Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). I. Comparison of sexual and doubled-haploid populations

Summary The genetic consequences of anther culture and chromosome-doubling techniques on burley tobacco (Nicotiana tabacum L.) were examined in this study. Three diploid populations, obtained from a burley tobacco inbred by conventional and anther-culture techniques, were compared. The first population consisted of 50 conventionally-selfed lines; the second population was made up of 35 doubled-haploid lines obtained from individual haploid plants by in vitro techniques (IVDH); and the third population consisted of 20 doubled-haploid lines whose chromosome complements had been doubled with colchicine (CDH). Comparisons of doubled-haploid lines with sexually-derived lines revealed significant differences for yield, maturity, leaf length, and alkaloid content. Yield reductions in the doubled-haploid populations averaged 8.5%. Significant differences observed between the IVDH and CDH populations indicate that the reported deleterious effects of colchicine contributed to the vigor reduction of doubled haploids. The anther derived lines in this study exhibited greater variation than did the sexual materials. This variation could provide useful variation for a breeding program. Variation exhibited by the sexual progeny of the highly inbred line, Kentucky 16, suggests that the differences among anther-derived materials are at least partially due to natural phenomena.Contribution from the department of Agronomy, University of Kentucky, Lexington, KY 40546-0091. This research was supported, in part, by a grant from R. J. Reynolds Tobacco Co. This paper (no 85-3-5) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). II. Evaluation of first- and second-cycle doubled haploids

Summary First- and second-cycle doubled haploids of burley tobacco (Nicotiana tabacum L.) were examined to ascertain the significance of source genotype on the performance of anther-derived material and to determine the magnitude of changes from the haploid-production and chromosome-doubling stages. The anther-derived lines evaluated in this study did not exhibit siginficant yield reductions. However, significant interactions between the cycle comparison and the source genotypes indicated that source genotype can affect the degree of vigor reduction observed. Significant variation was also observed among the second-cycle doubled haploids examined in this study. Partitioning of this variation indicates that most of it is due to alteration present at the haploid-production step instead of the chromosome-doubling step. In general, the variation present in the second-cycle doubled haploids was distributed around the values observed for the source first-cycle doubled haploids. Even though unexpected variation is often observed in anther-derived materials, these results suggest that reduced vigor is not intrinsically associated with androgenetic lines of burley tobacco. The most plausible explanations for the variation and vigor losses in anther-derived lines propose that natural phenomena, given the opportunity for expression via anther culture, are responsible. Spontaneous variants present in the gametic pool of inbreds or first-cycle doubled haploids offer one possible explanation for the performance observed in anther-derived lines of tobacco.Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091. This research was supported, in part, by a grant from R. J. Reynolds Tobacco Co. This paper (no. 85-3-29) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

A rapid and efficient microspore culture protocol was applied to produce homozygous progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F₁ hybrids with the resynthetic line ‘RS239’ were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of doubled haploid (DH) regenerants. The diploidization rate was subsequently verified by scoring flower morphology. In vitro colchicine treatment had a positive effect on induced diploidization, and was associated with the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, the effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large‐scale production of DHs in rapeseed as the genotype‐specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows efficient selection for DHs, thus saving labour and cost and in the laboratory and subsequent greenhouse phase.     

Anther-culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.)

An in vitro anther-culture method has been improved by using young mother plants and by using frequent subcultures, thus increasing the androgenic yield in different Capsicum annuum L. genotypes. An assortment of peppers was used, composed of 15 genotypes (four breeding lines, seven cultivars and four F₁ hybrids). A new system for qualifying the androgenic response was established. For use in practical breeding, a minimum of 5 % of plant regeneration was proposed as the criterion for a fair response. Accordingly, one excellent, one good and eight fair responses were identified among the genotypes investigated. As compared to the standard cultivar. 2 genotypes gave a significantly better response, i.e. ‘Fehérözön’ (75.8%) and ‘Szechuan 90716’ (21.0%). In comparative investigations, F₁ hybrids, produced from crosses between poor/non-responsive and responsive genotypes, showed a fair level of response, even the case of a poor response in donor parent. The ploidy level of the resulting plants was determined by flow-cytometric analysis.     

Interspecific cross‐hybrids of Nicotiana tabacum L. cv. (gla.) S ‘K326’ with Nicotiana alata

In order to introduce the Tomato Spotted Wilt Virus (TSWV) resistance from Nicotiana alata into Nicotiana tabacum, a cytoplasmic male sterility (CMS) line of N. tabacum (N. tabacum L. cv. (gla.) S ‘K326’), was successfully crossed with N. alata. Despite a high DNA content variability, F₁ hybrids could be classified in two subgroups, a major one encompassing fertile hybrids morphologically similar to their tobacco maternal parent but TSWV sensitive, and a minor one displaying sterile hybrids showing an intermediate phenotype and TSWV resistant. In order to elucidate the unexpected fertility recovery of the fertile F₁ plants, some N. alata fertility restoration ppr genes were cloned and were shown to be differentially expressed between parental lineages as well as between both F₁ subgroups, suggesting that N. alata contains fertility restoring allele able to overcome the CMS of N. tabacum.     

Relative efficiency of maize and Imperata cylindrica for haploid induction in Triticum durum following chromosome elimination‐mediated approach of doubled haploid breeding

Intergeneric hybridization in seven diverse durum wheat genotypes was carried out using two composite varieties of Himalayan maize, viz., Bajaura Makka and Early Composite, and a wild grass, Imperata cylindrica, as pollen sources. Observations related to various haploid induction parameters put forth I. cylindrica as significantly better pollen source for haploid induction in durum wheat over maize in terms of pseudoseed formation (46.93%), embryo formation (38.06%), haploid regeneration (40.42%) and haploid formation efficiency (7.44%). The line x tester analysis revealed that both male and female genotypes had significant effects on all haploid induction parameters except haploid formation frequency in later. Among the pollen sources, I. cylindrica emerged as best combiner based on GCA values when compared with the two Himalayan maize composites. Durum wheat genotype, A‐9‐30‐1 was recognized as the best general combiner followed by PDW 314. The present investigation proposed durum wheat × I. cylindrica as a superior technique over maize‐mediated system, and its large‐scale use can open a new horizon in the sphere of durum wheat doubled haploidy breeding programme.     

Efficient application of in vitro anther culture for different European winter wheat (Triticum aestivum L.) breeding programmes

The efficiency of our anther culture protocol was tested with high‐ and low‐responding genotypes, ‘Svilena’ and ‘Berengar’, and 93 F₁ winter wheat crosses in 2010 and 2011. Based on data for these genotypes, the effect of genotype influenced the number of embryo‐like structures, regenerated plantlets and green plantlets, while the number of albino plantlets was affected by genotype, year and environmental factors. Although genotype also influenced the production of green plantlets from breeding crosses, with green plantlets per 100 anthers ranging from 0.04 to 28.67, the average regeneration rate over all crosses was 5.3 green plantlets/100 anthers, which resulted in a total of 11 416 well‐rooted green plantlets. The survival rate of green plantlets following acclimatization was 97.21% in 2010 and 96.34% in 2011. In this study, the phenomenon of albinism and genotype dependency did not hinder the production of more than five thousand green plantlets each year. In our experiments, anther culture proved to be an efficient method in winter wheat breeding programmes with lower costs than alternative technologies.     

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.     

Oligogenic control of resistance to soil‐borne viruses SBCMV and WSSMV in rye (Secale cereale L.)

Rye production in European growing areas is constrained by the soilborne cereal mosaic virus (SBCMV) and the wheat spindle streak mosaic virus (WSSMV). To date, no European rye cultivars are known to exhibit resistance against these viruses. In this study, we pursued a quantitative trait locus (QTL) mapping strategy to identify genomic regions for resistance to SBCMV and WSSMV in rye. Three populations, each comprising 100 lines segregating for resistance to SBCMV and/or WSSMV, were evaluated for disease response at two years in three locations in Germany where soils are naturally infested with SBCMV and WSSMV. In the combined analysis across environments, one QTL for SBCMV resistance on chromosome 5R explained 31.9% of the phenotypic variation in one of the populations. For WSSMV resistance, one QTL explaining up to 64.0% of the phenotypic variation was detected on chromosome 7R in each of the three populations. On the Triticeae homoeologous group 5, we found evidence for synteny of the major QTL for SBCMV resistance between the wheat and rye genomes.     

Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

Identification and marker‐assisted introgression of QTL conferring resistance to bacterial leaf blight in cowpea (Vigna unguiculata (L.) Walp.)

Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is widespread in major cowpea [Vigna unguiculata (L.) Walp.] growing regions of the world. Considering the resource poor nature of cowpea farmers, development and introduction of cultivars resistant to the disease is the best option. Identification of DNA markers and marker‐assisted selection will increase precision of breeding for resistance to diseases like bacterial leaf blight. Hence, an attempt was made to detect QTL for resistance to BLB using 194 F_2 : 3 progeny derived from the cross ‘C‐152’ (susceptible parent) × ‘V‐16’ (resistant parent). These progeny were screened for resistance to bacterial blight by the leaf inoculation method. Platykurtic distribution of per cent disease index scores indicated quantitative inheritance of resistance to bacterial leaf blight. A genetic map with 96 markers (79 SSR and 17 CISP) constructed from the 194 F₂ individuals was used to perform QTL analysis. Out of three major QTL identified, one was on LG 8 (qtlblb‐1) and two on LG 11 (qtlblb‐2 and qtlblb‐3). The PCR product generated by the primer VuMt337 encoded for RIN2‐like mRNA that positively regulate RPM1‐ and RPS2‐dependent hypersensitive response. The QTL qtlblb‐1 explained 30.58% phenotypic variation followed by qtlblb‐2 and qtlblb‐3 with 10.77% and 10.63%, respectively. The major QTL region on LG 8 was introgressed from cultivar V‐16 into the bacterial leaf blight susceptible variety C‐152 through marker‐assisted backcrossing (MABC).     

Validation of a male‐linked gene locus (OGI) for sex identification in persimmon (Diospyros kaki Thunb.) and its application in F1 progeny

It is crucial to develop a rapid technique for identifying sexuality in the seedling stage of persimmon (Diospyros kaki Thunb.), and the elimination of male progeny has been regarded as an important strategy for enhancing breeding efficiency. In this study, phenotype characterization and genotyping of the male‐linked OGI marker were carried out using 205 accessions, including persimmon cultivars, F₁ progeny and nine related Diospyros species. All persimmon cultivars displayed consistent results regarding OGI amplification and sex phenotype. A total of 143 F₁ progeny were derived from 11 crosses, among which 95 individuals had flowered. In the flowering full‐sib families, the amplification of the OGI marker in agreement with the sex phenotype was obtained in 85 plants (89.5%). The segregation of OGI in ‘Huashi 1’ × ‘Luotian Tianshi’ and ‘Huashi 1’ × Male 3 F₁ populations fit a 1 : 1 ratio. Furthermore, high OGI transferability was observed in nine related species. Overall, the results indicated that the OGI locus could be used to distinguish male from female persimmon plants at an early stage.     

Development of AS‐PCR marker based on a key mutation confirmed by resequencing of Wx‐mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding

Soft rice with low amylose content (AC) ranging by 5–15% is a unique type with special eating and appearance quality and has become popular in the rice market. We resequenced the Wx‐mp, a key allele from Milky Princess, a Japanese low AC variety, and found that the +473 mutation in exon 4 is the key mutation in both Wx‐mp and its ancestor allele, Wx‐mq from Milky Queen. Based on this functional mutation, an allele‐specific PCR (AS‐PCR) marker was developed and proven in a breeding population derived from a cross between a Chinese late variety Nan Keng 46 (Wx‐mp/Wx‐mp) and an early line Ning 63121(Wx‐b/Wx‐b). Based on the marker‐aided selection by our newly developed AS‐PCR marker for Wx‐mp and the known ST10 marker for Stvb‐i, a total of 12 Wx‐mp homozygotes were selected from 198 F₂ progenies, and four of them were immune to rice stripe virus (RSV) with averagely 11.3 days earlier heading than Nan Keng 46 without significant change in grain yield.     

Drought‐induced osmotic adjustment and changes in carbohydrate distribution in leaves and flowers of cotton (Gossypium hirsutum L.)

Research has indicated osmotic adjustment as a mechanism by which leaves and roots of cotton plants overcome a drought period. However, the relevance of this mechanism in reproductive tissues of modern cultivars under drought has not been fully investigated. The objectives of this study were to measure osmoregulation and carbohydrate balance in reproductive tissues and their subtending leaves grown under water‐deficit conditions. Two cotton cultivars were grown under controlled environment and field conditions. Plants were exposed to water‐deficit stress at peak flowering, approximately 70 days after planting. Measurements included stomatal conductance, proline concentration, soluble carbohydrates and starch concentration, and water potential components. Stomatal conductance of drought‐stressed plants was significantly lower compared to control, while osmotic adjustment occurred in reproductive tissues and their subtending leaves by different primary mechanisms. Pistils accumulated higher sucrose levels, maintaining cell turgor in plants exposed to drought at similar levels to those in well‐watered plants. However, subtending leaves lowered osmotic potential and maintained cell turgor by accumulating more proline. Soluble carbohydrates and starch concentration in leaves were more affected by drought than those of floral tissues, with corresponding reduction in dry matter, suggesting that flowers are more buffered from water‐deficit conditions than the adjacent leaves.