Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.     

Chromosome pairing in meiosis of partially fertile wheat/rye hybrids

The normal course of meiosis depends on regular pairing of homologous chromosomes. In intergeneric hybrids, including those of wheat, there is no chromosome pairing because there are no homologs. In F₁ wheat/rye hybrids, pairing is largely prevented by the pairing homoeologous1 (Ph1) gene. In its presence, there are only rare instances of pairing; most chromosomes are univalent, and their orientation at metaphase I initiates different pathways of the meiotic cycle. The meiotic-like pathway includes a combination of the reductional and the equational + reductional steps at AI followed by the second division. The resulting gametes are mostly non-functional. The mitotic-like pathway involves equational division of univalents at AI and the absence of the second division. Any fertility of wheat/rye hybrids depends on the production of unreduced gametes arising from meiotic restitution (mitotic-like division). We examined the meiotic pairing in wheat/rye hybrids created from wheat lines with single rye chromosome substitutions and Ph1 present. This guaranteed F₁ meiosis with one pair of rye homologs. All hybrids formed bivalents, but proportions of meiocytes with bivalents varied. In the meiocytes where bivalents were present, there was a higher tendency for the meiotic-like pathway, while in meiocytes where bivalent pairing failed, the tendency was stronger for the mitotic-like pathway. Among the equationally dividing cells, we observed more than 90 % of meiocytes without bivalents, where rye homologs did not form bivalents, too. The data indicate a potential application of wheat/rye lines in producing genetic stocks of amphidiploids with designated genomic constitutions.     

Formation and function of phragmoplast during successive cytokinesis stages in higher plant meiosis

Cytoskeletal rearrangements were studied during meiotic telophase in a number of monocotyledonous plant species. Wild type and abnormal meiosis (in wide cereal hybrids, meiotic mutants and allolines) was analyzed. It was found that central spindle fibers that move centrifugally, along with newly-formed MTs, are the basis of phragmoplast formation and function in PMCs of monocotyledonous plant species with successive cytokinesis stages. A model for centrifugal movement of the meiotic phragmoplast is proposed; this model is a modification of the corresponding process during B-anaphase.     

Short periods of high temperature during meiosis prevent normal meiotic progression and reduce grain number in hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Exposure of wheat to high temperatures during male meiosis prevents normal meiotic progression and reduces grain number. We define a temperature-sensitive period and link heat tolerance to chromosome 5D.

Abstract

This study assesses the effects of heat on meiotic progression and grain number in hexaploid wheat (Triticum aestivum L. var. Chinese Spring), defines a heat-sensitive stage and evaluates the role of chromosome 5D in heat tolerance. Plants were exposed to high temperatures (30 or 35 °C) in a controlled environment room for 20-h periods during meiosis and the premeiotic interphase just prior to meiosis. Examination of pollen mother cells (PMCs) from immature anthers immediately before and after heat treatment enabled precise identification of the developmental phases being exposed to heat. A temperature-sensitive period was defined, lasting from premeiotic interphase to late leptotene, during which heat can prevent PMCs from progressing through meiosis. PMCs exposed to 35 °C were less likely to progress than those exposed to 30 °C. Grain number per spike was reduced at 30 °C, and reduced even further at 35 °C. Chinese Spring nullisomic 5D-tetrasomic 5B (N5DT5B) plants, which lack chromosome 5D, were more susceptible to heat during premeiosis–leptotene than Chinese Spring plants with the normal (euploid) chromosome complement. The proportion of plants with PMCs progressing through meiosis after heat treatment was lower for N5DT5B plants than for euploids, but the difference was not significant. However, following exposure to 30 °C, in euploid plants grain number was reduced (though not significantly), whereas in N5DT5B plants the reduction was highly significant. After exposure to 35 °C, the reduction in grain number was highly significant for both genotypes. Implications of these findings for the breeding of thermotolerant wheat are discussed.

     

Production of wheat-rye substitution lines based on winter rye cultivars with karyotype identification by means of C-banding,GISH, and SSR markers

The study presents a continuation of the research aimed at producing of wheat-rye substitution lines (2n = 42) based on the cross (Triticum aestivum L. × Secale sereale L.) × Triticum aestivum L., and using winter rye cultivars Vyatka and Vietnamskaya Mestnaya. In BC 1 F 5 two lines were identified, having karyotypes in which a pair of homologous wheat chromosomes was substituted by a homeologous pair of rye chromosomes. The chromosome composition of these lines was analyzed using C-banding, GISH, and SSR markers. It was demonstrated that karyotype of each line included a single pair of rye chromosomes and lacked wheat-rye translocations. The rye chromosomes were identified, and the chromosomes of wheat, at which the substitutions occurred, were determined. The lines generated by crosses with rye of Vyatka and Vietnamskaya Mestnaya cultivars were designated 1Rv(1A) and 5Rviet(5A), respectively. Chromosome identification and classification of the lines makes it possible to use them in breeding programs and genetic studies.     

小麦-黑麦代换系5A/5R与6A/6R杂交诱导同祖染色体配对与易位的研究

利用两个小麦-黑麦异源双代换系DS 5A/5R与DS 6A/6R杂交,探讨同祖染色体配对的可能性与创制小麦黑麦异源易位系.在方法上对杂种F1的减数分裂行为进行研究,观察5R与5A、6R与6A配对频率,探讨同祖染色体配对规律.实验结果看到杂交F1减数分裂中有22.91%的花粉母细胞有小麦染色体(ABD组)与黑麦染色体(R组)发生同祖配对.在F2及以后世代,通过染色体C分带、原位杂交检测,选择小麦-黑麦易位系.在F2代的45株中检测到9株有易位,易位频率为20%,是目前小麦-黑麦染色体易位频率最高的.染色体易位有的来源于同祖配对的交换,有的来源于单价体错分裂或断裂的重建.     

New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance

Powdery mildew is one of the serious diseases of wheat (Triticum aestivum L., 2n = 6 × = 42, genomes AABBDD). Rye (Secale cereale L., 2n = 2 × = 14, genome RR) offers a rich reservoir of powdery mildew resistant genes for wheat breeding program. However, extensive use of these resistant genes may render them susceptible to new pathogen races because of co-evolution of host and pathogen. Therefore, the continuous exploration of new powdery mildew resistant genes is important to wheat breeding program. In the present study, we identified several wheat-rye addition lines from the progeny of T. aestivum L. Mianyang11 × S. cereale L. Kustro, i.e., monosomic addition lines of the rye chromosomes 4R and 6R; a disomic addition line of 6R; and monotelosomic or ditelosomic addition lines of the long arms of rye chromosomes 4R (4RL) and 6R (6RL). All these lines displayed immunity to powdery mildew. Thus, we concluded that both the 4RL and 6RL arms of Kustro contain powdery mildew resistant genes. It is the first time to discover that 4RL arm carries powdery mildew resistant gene. Additionally, wheat lines containing new wheat-rye translocation chromosomes were also obtained: these lines retained a short arm of wheat chromosome 5D (5DS) on which rye chromosome 4R was fused through the short arm 4RS (designated 5DS-4RS·4RL; 4RL stands for the long arm of rye chromosome 4R); or they had an extra short arm of rye chromosome 4R (4RS) that was attached to the short arm of wheat chromosome 5D (5DS) (designated 4RS-5DS·5DL; 5DL stands for the long arm of wheat chromosome 5D). These two translocation chromosomes could be transmitted to next generation stably, and the wheat lines containing 5DS-4RS·4RL chromosome also displayed immunity to powdery mildew. The materials obtained in this study can be used for wheat powdery mildew resistant breeding program.     

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.     

Disruption of male meiosis in transgenic line tobacco res91

The course of meiosis in male-sterile trasgenic tobacco line res91 has been analysed. Cytological analysis included visualization of the spindle and phragmoplast. Abnormal meiosis was characterized with 1) cytomixis; 2) deformation of nuclei at prophase 2 in part of the cells, and lack of spindle formation in such cells at M2; 3) desorientation of spindles in meiosis 2. This set of abnormalities allows to suppose certain disturbancies of cytoskeleton during male meiosis in res91.     

Methomyl,imbraclaobrid and clethodim induced cytomixis and syncytes behaviors in PMCs of Pisum sativum L: Causes and outcomes

Cytomixis is a common phenomenon observed in meiotic cells such as anther which is influenced by various factors. Use of pesticides is a common practice in agriculture. However, it is not known whether pesticides can induce cytomixis in plant cells and induce genetic variation. To understand this, the present study was planned to assess the cytomixis and syncytes behaviors in PMCs of Pisum sativum L. Seeds of P. sativum (Family: Fabaceae) were treated with different concentrations of commonly used pesticides methomyl (ME), imbraclaobrid (IM) and clethodim (CL). Seeds were treated with various concentrations (0.1, 0.2, 0.3, 0.4 and 0.5% of ME, IM and CL prepared in water) for 1 and 3 h. Effect of pesticides on pollen fertility, frequency of cytomixis, and kind of cytomixis cells was assessed. In the cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), and various stages of meiosis (PI, MI, AI and TI) with cytomixis cells were observed. In addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) was assessed. During the microsporogenesis in P. sativum, the occurrence of cytomixis and syncytes at various stages of meiosis I were seen. The formation of cytoplasmic channels and direct fusing of pollen mother cells (PMCs) were both seen to cause cytomixis, with the former being more common than the latter. The percentage of PMCs with cytomixis and syncytes cells increased with increase in the concentration of pesticides. The result of the present investigation indicates that commonly used pesticides ME, IM, and CL have a significant effect on pollen fertility, frequency of cytomixis, and kind of cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), in addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) on P. sativum.     

Cytogenetic studies on microsporogenesis and male gametophyte development in autotriploid cucumber (Cucumis sativus L.): implication for fertility and production of trisomics

To understand the correlation between chromosomes behavior and fertility in autotriploid cucumber (Cucumis sativus L.), microsporogenesis in pollen mother cells (PMCs) and male gametophyte development were studied using improved staining and chromosome preparation techniques. Meanwhile, for more efficient selection of trisomics from the progeny of autotriploid-diploid crosses, fertilization rates of ovules from reciprocal crosses were counted to observe the transfer rate of gametes in the autotriploid cucumber. Variable chromosome configurations, e.g. multivalents, quadrivalents, trivalents, bivalents and univalents were observed in the most PMCs of the autotriploids at metaphase I. Chromosome lagging and bridges at anaphase in both meiotic divisions resulted from irregular chromosome separation and asynchronization was frequently observed as well, which led to formation of micronuclei and inviable gametes. The frequency of normal PMCs in autotriploids at the stage of tetrad was only 40.6%. Among those normal microspores, most of them (91.2%) could develop into normal gametophytes with 2 cells and 3 germ pores. Stainability and germination rate of pollen grains were only 18.8 and 13.5%, respectively. However, chromosomes separated to form gametes with 8 chromosomes at anaphase I, suggesting a possible method for the production of primary trisomics from the progeny of autotriploid-diploid crosses. Fruit set of 3n × 2n and 2n × 3n were 80 and 70%, respectively. It obtained an average of 6.2 plump seeds per fruit in 3n × 2n, while 4.9 in 2n × 3n crosses. Transfer rates of gametes through the gastrula or the pollen in autotriploids were 13.4 and 10.4%, respectively. Some aneuploid gametes (n + 1 = 8, n + 2 = 9) also have capability of setting seed and sexual reproduction besides normal gametes containing whole chromosome sets (n = 7, 2n = 14). Further, some primary trisomic plants were selected from the progeny of autotriploid-diploid crosses. Based on the results obtained we suggest that abnormal meiosis in PMCs was the cytogenetic reason for low fertility of autotriploid cucumber pollen. 3n × 2n cross was more efficient for selecting primary trisomic plants in cucumber.     

Meiosis in a triploid hybrid of <Emphasis Type="Italic">Gossypium</Emphasis>: high frequency of secondary bipolar spindles at metaphase II

Studies on meiosis in pollen mother cells (PMCs) of a triploid interspecific hybrid (3x = 39 chromosomes, AAD) between tetraploid Gossypium hirsutum (4n = 2x = 52,AADD) and diploid G. arboreum (2n = 2x = 26,AA) are reported. During meiotic metaphase I, 13 AA bivalents and 13 D univalents are expected in the hybrid. However, only 28% of the PMCs had this expected configuration. The rest of the PMCs had between 8 and 12 bivalents and between 12 and 17 univalents. Univalents lagged at anaphase I, and at metaphase II one or a group of univalents remained scattered in the cytoplasm and failed to assemble at a single metaphase plate. Primary bipolar spindles organized around the bivalents and multivalents. In addition to the primary spindle, several secondary and smaller bipolar spindles organized themselves around individual univalents and groups of univalents. Almost all (97%) of the PMCs showed secondary spindles. Each spindle functioned independently and despite their multiple numbers in a cell, meiosis I proceeded normally, with polyad formation. These observations strongly support the view that in plant meiocytes bilateral kinetochore symmetry is not required for establishing a bipolar spindle and that single unpaired chromosomes can initiate and stabilize the formation of a functional bipolar spindle.     

The effect of Secale cereale L. heterochromatin on wheat chromosome pairing

Metaphase-I chromosome association in PMCs of five F₁ hybrids 6x-triticale x T. turgidum (2n=5x=35 and genomes AABBR), and 13 plants from their backross or self offspring is reported. In wheat 18 chromosome arms and in rye 14 arms were recognized after C-banding and individually studied. Plants of backcross and F₂ showed variability for number and type of rye chromosomes, having in common the 28 durum wheat chromosomes (AABB). By testing meiotic association in plants with different rye chromosome constitutions, significant negative correlations were found. A clear negative effect of rye heterochromatin on pairing in wheat chromosomes is observed, the influence being more pronounced for large arms than for the short ones.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

Molecular cytogenetic characterization of a new wheat-rye 1BL•1RS translocation line expressing superior stripe rust resistance and enhanced grain yield

Main conclusion

A new wheat-rye 1BL?1RS translocation line, with the characteristics of superior stripe rust resistance and high thousand-kernel weight and grain number per spike, was developed and identified from progenies of wheat-rye- Psathyrostachys huashanica trigeneric hybrids.

Abstract

The wheat-rye 1BL?1RS translocation line from Petkus rye has contributed substantially to the world wheat production. However, due to extensive growing of cultivars with disease resistance genes from short arm of rye chromosome 1R and coevolution of pathogen virulence and host resistance, these cultivars successively lost resistance to pathogens. In this study, a new wheat-rye line K13-868, derived from the progenies of wheat-rye-Psathyrostachys huashanica trigeneric hybrids, was identified and analyzed using fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), acid polyacrylamide gel electrophoresis (A-PAGE), and molecular markers. Cytological studies indicated that the mean chromosome configuration of K13-868 at meiosis was 2n = 42 = 0.14 I + 18.78 II (ring) + 2.15 II (rod). Sequential FISH and GISH results demonstrated that K13-868 was a compensating wheat-rye 1BL?1RS Robertsonian translocation line. Acid PAGE analysis revealed that clear specific bands of rye 1RS were expressed in K13-868. Simple sequence repeat (SSR) and rye 1RS-specific markers ω-sec-p1/ω-sec-p2 and O-SEC5′-A/O-SEC3′-R suggested that the 1BS arm of wheat had been substituted by the 1RS arm of rye. At the seedling and adult growth stage, compared with its recurrent wheat parent SM51 and six other wheat cultivars containing the 1RS arm in southwestern China, K13-868 showed high levels of resistance to stripe rust (Puccinia striiformis f. sp. tritici, Pst) pathogens prevalent in China, which are virulent to Yr10 and Yr24/Yr26. In addition, K13-868 expresses higher thousand-kernel weight and more grain number per spike than these controls in two growing seasons, suggesting that this line may carry yield-related genes of rye. This translocation line, with significant characteristics of resistance to stripe rust and high thousand-kernel weight and grain number per spike, could be utilized as a valuable germplasm for wheat improvement.

     

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Formins regulate phragmoplast expansion, microtubule turnover rate, actin nucleation, and cell plate membrane remodeling during cytokinesis.     

Introgression of the reduced height gene <Emphasis Type="Italic">Rht10</Emphasis> from the wheat cultivar Ai-Bian 1 into the triticale genotype

A gene determining reduced height, Rht10, from the wheat cultivar Ai-Bian 1 was introgressed into the triticale genotype. Initially, Ai-Bian 1 was crossed with the wheat cultivar Chinese Spring (CS), a carrier of Kr genes, to overcome the uncrossability of this cultivar with rye. Amphidiploids were produced by hybridizing the F₂ (CS × Ai-Bian 1) plants displaying reduced height (at the level of Ai-Bian 1) with rye. Free pollination of F₁ (F₂ of CS × Ai-Bian 1) × Saratovskaya 7 with triticale pollen gave fertile viable hybrids; the majority of hybrids were phenotypically closer to octoploid triticale; however, the variants intermediate between octo-and hexaploids were also present. The height of amphidiploids varied from 40 to 90 cm, and the grain yield per spike amounted on the average to 11.7–24.7 grains, which exceeded essentially this value in F₁ plants.     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.