent years, CFW has been spreading across northern Chinad. Thus, development of resistant cultivars is necessary to control CFW. Here we report microspore culture from a cross of two elite cabbage inbred lines, 96–100 (highly resistant to CFW) and 01–20 (highly susceptible to CFW), in order to obtain doubled haploids (DHs) to cultivate cabbage materials for resistance breeding. A total of 196 DHs were obtained in 2011 and 2012 and three elite DH lines were selected from them through agronomic trait evaluation as well as marker-assisted selection. These three elite DH lines were crossed with elite cabbage inbred lines to create hybrids, which were then evaluated for their resistance and agronomic traits. Three excellent hybrids were selected. The use of microspore culture and marker-assisted selection greatly shortened the time required to obtain these excellent hybrids. These resistant varieties may become an effective control of CFW in diseased areas.     

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.     

Lack of durability of resistance to cereal rusts in wheat when selection is for complete resistance

Twenty-one bread-wheat entries were selected after careful screening for complete or near-complete resistance to yellow rust (Puccinia striiformis), stem rust (P. graminis), and leaf rust (P. recondita). In 1987, the 21 entries were intercrossed in a near-half diallel scheme. The resulting 190 F₂ populations were advanced to F₇ under selection for complete resistance to the three rusts and for good agronomic types. In 1992 the 21 parents and 140 selected F₇ lines were assessed for their resistance to the three rusts. Of the 21 parents, 12 showed a breakdown of yellow rust resistance, five a breakdown of stem rust resistance and two a breakdown of leaf rust resistance. In addition, several of the 140 selected F₇ lines, all still resistant in F₆, had become susceptible to one or more of the rusts. It appears that a progression towards more complex races, especially of yellow rust, is inevitable for the wheat-cereal rust patho-systems when the selection is for complete or near-complete resistance.     

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.     

Combining Ability of Doubled Haploids in Coffea canephora P.

Fifty-five doubled haploids (DH) of Coffea canephora were crossed with either heterozygous genotypes or DH in order to study their combining ability. Three agronomic trials were established. Marked hybrid vigour was observed for all characters analyzed including yield. Large differences were evident among top-crosses involving different DH produced from the same parental clone reflecting the high level of heterozygosity of clones. Factorial mating design analysis indicated that all genetic variance was attributable to additive effects in estimates of yield as well as plant height and leaf characteristics. The general combining ability variance component was also predominant for stem girth and susceptibility to leaf rust, although effects due to interaction were detected. Some hybrid combinations had yield comparable to standard clonal varieties. The implications of such results for breeding of Coffea canephora are discussed. Particularly, the development of F₁ hybrid varieties is envisaged.     

Simulations and practical problems of applying multiple marker assisted selection and doubled haploids to wheat breeding programs

Marker assisted selection (MAS) and wheat doubled haploids (DH) are relatively new technologies, recently applied to wheat breeding programs. Simulations demonstrate that DHs increase the efficiency of MAS, and offer faster strategies for combining large numbers of genes with a minimum number of marker tests. When small numbers of marked loci (1-3) are selected simultaneously, selection of DH progeny is 5-6 times more efficient than selecting F₄ derived families. Combining 4-8 marked loci, screening of F₂ plants and using only those plants homozygous or segregating for all of the marked loci as parents for DH production (10-31% of F₂ plants) is 3-10 times as efficient as using F₁ plants. A number of protocols have been proposed involving sib-matings and selection to fix some genes, with further selection in the second generation to improve the proportion of useful DH lines. In one scheme (recombinant F₂ selection) all F₂ plants, either homozygous or heterozygous for the marked alleles, are intercrossed at random and the recurrent F₁ plants still having these alleles are used for DH production. An alternative strategy (recurrent DH selection) is to select from an initial DH population and intercross those lines having most favourable marked loci with a second cycle of DHs to fix all favourable marked loci. Combining more than 12 marked gene loci does not seem feasible, due to the very large numbers of F₂s (>2000) required. This has implications when using MAS for quantitative trait loci, where many minor gene loci would have to be combined. Direct selection for some multi-genic quantitative traits amongst the DH lines may be more efficient than using MAS where recurrent selection is used. At the Cereal Research Centre, the practical problems of using these protocols as part of the spring wheat breeding program are being evaluated. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of two crossing and four selection schemes for yield, yield traits, and slow rusting resistance to leaf rust in wheat

The most important breeding objectives in crop improvement are improving grain yield, grain quality, and resistances to various biotic and abiotic stresses. The objectives of our study were to compare two crossing and four selection schemes for grain yield, yield traits, and slow rusting resistance to leaf rust (Puccinia recondita) based on additive genes in wheat (Triticum aestivum), and to identify the most efficient crossing and selection methodologies in terms of genetic gains and cost efficiency. Segregating populations were derived from 18 simple crosses and the same number of top (three-way) crosses. Half of the crosses were derived from Yecora 70 and the other half from Veery #10 as the common leaf rust susceptible parents. The four selection schemes were: pedigree, modified bulk (F₂ and F₁-top as pedigree, selected lines in F₃, F₄, F₂-top, F₃-top as bulk; and pedigree in F₅ and F₄-top populations), selected bulk (selected plants in F₂, F₃, F₄, F₁-top, F₂-top and F₃-top as bulk; and pedigree in F₅ and F₄-top populations), and nonselected bulk (bulk in F₂, F₃, F₄, F₁-top, F₂-top and F₃-top; and pedigree in F₅ and F₄-top populations). A total of 320 progeny lines, parents and checks were tested for grain yield, other agronomic traits and leaf rust resistance during the 1992/93 and 1993/94 seasons in Ciudad Obregon (Sonora State, Mexico) which represents a typical high yielding irrigated site. The influence of the type of cross and the selection scheme on the mean grain yield and other traits of the progenies was minimal. The selection of parents was the most important feature in imparting yield potential and other favourable agronomic traits. Moreover, the highest yielding lines were distributed equally. Progeny lines derived from Veery #10 crosses had significantly higher mean grain yield compared to those derived from the Yecora 70 crosses. Furthermore, a large proportion of the highest yielding lines also originated from Veery #10 crosses. Mean leaf rust severity of the top cross progenies was lower than that of the simple cross progenies possibly because two parents contributed resistance to top cross progenies. Mean leaf rust severity of the nonselected bulk derivatives was twice that of lines derived from the other three schemes. Selected bulk appears to be the most attractive selection scheme in terms of genetic gains and cost efficiency. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of Spring Barley Lines Produced by Single Seed Descent, Pedigree Inbreeding and Doubled Haploidy

Samples of inbred lines of Hordeum vulgare produced by pedigree inbreeding, doubled haploidy and single seed descent have been compared for a range of agronomic characters. Differences between the means of the in bred s produced by single seed descent and pedigree breeding were due to differing levels of inbreeding. The present study confirmed chat recombinant inbred families which out-perform the higher scoring parent may be produced by either method. Caution should, however, be exercised when simply inherited characters controlled by major genes are fixed in early generations. Differences between the doubled haploid and single seed descent samples for the character thousand grain weight were attributable to coupling linkages involving epistatic genes. The production and evaluation of random inbred lines should be considered as an alternative to pedigree methods of barley breeding.     

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     

玉米DH系的表现及其同工酶电泳分析

以孤雌生殖诱导系高诱1号诱导的单倍体植株为材料,由单倍体加倍后获得的DH系性状上表现高度一致,其中一些DH系的长势和果穗性状达到了育种上的要求.DH系的过氧化物酶和酯酶同工酶电泳表明,部分DH系具有母本谱带,部分具有父本谱带,由此说明,孤雌生殖诱导系诱导单倍体具有一定的随机性.     

Doubled Haploids of Cocoa (Theobroma cacao L.) 2. Observations of Monogenic and Polygenic Characters

The expression of monogenic and polygenic characters has been studied in families of spontaneous haploids of cocoa which nave been doubled with colchicine. Segregation of six enzyme markers, heterozygous in the various parent clones, has been analysed in the doubled haploids for alcohol dehydrogsnase (ADH), malate dehydrogenase locus B (MDH-B), isocitrate dehydrogenase (IDH), acid phosphatase (ACP), phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI). Nine of the eleven families showed a Mendelian-type segregation, two presented distortions but all alleles were found in the haploid progenies. Polygenic characters were expressed with great variability: A few DHs showed similar or even higher values than their parents for individual characters, but the majority were lower, and large depressions could be seen for the group of characters studied. These results have consequences for the use of doubled haploids of cocoa which must both give a sure production of hybrid seeds and be good progenitors; no prediction of their value as progenitors seems possible after observation of their own characteristics and the only selection made from these plants will be a propos their characters directly involved in seed production. These selected doubled haploids will subsequently have to be tested as progenitors.     

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.     

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).     

Factors affecting oat haploid production following oat × maize hybridization

Doubled haploids (DHs) are becoming increasingly important in crop breeding programmes but methods for producing oat DHs remain inefficient. In this study haploid and DH oat plants were produced using the oat × maize hybridization method. Factors influencing the rate of caryopsis and haploid embryo production including genotype, post‐pollination plant growth regulator application and temperature were investigated. The four growth regulators tested showed significant differences in their capacity to induce caryopsis formation with dicamba producing the highest numbers of caryopses, followed by picloram, 2,4‐dichlorophenoxyacetic acid (2,4‐D) and gibberellic acid (GA₃). No significant differences were observed between these growth regulators for their effect on embryo production. The concentration of dicamba was also important and was found to influence caryopsis but not embryo production, with 50 and 100 mg/l dicamba producing significantly more caryopses than 25 or 5 mg/l. Temperature had a significant impact on both caryopsis and embryo production with the magnitude and direction of response depending on genotype. Rates of haploid embryo production observed were between 0.8% and 6.7% of the pollinated florets. The proportion of haploids, which survived and were successfully doubled with colchicine following transfer to soil was between 72% and 81%.     

Evaluation of quantitative resistance to yellow rust (Puccinia striiformis f. sp. hordei) in the ICARDA/CIMMYT barley breeding programme

The resistance to yellow rust (Puccinia striiformis f. sp. hordei) of 500 advanced barley lines from the ICARDA/CIMMYT breeding programme in Mexico was evaluated on seedlings in the greenhouse and on adult plants in the field. A high frequency of advanced lines (85.8%) showed a susceptible reaction (infection type ≥ 7) on seedlings after inoculation with isolate Mex-1, representing a Mexican variant of race 24. This indicates the absence of effective hypersensitive resistance. In addition, the same advanced lines showed a large variation in disease severity in the field, ranging from 0 to 95%. More than 76% of the advanced lines with a susceptible reaction in the seedling stage demonstrated low disease severity (10% or less in the adult plant). Consequently, these advanced lines possess high levels of quantitative resistance. Two aspects in the ICARDA/CIMMYT barley breeding programme may explain the large number of advanced lines with high levels of quantitative resistance. First, a recurrent selection approach is applied when advanced (F₅) lines reaching homozygosity are intercrossed. Second, low levels of disease are accepted in the selection process instead of selecting the ultimate green plant. Both aspects combined allow the accumulation of quantitative resistance. Certain cultivars released from South-American national programmes in the late 1970s and early 1980s in Peru (UNA-80), Bolivia (IBTA 80) and Ecuador (Teran) are still resistant, demonstrating the durable nature of quantitative resistance to yellow rust.     

Rapid introduction of disease resistance from rye into common wheat by anther culture of a 6x triticale × nulli-tetrasomic wheat

Powdery mildew (caused by Erysiphe graminis) and yellow rust (caused by Puccinia striiformis) are the two most serious wheat diseases found in China. Rye chromosomes, carrying genes for resistance to these diseases, were introduced into common wheat in two generations using chromosome engineering and anther culture. The F₁ hybrids from a cross involving a hexaploid triticale (×Triticosecale Wittmack) בChinese Spring’ nulli‐tetrasomic N6DT6A wheat aneuploid line were anther cultured and doubled‐haploid plants were regenerated. Using genomic in situ hybridization, C‐banding and biochemical marker analyses, one of the anther‐cultured lines (ZH‐1)studied in detail, proved to be a doubled‐haploid with one rye chromosome pair added (1R) and a homozygous 6R/6D substitution (2n= 44). The line was tested for expression of disease resistance and found to be highly resistant to powdery mildew and moderately resistant to yellow rust.     

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.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.