Use of asynchrony in flowering for easy and economical polyhaploid induction in wheat following Imperata cylindrica‐mediated chromosome elimination approach

Wheat × Imperata cylindrica‐mediated approach of doubled haploidy breeding requires hand emasculation followed by pollination with I. cylindrica pollen. The pace of this endeavour can be enhanced by utilizing asynchronous flowering of wheat spikes by direct pollination without emasculation followed by morphological marker–assisted screening of selfed and crossed seeds. The emasculated and un‐emasculated spikes of 13 spring and six winter wheat genotypes and two triticale × wheat derivatives were pollinated with I. cylindrica pollen. The response of different genotypes for production of crossed and selfed seeds with direct pollination varied significantly within and between groups for spring and winter wheats, whereas triticale × wheat derivatives responded similarly to each other but significantly different from spring and winter wheats. Although, the proportion of pseudoseed formation was lower in case of direct pollination, yet in some genotypes, it was comparable to that of pollination after emasculation. Moreover, the response for haploid embryo induction frequency was similar in both the cases. The method of direct pollination can be utilized for easy and economical induction of haploids.     

Efficient haploid induction in wheat by using pollen of Imperata cylindrica

Intergeneric hybridization between wheat ( Triticum aestivum L.) and a wild weedy species, Imperata cylindrica (2n = 20) resulted in the recovery of a high frequency of wheat haploids, which were obtained through the elimination of I. cylindrica chromosomes. Cytological analysis of the root tips revealed the somatic chromosome count of the regenerants equalled 21. Haploid regenerants were also obtained in all the crosses of wheat F1s with I. cylindrica , implying the genotype nonspecific nature of wheat x I. cylindrica hybridization. Variation among wheat F1 hybrids was observed with respect to seed formation (44.9-84.5%), embryo formation (15.1-47.7%) and regeneration (27.0-75.0%) in crosses with I. cylindrica. Comparisons based on the efficiency of I. cylindrica and maize ( Zea mays ) as pollen sources indicated that Imperata-mediated haploid production is equally efficient. There is natural coincidence of flowering period of I. cylindrica with that of wheat under sub-temperate conditions which is advantageous compared with maize, which cannot be grown during the winter season in the sub-temperate regions.     

Review of doubled haploid production in durum and common wheat through wheat × maize hybridization

Production of doubled haploids (DHs) is an important methodology to speed the process of breeding and development of mapping populations in crops. The procedure for DH production includes two major steps: haploid induction and chromosome doubling. In recent years, wide hybridization between wheat and maize has become a main approach for haploid production in wheat. In this method, the maize chromosomes are completely eliminated during the early development of the hybrid seeds after wheat spikes were pollinated with maize pollen. Numerous wheat cultivars and mapping populations have been developed using wheat–maize hybridization. In this study, we review the procedures of DH production of durum and common wheat via wide hybridization with maize, the factors which affect the efficiency of DH production, and the mechanism of selective elimination of the maize genome during the early development of the hybrid embryos. We also report a highly efficient protocol for DH production in durum and common wheat, which was established based on the optimal conditions for each of the factors that affect the efficiency of DH production.     

Germplasm enhancement of maize: a look into haploid induction and chromosomal doubling of haploids from temperate‐adapted tropical sources

The allelic diversity (AD) project of the Germplasm Enhancement of Maize (GEM) programme utilized the double haploid (DH) breeding method to expedite development and release of lines derived from 300 exotic maize races. Using 18 races in this study, differential effects on haploid induction rates (HIRs) and doubling rates (DRs) by the recurrent parents PHB47 and PHZ51, the elevation that the race is traditionally grown at, and by the race itself were addressed in this study. Races from the AD project were grouped by elevation of their origin, high, middle or low altitude. Six races per elevation were randomly selected and backcrossed using both recurrent parents to generate 36 populations. Ten replications were randomized in a complete randomized design for two growing seasons. The recurrent parent effect was significant, with PHB47 having a higher HIR than PHZ51. Effect of elevation was significant with higher HIR associated with low‐elevation origin, and race also proved to be significant. Effects of elevation, recurrent parent and race were not significant for DR.     

Assessing diversity in Triticum durum cultivars and breeding lines for high versus low cadmium content in seeds using the CAPS marker usw47

Cadmium (Cd) is a toxic heavy metal that occurs naturally in soils. Durum wheat is known to accumulate generally more Cd than other cereal crops. The uptake of Cd in durum wheat is governed by the gene Cdu1, which co‐segregates with several DNA markers, such as the co‐dominant marker usw47 and the dominant marker ScOPC20. A panel of 314 durum wheat cultivars or lines originating from 16 countries or regions were assessed with usw47. The plant material was mainly comprised of cultivars and modern breeding lines. From this set, 165 durum wheat lines were classified as low‐Cd accumulators, 144 high‐Cd accumulators and five were heterogeneous. A smaller subset of 16 cultivars had previously been evaluated for Cd accumulation in replicated field trials. Lines with the high‐Cd allele showed a 2.4‐fold higher Cd content in the seeds than lines with the low‐Cd allele. We also compared the utility of markers usw47 and ScOPC20 as selection tools. Marker‐assisted selection appears as a robust and practicable tool for breeding durum cultivars with low‐Cd content.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.     

Relative efficiency of different Gramineae genera for haploid induction in triticale and triticale x wheat hybrids through the chromosome elimination technique

The study was undertaken to evaluate the relative efficiency of different Gramineae genera for haploid induction in triticale (x Triticosecale) and triticale × wheat (Triticum aestivum) hybrids through the chromosome elimination (wheat × maize, Zea mays) system. Eight intergenotypic triticale and 15 triticale x wheat crosses were subjected to hybridization with nine different Gramineae genera viz., Z. mays, Sorghum bicolor, Pennisetum americanum, Setaria italica, Festuca arundinacea, Imperata cylindrica, Cynodon dactylon, Lolium temulentum and Phalaris minor in two separate experiments. This was followed by in vivo auxin treatment of the crossed spikes and subsequent rescue of the haploid embryos to regenerate green haploid plantlets. All the triticale and triticale x wheat crosses resulted in seed set in variable frequencies when hybridized with maize, I. cylindrica, pearl millet and sorghum. Seed set was also obtained with S. italica, F. arundinacea and P. minor in a few crosses in both groups. In general, all the triticale x wheat crosses, except for one in each case, resulted in embryo formation and green haploid plantlet regeneration when hybridizations were carried out with maize and I. cylindrica. However, the latter outperformed the former in embryo formation (25.48% vs. 20.0%) and regeneration (34.17% vs. 15.10%) frequencies, the differences being significant for regeneration frequencies. In the case of triticale hybrids, no significant differences between maize and I. cylindrica were observed for the three parameters of haploid induction. Embryo formation and regeneration were also observed in some of the triticale as well as triticale × wheat F₁ hybrids when hybridized with sorghum and pearl millet.     

Evaluation of a semi‐controlled test as a selection tool for frost tolerance in durum wheat (Triticum durum)

Most durum wheat (Triticum durum) varieties possess only low winter hardiness due to their frost susceptibility. In North America and Central Europe, durum wheat is therefore typically sown in spring to circumvent the local winter conditions. However, the yield potential of durum in these regions could be much better exploited if durum varieties with increased frost tolerance were available, which could be sown in autumn. A factor limiting breeding for increased frost tolerance is the variation in the occurrence of frost stress across years. The ‘Weihenstephaner Auswinterungsanlage’ is a semi‐controlled test that exposes the plants to all weather conditions. Snow coverage of the plants, serving as frost protection, is prevented by the movable glass lid of the semi‐controlled test. In this study, different scorings for frost tolerance based on this semi‐controlled test were evaluated and compared with frost tolerance data in the field. Our results illustrate the potential of the ‘Weihenstephaner Auswinterungsanlage’ as an indirect selection tool for frost tolerance in durum breeding programmes, especially when regular frost tolerance data from the field are not available.     

Influence of winter and spring wheat genetic backgrounds on haploid induction parameters and trait correlations in the wheat × maize system

The interactive influence of winter and/or spring wheat genetic background on haploid induction parameters and trait correlation was studied by hybridizing five elite and diverse genotypes each of winter and spring wheat and their F₁s (winter × winter, spring × spring, and winter × spring, generated in a diallel design excluding reciprocals) with a single genotype of maize. Data were recorded with respect to per cent seed formation, embryo formation, and regeneration. High genetic variability was present among the wheat genotypes (parents + F₁s) for the three haploid induction parameters. Significant differences were obtained within and between different groups viz., spring wheats, winter wheats, spring × spring wheats, winter × winter wheats, and winter × spring wheats with respect to the three haploid induction parameters based on ANOVA. The winter genotypes (winter parents and winter × winter wheat hybrids) responded better than the spring groups (spring wheat parents, spring × spring and winter × spring wheat hybrids) with respect to embryo formation and winter × spring wheat hybrids yielded significantly the highest numbers of regenerants. Correlation studies amongst the haploid induction parameters indicated that the genes controlling seed formation and haploid plantlet regeneration are negatively correlated when the genetic backgrounds of both ecotypes are combined in winter × spring hybrids. Haploid embryo formation had no association with seed formation and regeneration in all genetic backgrounds, suggesting independent inheritance.     

S‐locus diversity and cross‐compatibility of wild Prunus avium for timber breeding

Prunus avium is primarily cultivated for its fruit, sweet cherries. However, it is also used to produce high‐quality timber. In a P. avium seed orchard, gametophytic self‐incompatibility is a restriction for free pollen flow and should be considered when establishing basic forest materials. In this study, S‐locus diversity and cross‐incompatibility of wild cherry individuals in clonal banks established for breeding for timber production were investigated. Wild cherry trees (140) with outstanding forest growth habit, collected in northern Spain, grafted and planted in two clonal banks, were genotyped at the S‐locus. The self‐incompatibility S‐locus genes, S‐RNase and SFB, were analysed by PCR. Twenty‐two S‐haplotypes, resulting in 72 different S‐genotypes, were identified. The genotypes were grouped into 33 incompatibility groups and 39 unique genotypes. This initial S‐locus analysis revealed large genetic diversity of wild cherry trees from the Spanish northern deciduous forest, and provides useful information for seed orchard design. Wild P. avium displays significantly more genetic diversity than what is detected in local cultivars, revealing a narrowing of genetic diversity during local domestication.     

Survey and new insights in the application of PCR‐based molecular markers for identification of HMW‐GS at the Glu‐B1 locus in durum and bread wheat

Wheat, among all cereal grains, possesses unique characteristics conferred by gluten; in particular, high molecular weight glutenin subunits (HMW‐GS) are of considerable interest as they strictly relate to bread‐making quality and contribute to strengthening and stabilizing dough. Thus, the identification of allelic composition, in particular at the Glu‐B1 locus, is very important to wheat quality improvement. Several PCR‐based molecular markers to tag‐specific HMW glutenin genes encoding Bx and By subunits have been developed in recent years. This study provides a survey of the molecular markers developed for the HMW‐GS at the Glu‐B1 locus. In addition, a selection of molecular markers was tested on 31 durum and bread wheat cultivars containing the By8, By16, By9, Bx17, Bx6, Bx14 and Bx17 Glu‐B1 alleles, and a new assignation was defined for the ZSBy9_aF1/R3 molecular marker that was specific for the By20 allele. We believe the results constitute a practical guide for results that might be achieved by these molecular markers on populations and cultivars with high variability at the Glu‐B1 locus.     

The potential of genomic‐assisted breeding to improve Fusarium head blight resistance in winter durum wheat

Durum wheat is the most important tetraploid wheat mainly used for semolina and pasta production, but is notorious for its high susceptibility to Fusarium head blight (FHB). Our objectives were to identify and characterize quantitative trait loci (QTL) in winter durum and to evaluate the potential of genomic approaches for the improvement of FHB resistance. Here, we employed an international panel of 170 winter and 14 spring durum lines, phenotyped for Fusarium culmorum resistance at five environments. Heading date, plant height and mean FHB severity showed significant genotypic variation with high heritabilities and FHB resistance was negatively correlated with both heading date and plant height. The dwarfing gene Rht‐B1 significantly affected FHB resistance and the genome‐wide association scan identified eight additional QTL affecting FHB resistance, explaining between 1% and 14% of the genotypic variation. A genome‐wide prediction approach yielded only a slightly improved predictive ability compared to marker‐assisted selection based on the four strongest QTL. In conclusion, FHB resistance in durum wheat is a highly quantitative trait and in breeding programmes may best be tackled by classical high‐throughput recurrent phenotypic selection that can be assisted by genomic prediction if marker profiles are available.     

Maize participatory breeding in Portugal: Comparison of farmer's and breeder's on‐farm selection

“VASO” is a Portuguese participatory maize breeding project (1984), where several maize landraces such as “Pigarro” have been selected both by a farmer's (phenotypic recurrent selection) and a breeder's approach (S2 lines recurrent selection). The objectives of this study were to determine the phenotypic and genotypic responses to participatory selection using these two different approaches, to clarify to which extent both selection methods preserve genetic diversity, and conclude what is the preferred method to apply in sustainable farming systems. The results, obtained via ANOVA, regression analyses and molecular markers, indicate that for both selection methods, genetic diversity was not significantly reduced, even with the most intensive breeder's selection. Although there were some common outputs, such as the determinated versus indeterminated ears, cob and ear weight ratio per ear and rachis 2, specific phenotypic traits evolved in opposite directions between the two selection approaches. Yield increase was only detected during farmer selection, indicating its interest on PPB. Candidate genes were identified for a few of the traits under selection as potential functional markers in participatory plant breeding.     

QTL mapping for six ear leaf architecture traits under water‐stressed and well‐watered conditions in maize (Zea mays L.)

Morphological traits for ear leaf are determinant traits influencing plant architecture and drought tolerance in maize. However, the genetic controls of ear leaf architecture traits remain poorly understood under drought stress. Here, we identified 100 quantitative trait loci (QTLs) for leaf angle, leaf orientation value, leaf length, leaf width, leaf size and leaf shape value of ear leaf across four populations under drought‐stressed and unstressed conditions, which explained 0.71%–20.62% of phenotypic variation in single watering condition. Forty‐five of the 100 QTLs were identified under water‐stressed conditions, and 29 stable QTLs (sQTLs) were identified under water‐stressed conditions, which could be useful for the genetic improvement of maize drought tolerance via QTL pyramiding. We further integrated 27 independent QTL studies in a meta‐analysis to identify 21 meta‐QTLs (mQTLs). Then, 24 candidate genes controlling leaf architecture traits coincided with 20 corresponding mQTLs. Thus, new/valuable information on quantitative traits has shed some light on the molecular mechanisms responsible for leaf architecture traits affected by watering conditions. Furthermore, alleles for leaf architecture traits provide useful targets for marker‐assisted selection to generate high‐yielding maize varieties.     

Identification and characterization of sources for photo‐ and thermo‐insensitivity in Vigna species

Cultivation of the same varieties of mungbean and blackgram across different seasons and locations is constrained by their photo‐ and thermo‐sensitive behaviour. Developing insensitive genotypes, which can fit well across all seasons, requires robust donors which would provide genes imparting this trait. This study was undertaken to identify such donors in the Vigna species. Forty‐eight accessions belonging to 13 Vigna species and eight released cultivars were evaluated under natural field conditions. Among these, two accessions, viz. V. umbellata (IC251442) and V. glabrescens (IC251372) were found photo‐ and thermo‐insensitive as these were able to flower and set pods at temperatures as high as 43.9°C and as low as 2.7°C. Pollen viability studies indicated viable pollen (>75% at 2.7°C and >85% at 41.9°C) and normal pollen tube growth at both the extremes of temperature. The identified V. glabrescens accession has long, constricted pods and dark green, mottled seeds while V. umbellata has smooth, curved pods and shining, oval, large seeds. Both these accessions can be utilized in developing photo–thermo insensitive genotypes in cultivated Vigna species.     

Phenotypic evaluation of floral and flowering traits with relevance for hybrid breeding in wheat (Triticum aestivum L.)

Hybrid breeding is a promising approach to increase the yield potential in wheat (Triticum aestivum L.). The profitability of wheat hybrids highly depends on a cost‐efficient system for hybrid seed production for which an adequate outcrossing in the male pool is of utmost importance. Employing a set of 51 elite winter wheat lines, we developed and evaluated phenotyping methods for floral and flowering traits with relevance for improved cross‐pollination. We observed significant genotypic variances and high heritabilities for most traits, including important traits like pollen mass and anther extrusion. Our results suggest the utility of the developed phenotyping approaches for applied plant breeding and the potential of the traits to assist in the design of the male ideotype for increased cross‐fertilization.     

Marker‐assisted breeding of Pi‐1 and Piz‐5 genes imparting resistance to rice blast in PRR78, restorer line of Pusa RH‐10 Basmati rice hybrid

Rice blast, caused by fungus Magnaporthe grisea, is a serious disease causing considerable economic damage worldwide. Best way to overcome disease is to breed for disease‐resistant cultivars/parental lines of hybrids. Pusa RH10, first aromatic, fine‐grain rice hybrid released and cultivated extensively in India. Hybrid and its parental lines, Pusa 6A and PRR78, are highly susceptible to blast. CO39 pyramid carrying two dominant, broad‐spectrum blast‐resistance genes, viz. Pi‐1 and Piz‐5, used as a donor parent to introgress these genes into PRR78 using marker‐assisted backcrossing (MABC). Microsatellite markers RM5926 and AP5659‐5 tightly linked to Pi‐1 and Piz‐5 genes, respectively, were used for foreground selection to derive introgression lines. Further, these lines were evaluated for agronomic performance, disease reaction and cooking quality traits along with PRR78. Most of the improved lines were on par with PRR78 for all traits evaluated except gelatinization temperature. Recurrent parent genome percentage (RPG) study also revealed similarity of these lines with PRR78. Hybrids derived using improved PRR78 lines were superior over Pusa RH10 in terms of yield.