Ploidy of broccoli regenerated from microspore culture versus anther culture

The use of microspore or anther culture to generate doubled-haploids (DH) is an important adjunct to broccoli breeding) Regenerated populations from broccoli anther culture are usually mixtures of ploidy. However, ploidy composition of populations derived from microspore culture has not been reported. The purpose of the present study was to characterize regenerants derived from microspore culture, to evaluate factors influencing these characteristics and to compare results with those from anther culture. Eight populations, four from each culture method, were generated simultaneously using the same four F₁ hybrids as donor parents. The ploidy level of all regenerants was determined by DNA flow cytometry: the majority of them were diploid. As in anther culture, a mixture of ploidy was observed in all populations derived from microspore culture. Ploidy variation was more frequent among clonal families from anther culture (10%) than microspore culture (5%)‘Everest’ was the most productive donor parent with both methods, while ‘Greenbelt’ and ‘Major’ were least productive in anther and microspore culture, respectively. Genotype specificity for the total number of regenerated plants and ploidy composition occurred in both culture methods.     

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.     

Effect of parental genotypes and colchicine treatment on the androgenic response of wheat F1 hybrids

The effect of the parental genotypes and colchicine treatment on the androgenic response of wheat (Triticum aestivum L.) F1 hybrids was studied. For this, anthers from three F1 hybrids and their parents were cultured on W14 initiation medium and W14 supplemented with 0.03% colchicine. The number of responding anthers, microspore‐derived structures/100 anthers, green plants/embryos cultured, green plants/100 anthers and albino plants/100 anthers were recorded. It was observed that embryo formation and plant regeneration ability were genetically controlled and genotype dependent. In both treatments the variety Kavkaz had a significantly higher percentage of responding anthers, microspore‐derived structures and green plants/100 anthers than the other genotypes. On the other hand, the variety Myconos also demonstrated high microspore‐derived structure production and green plant regeneration when treated with colchicine. The good response observed in these two varieties indicates the importance of colchicine treatment only for certain genotypes. Green plant production capacity of the hybrids was intermediate to that of the parental varieties. As one parent with a high or even an intermediate response to anther culture could lead to the production of sufficient (for breeding purposes) green plants from the F₁ hybrids, it was concluded that screening the inbred lines for the response to anther culture with and without colchicine treatment could contribute to utilization of breeding material with a low response to anther culture via the proper hybrid combinations.     

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.     

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.     

QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice

Anther culturability of rice is a quantitative trait controlled by nuclear‐encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F₂ populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F₂ populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

Inheritance of coffee leaf rust resistance and identification of AFLP markers linked to the resistance gene

The most important disease of Coffea arabica is coffee leaf rust caused by the fungus Hemileia vastatrix. The purpose of this study was to characterize the inheritance of coffee resistance gene(s) to race II of this pathogen and to identify and map molecular markers linked to this trait. Different populations were used: F₂ (160 plants), BCr (20), and BCs (135), derived from a cross between the resistant genotype Híbrido de Timor UFV 427-15 and the susceptible cultivar Catuaí Amarelo UFV 2143-236 (IAC 30). The segregation analysis showed that the resistance of Híbrido de Timor to race II of the H. vastatrix is conferred by a single dominant gene. The amplification of 176 AFLP (Amplified fragment length polymorphism) primer combinations using bulked segregant analysis (BSA) allowed the identification of three molecular markers linked to the resistance gene. Genetic mapping of these three markers in the F₂ population indicated that they are distributed on both sides, flanking the resistance gene. The markers E.CTC/M.TTT405 and E.CGT/M.TGT300 were found linked to the resistance gene at 8.69 cM (LOD 18.91) and 25.10 cM (LOD 5.37), respectively, while E.CCT/M.TTC230 was localized on the other side of the gene, at 20.50 cM (LOD 6.15). These markers are the first rust resistance markers identified in Híbrido de Timor and can be useful for marker assisted selection in coffee breeding programs.     

Mapping of powdery mildew and leaf rust resistance genes on the wheat-rye translocated chromosome T1BL·1RS using molecular and biochemical markers

Powdery mildew and leaf rust resistance genes on the 1RS arm of the T1BL·1RS translocated chromosome were mapped in relation to the Sec‐1 locus and AFLP and restriction fragment length polymorphism markers, respectively, employing segregating F₃ populations. Integration of molecular markers indicated that Pm17 lies between the Lr26 and Sec‐1 loci, with both resistance genes allocated distally to the Sec‐1 locus in the satellite of the 1RS arm.     

DNA marker-assisted selection to pyramid rust resistance genes in “carioca” seeded common bean lines

This work reports a gene pyramiding approach assisted by DNA markers used to develop “carioca” seeded common bean (Phaseolus vulgaris L.) elite lines harboring three different rust resistance genes. Rust is among the most destructive diseases that attack P. vulgaris and cause serious damage worldwide. The rust resistance genes Ur-5 (from ‘Mexico 309’), Ur-11 (from ‘BelMiDak RR-3’), and Ur-14 (from ‘BRS Pioneiro’, a “carioca” seeded cultivar derived from the resistance source ‘Ouro Negro’) were combined in the “carioca” seeded bean cultivar ‘Rudá’. Firstly, two different backcross programs were conducted separately to produce progenies harboring individually the Ur-5 and Ur-11 genes. Molecular fingerprinting analysis was used to select plants genetically similar to ‘Rudá’ in the backcross cycles to accelerate the recurrent-background recovery. The obtained progenies were initially intercrossed and then crossed with ‘BRSMG Pioneiro’ (Ur-14). The final F₁ plants derived from these crosses were screened with DNA markers linked to the three rust resistance genes: SI19 (Ur-5), SAE19 (Ur-11) and OPX11 (Ur-14). The plants selected as harboring all the alleles of interest were used to obtain the next generations. The selection based on DNA markers was conducted up to the F_4:5 generation. We were able to select F_4:7 progenies showing all the DNA markers associated to the genes of interest and resistant to all specific races of U. appendiculatus used for phenotypically detecting each one of the rust resistance genes. Yield evaluations show that these selected lines are as productive as the recurrent parent ‘Rudá’ and high-performing control cultivars grown in Brazil.     

Molecular mapping of a stripe rust resistance gene in wheat cultivar Wuhan 2

Stripe rust (or yellow rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat worldwide. Growing resistant cultivars is the best approach to control the disease. To identify and map genes for stripe rust resistance in wheat cultivar ‘Wuhan 2', an F₂ population was developed from a cross between the cultivar and susceptible cultivar Mingxian 169. The parents, 179 F₂ plants and their derived F_2:3 lines were evaluated for responses to Chinese races CYR30 and CYR31 of the pathogen in a greenhouse. A recessive gene for resistance was identified. DNA bulked segregant analysis was applied and resistance gene analog polymorphism (RGAP) and simple sequence repeat (SSR) techniques were used to identify molecular markers linked to the resistance gene. A genetic map consisting of five RGAP and six SSR markers was constructed. The recessive gene, designated Yrwh2, was located on the short arm of chromosome 3B and flanked by SSR markers Xwmc540 and Xgwm566 at 5.9 and 10.0 cM, respectively. The chromosomal location of the resistance gene and its close marker suggest that the locus is different from previously reported stripe rust resistance genes Yr30, QYr.ucw-3BS, Yrns-B1, YrRub and QYrex.wgp-3BL previously mapped to chromosome 3B. Yrwh2 and its closely linked markers are potentially useful for developing stripe rust resistance wheat cultivars if used in combination with other genes.     

Association of leaf rust and powdery mildew resistance in a recombinant derived from a Hordeum vulgare × Hordeum bulbosum hybrid

While studying powdery mildew resistance in a recombinant line (code 81882) derived from a Hordeum vulgare (cv. ‘Vada’) ×Hordeum bulbosum hybrid, a low infection type of resistance to leaf rust was observed. To determine the mode of inheritance of the leaf rust resistance and whether there was linkage between the two resistances, F₂ and F₃ progenies from crosses between 81882 and ‘Vada’ were inoculated with the leaf rust and powdery mildew pathogens. Southern blots were prepared using restricted DNA extracted from leaves of 82 F₂ plants and four chromosome 2HS sequences were hybridized with the blots to define the length of the introgression. The leaf rust resistance appears to be inherited as a single dominant gene on chromosome 2HS, which co-segregates with the powdery mildew resistance. There was an almost complete association between the resistances and the respective molecular markers, but it is likely that the strong linkage results from the frequent inheritance of the introgressed H. bulbosum DNA as an intact segment of chromatin with only low levels of recombination within the segment.     

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F₂ populations were developed by self-pollinating 3 black rot resistant F₁ plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F₂ plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F₂ plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of partial resistance to coffee leaf rust ( Hemileia vastatrix Berk & Br.) introgressed into the cultivated Coffea arabica L. from the diploid C. canephora species

The objective of this study was to identify polymorphic molecular markers associated with partial resistance to coffee leaf rust, Hemileia vastarix. A segregating F ₂ population derived from a cross between the susceptible Coffea arabica cv. Caturra and a C. canephora-introgressed Arabica line exhibiting high partial resistance was analyzed. Rust resistance measured as rust incidence (RI) and defoliation (DEF) was evaluated in field conditions in three consecutive years (2003–2005). During the 2003 season, which was characterized by favorable conditions for a rust epidemic, the F ₂ plants exhibited different levels of resistance ranging from very susceptible (50.1% for DEF and 49.5% for RI) to highly partial resistance (9.1% for DEF and 3.7% for RI). Molecular analysis enabled identification of seven polymorphic markers (5 AFLP and 2 SSR) exhibiting significant association with partial resistance. Coexistence of resistance homozygous alleles (RR) at codominant SSR loci was correlated with high resistance. This study is the first attempt to develop PCR-based sequence specific markers linked to partial rust resistance in coffee.     

Development of SCAR markers for identification of stem rust resistance gene Sr31 in the homozygous or heterozygous condition in bread wheat

The stem rust resistance gene Sr31, transferred from rye (Secale cereale) into wheat (Triticum aestivum L.) imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) found in India. Wheat genotypes including carriers and non‐carriers of the Sr31 gene were analysed using arbitrary primed polymerase chain reaction (AP‐PCR). AP‐PCR markers viz. SS30.2_580(H) associated with the Sr31 gene and SS26.1₁₁₀₀ associated with the allele for susceptibility were identified. Linkage between the markers and phenotypes was confirmed by analysing an F₂ population obtained from a cross between a resistant and a susceptible genotype. The markers were tightly linked to the respective alleles. Both the AP‐PCR markers were converted into sequence characterized amplified region (SCAR) markers, viz. SCSS30.2₅₇₆ and SCSS26.1₁₁₀₀ respectively. The markers were validated in two more segregating populations and 49 wheat genotypes. Using both markers it was possible to distinguish the homozygous from the heterozygous carriers of the Sr31 gene in the F₂ generation. The markers developed in this study can be used for pyramiding of the Sr31 gene with other rust resistance genes and in marker‐assisted selection.     

Glucosinolates Determined by HPLC in the Seeds of Microspore-Derived Homozygous Lines of Rapeseed (Brassica napus L.)*

Microspore culture was employed to measure the relative efficiencies of anther culture and isolated microspore culture for the regeneration of embryoids and plants of Brassica napus. The yield of embryoids and plants was at least 10-fold greater from isolated microspores than from anther cultures. Approximately 1400 microspore-derived homozygous line's, the parental varieties and the corresponding F₂ plants were grown in a field trial. Important agricultural characteristics, such as morphological homogeneity, growth rate, onset of flowering and seed setting were evaluated subjectively and seed yield and glucosinolate content of individual plants were determined. The relative concentrations of up to S different glucosinolates in these seeds were measured via an automated high performance liquid chromatography (HPLC) system. The alkenyl and indole glucosinolates, the two most important categories of glucosinolates, were found in varying proportions and were independently determined in these line's. Our results do not support the previously suggested connection between low concentrations of glucosinolates and weak growth and/or poor seed yield. Additionally, no evidence was found that the lines derived from isolated microspore culture were subjected to unexpected selection pressures that might adversely affect the diversity of the lines obtained. These results demonstrate that microspore culture is a powerful tool not only for genetic analysis bur also for practical plant breeding.     

Genetic basis of seedling-resistance to leaf rust in bread wheat 'Thatcher'

The bread wheat cultivar ‘Thatcher’ is documented to carry the gene Lr22b for adult‐plant resistance to leaf rust. Seedling‐resistance to leaf rust caused by Puccinia triticina in the bread wheat cultivar ‘Thatcher’, the background parent of the near‐isogenic lines for leaf rust resistance genes in wheat, is rare and no published information could be found on its genetic basis. The F₂ and F₃ analysis of the cross ‘Agra Local’ (susceptible) × ‘Thatcher’ showed that an apparently incompletely dominant gene conditioned seedling‐resistance in ‘Thatcher’ to the three ‘Thatcher’‐avirulent Indian leaf rust pathotypes – 0R8, 0R8‐1 and 0R9. Test of allelism revealed that this gene (temporarily designated LrKr1) was derived from ‘Kanred’, one of the parents of ‘Thatcher’. Absence of any susceptible F₂ segregants in a ‘Thatcher’ × ‘Marquis’ cross confirmed that an additional gene (temporarily designated LrMq1) derived from ‘Marquis’, another parent of ‘Thatcher’, was effective against pathotype 0R9 alone. These two genes as well as a second gene in ‘Kanred’ (temporarily designated LrKr2), which was effective against all the three pathotypes, but has not been inherited by ‘Thatcher’, seem to be novel, undocumented leaf rust resistance genes.     

小麦抗病品系5R625抗叶锈病基因的分子鉴定

小麦品系5R625苗期和田间均对小麦叶锈病有良好抗性,但其所携带的抗病基因还不清楚。利用36个携带已知抗叶锈病基因的对照品系和15个中国小麦叶锈菌小种对5R625携带的抗病基因进行了苗期人工接种鉴定和基因推导,结果 5R625对这15个叶锈菌生理小种的侵染型与Lr9、Lr19、Lr24、Lr28、Lr39、Lr47、Lr51、Lr53相同。利用5R625和感病品种郑州5389的杂交后代F1、F2和F2:3群体对5R625的抗病性进行了遗传分析,苗期和成株期的分析结果均表明5R625对小麦叶锈菌的抗性由1个显性基因控制。进一步利用F2:3家系和分子标记方法将该基因定位在3DL染色体上。与5R625携带的抗病基因连锁的5个分子标记中,STS标记24-16和SCAR标记OP-J09此前已经被证明与已知抗叶锈病基因Lr24共分离,因此,推测5R625携带的抗病基因与Lr24可能为同一基因。     

Leaf and stem rust resistance of hexaploid wheat cultivars salzmünder bartweizen and weique

Summary Wheat cultivars Salzmünder Bartweizen and Weique gave a similar pattern of reaction type when tested with 27 different races of stem rust and 17 races of leaf rust from Canada and Czechoslovakia. Both cultivars were resistant to all races of stem rust and most of the races of leaf rust. Genetic studies indicated that the two varieties have the same genes for rust resistance, with the genes for leaf and stem rust being closely linked.Transmission of the genes for rust resistance was irregular and appeared to be associated with meiotic irregularities in chromosome pairing. In all F₂ populations which deviated significantly from a 3:1 ratio, a deficiency of resistant plants was noted. The greatest deficiency was in the F₂ of crosses involving Salzmünder Bartweizen, of which the F₁ hybrid also had the greatest meiotic irregularities.Chimeral plants, of which the longitudinal half of the leaf showed a resistant reaction and the other half a susceptible reaction, were observed in crosses with Salzmünder Bartweizen.     

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.