Spike morphology alternations in androgenic progeny of hexaploid triticale (× Triticosecale Wittmack) caused by nullisomy of 2R and 5R chromosomes

Induction of androgenesis, followed by chromosome doubling, is a crucial method to obtain complete homozygosity in one-generation route. However, in vitro androgenesis can result in various genetic and epigenetic changes in derived triticale plants. In this study, we evaluated chromosome alternations and we associated them with the changes of spike morphology in androgenic progeny of triticale. We karyotyped offspring plants that derived from double haploid plants using fluorescence in situ hybridization techniques. We distinguished four major groups of karyotypes: double ditelosomics, nullisomics N2R, nullisomics N5R, and triticale plants with a complete set of chromosomes. It is known that more than half of QTLs connected with androgenic response are located in R-genome of triticale but 2R, 5R, and 6R chromosomes are not included. We hypothesized that the reason why only aberrations of chromosomes 2R and 5R appear during androgenesis of triticale is that because these chromosomes are not involved in the stimulation of androgenic response and the following regeneration of plants is not disrupted. Concerning the established groups, we evaluated following quantitative traits: spike length, number of spikes per plant, number of spikelets per spike, and number of grains per spike. The nullisomy of chromosome 2R and 5R resulted in vast changes in spike architecture of triticale plants, which can be correlated with the location of major QTLs for spike morphology traits on these chromosomes. The spikes of nullisomic plants had significantly decreased spike length which correlated with the reduction of number of spikelets per spike and number of grains per spike.

     

Meiotic pairing in hybrids between tetraploid Triticale and related species: new elements concerning the chromosome constitution of tetraploid Triticale

Summary Two F5 strains of tetraploid triticale (2n= 4x=28), obtained from 6x triticaleX2 rye progenies, were crossed with diploid and tetraploid rye, some durum and bread wheats, and various 8x and 6x triticale lines. Meiosis in the different hybrid combinations was studied. The results showed that the haploid complement of these triticales consists of seven chromosomes from rye and seven chromosomes from wheat. High frequencies of PMCs showing trivalents were observed in hybrids involving the reference genotypes of wheat and triticale. These findings proved that several chromosomes from the wheat component have chromosome segments coming from two parental wheat chromosomes. The origin of these heterogeneous chromosomes probably lies in homoeologous pairing occurring at meiosis in the 6x triticaleX2x rye hybrids from which 4x triticale lines were isolated. A comparison among different hybrids combinations indicated that the involvement of D-genome chromosomes in homoeologous pairing is quite limited. In contrast, meiotic patterns in 4x triticale X 2x rye hybrids showed a quite high pairing frequency between some R chromosomes and their A and B homoeologues.     

Effect of rye chromosomes on features of androgenesis in wheat-rye substituted lines of Triticum aestivum L. sort Saratovskaya 29/Secale cerale L. sort Onokhoiskaia and Triticale]

The characteristic features of androgenesis in six wheat-rye substitution lines Triticum aestivum L. (cv. Saratovskaya 29)/Secale cereale L. (cv. Onokhoiskaya) and triticale (2n = 56) using anther culture at different concentrations of 2,4-D in the growth medium were studied. Under variable cultivation conditions, the significant effect of genotypic diversity on the variability of such androgenesis parameters as the frequency of productive anthers, the frequency of embryoid formation, and the frequency of total regenerated plantlets, was shown. It was demonstrated that chromosomes 1R, 3R, and 7R stimulated the formation of androgenous embryoids, while chromosome 5R produced an opposite effect. In triticale and substitution lines, the regeneration ability of androgenous embryoids induced by elevated 2,4-D concentrations was inhibited. Chromosome 1R of the Onokhoiskaya cultivar was suggested to contain genes suppressing regeneration of green plantlets, while chromosome 3R, conversely, stimulated their formation. Chromosomes 1R, 2R, 3R, and 7R of the Onokhoiskaya cultivar did not inhibit the spontaneous formation of androgenous hexaploids in the substitution lines.     

The Effect of Rye Chromosomes on the Characteristic Features of Androgenesis in Wheat–Rye Substitution Lines Triticum aestivumL. (cv. Saratovskaya 29)/Secale cerealeL. (cv.Onokhoiskaya) and Triticale

The characteristic features of androgenesis in six wheat–rye substitution lines Triticum aestivumL. (cv. Saratovskaya 29)/Secale cerealeL. (cv. Onokhoiskaya) and triticale (2n= 56) using anther culture at different concentrations of 2,4-D in the growth medium were studied. Under variable cultivation conditions, the significant effect of genotypic diversity on the variability of such androgenesis parameters as the frequency of productive anthers, the frequency of embryoid formation, and the frequency of total regenerated plantlets, was shown. It was demonstrated that chromosomes 1R, 3R, and 7R stimulated the formation of androgenous embryoids, while chromosome 5R produced an opposite effect. In triticale and substitution lines, the regeneration ability of androgenous embryoids induced by elevated 2.4-D concentrations was inhibited. Chromosome 1R of the Onokhoiskaya cultivar was suggested to contain genes suppressing regeneration of green plantlets, while chromosome 3R, conversely, stimulated their formation. Chromosomes 1R, 2R, 3R, and 7R of the Onokhoiskaya cultivar did not inhibit the spontaneous formation of androgenous hexaploids in the substitution lines.     

Quantitative trait loci associated with androgenic responsiveness in triticale (×Triticosecale Wittm.) anther culture

Quantitative trait loci (QTLs) associated with androgenic responsiveness in triticale were analyzed using a population of 90 DH lines derived from the F1 cross between inbred line ‘Saka 3006’ and cv. ‘Modus’, which was used in a number of earlier studies on molecular mapping in this crop. Using Windows QTL Cartographer and MapQTL 5.0, composite interval mapping (CIM) and association studies (Kruskal–Wallis test; K–W) for five androgenesis parameters (androgenic embryo induction, total regeneration and green plant regeneration ability, and two characteristics describing final androgenesis efficiency) were conducted. For the studied components of androgenic response, CIM detected in total 28 QTLs which were localized on 5 chromosomes from A and R genomes. Effects of all QTLs that were identified at 2.0 or above of the LOD score explained 5.1–21.7?% of the phenotypic variation. Androgenesis induction was associated with seven QTLs (LOD between 2.0 and 5.8) detected on chromosomes 5A, 4R, 5R and 7R, all of them confirmed by K–W test as regions containing the markers significantly linked to the studied trait. What is more, K–W test revealed additional markers on chromosomes: 5A, 2BL, 7B and 5R. Both total and green regeneration ability were controlled by genes localized on chromosome 4A. Some of the QTLs that affected final androgenesis efficiency were identical with those associated with androgenic embryo induction efficiency, suggesting that the observed correlation may be either due to tight linkage or to pleiotropy. Key message Five regions of the triticale genome were indicated as revealing significant marker/trait association. Markers located in these regions are potentially useful for triticale breeding through marker-assisted selection.     

Aneuploidy frequency in sperm of fertile men

Analysis of sperm aneuploidy in 11 healthy men using two-or three-color FISH permitted to determine the average frequency of disomy for chromosomes 13 and 21 (0.11% and 0.2%, respectively), disomy for chromosome 18 (0.05%) and to reveal gonosomal aneuploidy variants and their frequency. The frequency of XX disomy was 0.04%; XY, 0.17%; YY, 0.06%; and gonosomal nullisomy, 0.29%. We assessed the frequency of meiotic nondisjunction of 13, 21, 18, X, and Y chromosomes and the frequency of XX, XY, and YY diploid spermatozoa. The XY variant prevailed in gonosomal aneuploidy and diploidy and was associated with abnormal chromosomal segregation in meiotic anaphase I. The contribution of human sperm chromosomal imbalance to early embryonic lethality and to some forms of chromosomal abnormalities in the off-spring is discussed.     

Loss of chromosomes 2R and 5RS in octoploid triticale selected for agronomic traits

The advanced lines of octoploid triticale which have been bred for nearly a half century in China show significant improvements in agronomic traits such as plant height, fertility, threshability, maturity and seed plumpness, although no intentional cytological selection had been performed. In this study, eight primary and six advanced lines were analyzed by fluorescence and genomic in situ hybridization to elucidate their chromosome constitutions. In the advanced lines, about 70% of the plants examined had 2n = 56 chromosomes (range: 50 to 58). Almost all advanced lines, however, had lost rye chromosome 2R and the short arm of 5R (5RS). The exceptions were lines Y1005 and Y4683: The former had lost only the 2R chromosome and the latter only 5RS. The reduction of rye chromosomes was compensated by an extra pair of 2D or A-genome (possibly 2A) chromosomes in plants with 2n = 56. This suggests that the loss of 2R and 5RS chromosomes contributes to the improvement of octoploid triticale. Since the plants with chromosome 2R are non-free threshing and chromosome 2D of synthetic wheat is known to carry the Tg (tenacious glumes) gene, it is possible that chromosome 2R carries a gene affecting the threshability, and we carried out selection to remove it. We also discuss the possible relationshipbetween 5RS and the genetic stability of octoploid triticale.     

Introduction of d-genome chromosomes from Aegilops squarrosa L. into tetraploid triticale (AB)(AB)RR (2n=28)

Summary Tetraploid triticale with the genome constitution (ABD) (ABD)RR (2n=4x=28) selected from the progenies of DDRR x (AB)(AB)RR hybrids (D(AB)RR) were karyotyped using C-banding. The aneuploidy frequency was 10.7% with 4.4% hypoploids and 6.3% hyperploids in the F₅. Among 67 plants having 28 chromosomes, 41.8% had a stabilized karyotype, while 58.2% were unstabilized with at least one homoeologous group segregating for A-, B- or D-genome chromosomes. The stabilized plants represented ten different karyotypes that contained one to five disome substitutions of D-genome chromosomes for A- or B-genome chromosomes. Two (BD) (BD)RR tetraploids had no A-genome chromosomes. The average number of D substitutions was 3.0 per line. Of the seven substitutions possible only one, 4D(4B), was not present. In the progeny of plants selected for fertility a selection pressure acted against wheat chromosomes 1B, 3B, 4D and 7D. The most favoured chromosome constitution of the (ABD) mixed genome was 1D, 2A, 3D, 4B, 5B, 6A and 7B. Plants of that karyotype but with a heterologous pair of chromosomes 5B and 5D had the best seed set. Evolutionary and breeding aspects of tetraploid triticale are discussed.     

Comparative Effects of Rye Chromosomes 1R and 5R on Androgenesis in Cultured Anthers of Wheat–Rye Substitution Lines as Dependent on the Line Origin

The effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat–rye substitution lines was studied as dependent on the cultivar origin of the rye chromosomes and on the wheat genome (A or D) subjected to substitution. Chromosome 1R stimulated embryogenesis in anther cultures, while chromosome 5R suppressed it regardless of whether the corresponding wheat chromosomes were substituted in the A or D genome. The effect of chromosome 1R on embryogenesis proved to depend on its cultivar origin. Along with rye chromosome 1R, wheat chromosome 1A was shown to substantially affect total seedling regeneration. Regeneration of green seedlings was dramatically affected both by rye chromosome 1R and by wheat chromosome 1D. The results supported the published data that individual androgenesis parameters (embryogenesis, total plant regeneration, green plant regeneration) are controlled by different genetic mechanisms.     

Isolated microspore culture of Canadian 6× triticale cultivars

Isolated microspore culture was conducted on nine Canadian triticale cultivars (X Triticosecale Wittmack) using two induction media developed for wheat, with or without 100 g l⁻¹ Ficoll. Significant interactions were observed for the number of embryos and calluses induced, green and albino plantlets regenerated and fertility of green plants. Ficoll was beneficial in both media to increase numbers of embryos and green plants for all cultivars. Overall, medium NPB99 supplemented with ficoll provided the most suitable condition for most cultivars. AC Alta performed slightly better on CHB3 supplemented with Ficoll. Only cv. Wapiti was not amenable to androgenesis. The cultivars AC Certa, AC Copia, AC Alta, Sandro, Ultima, Frank, Pronghorn and Banjo produced respectively, 10, 9, 6, 5, 4, 3, 3 and 1 green plants per Petri dish (35,000 microspores), on their optimum treatment. Twenty-two percent of total lines produced were fertile, and considered doubled haploids. The application of isolated microspore culture to triticale, opens new possibilities in breeding triticale, for the utilization of in vitro selection and genetic engineering.     

Comparative effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat-rye substitution lines dependending on the line origin

The effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat--rye substitution lines was studied as dependent on the cultivar origin of the rye chromosomes and on the wheat genome (A or D) subjected to substitution. Chromosome 1R stimulated embryogenesis in anther cultures, while chromosome 5R suppressed it regardless of whether the corresponding wheat chromosomes were substituted in the A or D genome. The effect of chromosome 1R on embryogenesis proved to depend on its cultivar origin. Along with rye chromosome 1R, wheat chromosome 1A was shown to substantially affect total seedling regeneration. Regeneration of green seedlings was dramatically affected both by rye chromosome 1R and by wheat chromosome 1D. The results supported the published data that individual androgenesis parameters (embryogenesis, total plant regeneration, green plant regeneration) are controlled by different genetic mechanisms.     

Influence of different concentrations of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale

In this work, the effect of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale was studied. The study of androgenesis was conducted on two model samples YaTKh-327-11 and YaTKh-18-11. The research revealed that the addition of the antioxidant ascorbic acid at a concentration of 2–8 mg/L into the culture medium to induce embryogenesis enhanced the formation of androgen structures and regeneration of green plants. The addition of gibberellic acid into the culture medium to induce embryogenesis gave negative effects. The average yield of green plants in the anther culture of spring triticale was 2 pcs/100 anthers. According to the research, 277 green plants were received and spontaneous doubling of chromosomes was recorded in 26.7%.     

小麦-黑麦附加系的创制及5R抗白粉病新基因的发现

以重复序列pAS1和pSc119.2为探针,对八倍体小黑麦×普通小麦的F5代植株进行了FISH分析,同时对这些材料进行了田间抗病性鉴定。从中鉴定出了1R、2R、3R、4R、5R、6R、7R单体附加系和1R、2R二体附加系,1R和4R附加系出现频率相对较高。5R和6R单体附加系对白粉病免疫,推测5R染色体上带有新的白粉病抗性基因。此外,还检测到不少植株染色体组发生了变异,且小麦4B染色体优先缺失。     

Doubled haploids in production of male sterility maintaining triticale (<Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) lines

The androgenetic response of several selected male sterility-maintainer genotypes of triticale was investigated. Androgenesis induction was obtained in all cultivars, but a large genotypic variation in green plant regeneration was observed. The number of regenerated triticale plants varied from 0.1 to 4.7 green plants per spike, depending on genotype. Spontaneous doubling of chromosomes varied from 14 to 60% for particular genotypes and, on average, reached the value of 34% for all genotypes. Fertile DH lines obtained in this study will find practical application in the development of triticale male sterile lines that are desirable in hybrid breeding.     

Organelle antioxidants improve microspore embryogenesis in wheat and triticale

Low frequency of green plant production and albinism limits the use of isolated microspore culture (IMC) in cereal breeding programs. The present study was conducted in triticale and bread wheat IMC to increase the production of green plants and minimize albinism. NPB-99?+?10% Ficoll induction medium was supplemented with mitochondrial or plastid antioxidants, in a completely random design, to evaluate their contribution to successful microspore embryogenesis and green plant production. Each group of antioxidants was tested independently: first in triticale and then validated in various spring wheat genotypes. While the response differed by wheat genotype, induction medium supplemented with proline (10 mM) yielded a greater number of embryos/embryo-like structures and green plants in both triticale and wheat. No differences were found with respect to albinism in triticale or wheat except for the cv. Sadash. Among plastid antioxidants tested, glutathione (2 μM) proved to be the best antioxidant to increase embryo and green plant production. Salicylic acid also helped to reduce the number of albino plants in triticale and the wheat genotype SWS366. Overall, induction medium supplemented with proline or glutathione enhanced microspore embryogenesis in both triticale and wheat and increased the number of green plants in the recalcitrant genotypes.     

Assessment of the Combining Ability of Triticale and Secalotriticum with Respect to in Vitro Androgenesis Characteristics

The combining ability of cultivars and strains of triticale and secalotriticum with respect to the characteristics of androgenesis in anther culture has been analyzed (the 6 × 3 polytester design and parental forms). Nonadditive effects of gene interaction have been found to be prevalent in the genetic control of the characters analyzed in plants with the given genotypes. However, the characteristics of androgenesis were the highest in those hybrid combinations in which genotypes with high values of general combining ability (GCA) and positive effects of specific combining ability (SCA) participated. A program of the analysis of combining ability in triticale and secalotriticum is proposed. The program is aimed at detecting hybrid combinations with the best androgenetic capacities and takes into account both additive and nonadditive effects.     

Evaluation of segregation patterns of 21;21 Robertsonian translocation along with sex chromosomes and interchromosomal effects in sperm nuclei of carrier by FISH technique

Meiotic segregation patterns of carriers of Robertsonian translocations (RT) are important for assessing the risk of unbalanced forms. We investigated the ratio of sperm with t(21;21) to sperm with nullisomy for chromosome 21; the segregation of the t(21;21) along with sex chromosomes, and also interchromosomal effects on chromosome 10 by using three color fluorescence in situ hybridization (FISH) with telomere specific (Tel 21q) and centromere-specific alpha satellite probes for chromosomes X, Y, and 10. The percentage of cosegregation of t(21;21) with sex chromosomes (49.50%) and without sex chromosomes (46.98%) was not significant. There are no significant differences between the percentages of cosegregation of t(21;21) with chromosome X (23.36%) and with chromosome Y (26.16%). No evidence of an interchromosomal effect on chromosome 10 was detected, the percentage of chromosome 10 aneuploidy being similar to that in controls. In addition, the frequency of diploid sperm nuclei was not significantly higher in the carrier (0.32%) than in the controls (0.44%) (P > 0.05). The sex ratio was similar within the carrier and the controls and between the carrier and the control. Three color-FISH analysis, using different probe combinations, seems a rapid and accurate tool for direct analysis of meiotic segregation product.     

Meiotic behaviour of tetraploid wheats (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L.) and their synthetic hexaploid wheat derivates influenced by meiotic restitution and heat stress

Meiotic restitution is considered to be a common mechanism of polyploidization in plants and hence is one of the most important processes in plant speciation. Meiotic behaviour of plant chromosomes is influenced by both genetic and environmental factors. In this study, the meiotic behaviour of cereal crops was investigated, which includes tetraploid wheat genotypes (with and without the meiotic restitution trait) and their derivates (synthetic hexaploid wheats and a doubled haploid (DH) line), grown at two planting dates in the field. In addition, two local landraces of emmer wheat (Triticum turgidum ssp. dicoccum), one wheat cultivar (Chinese spring), one DH triticale cultivar (Eleanor) and one rye accession were included. Immature spikes of mid-autumn and end-winter sowing plants were collected in April and May 2008, respectively, fixed in Carnoy’s solution and stained with hematoxylin. Pollen mother cells (PMCs) from anthers at different stages of meiotic process were analysed for their chromosomal behaviour and irregularities. Meiotic aberrations such as laggards, chromosome bridges, micronuclei, abnormal cytokines, chromatin pulling and meiotic restitution were observed and the studied genotypes were accordingly ranked as follows: triticale > synthetic hexaploid wheats > tetraploid wheats possessing meiotic restitution > tetraploid wheats lacking meiotic restitution > rye. The results indicated that the samples that had been planted in the autumn, thus experiencing an optimum temperature level at the flowering stage, exhibited less meiotic irregularities than winter planting samples that encountered heat stress at the flowering period.     

In vitro androgenesis response of durum wheat disomic substitution

The variety 'Langdon' and its substitution series were used to evaluate the effect of each substituted chromosome of the A and B genomes on the in vitro androgenetic potential. This study showed the implication of chromosomes 1B and 5B in repressing embryogenesis. Genes located on these chromosomes seem to have an inhibitor effect. The substitution of these chromosomes by their homeologous ones from the D genome increased the number of embryos while with the presence of the original genes the number of embryos was less than in the control. Chromosome 5B is also especially involved in the regeneration of green plants. The genetic control is inhibitory; this explains the difficulty of obtaining good levels of in vitro androgenesis in durum wheat. In this study no effect of the D genome on the androgenetic response of the substitution lines was observed.     

Evaluation of the combined ability of triticale and secalotriticum by signs of androgenesis in vitro

The combining ability of cultivars and strains of triticale and secalotriticum with respect to the characteristics of androgenesis in anther culture has been analyzed (the 6 x 3 polytester design and parental forms). Nonadditive effects of gene interaction have been found to be prevalent in the genetic control of the characters analyzed in plants with the given genotypes. However, the characteristics of androgenesis were the highest in those hybrid combinations in which genotypes with high values of general combining ability (GCA) and positive effects of specific combining ability (SCA) participated. A program of the analysis of combining ability in triticale and secalotriticum is proposed. The program is aimed at detecting hybrid combinations with the best androgenetic capacities and takes into account both additive and nonadditive effects.