Molecular Analysis of Leaf Rust-Resistant Introgression Lines Obtained by Crossing of Hexaploid Wheat Triticum aestivum with Tetraploid Wheat Triticum timopheevii

Twenty-four Triticum aestivum×T. timopheevii hybrid lines developed on the basis of five varieties of common wheat and resistant to leaf rust were analyzed by the use of microsatellite markers specific for hexaploid wheat T. aestivum. Investigation of intervarietal polymorphism of the markers showed that the number of alleles per locus ranged from 1 to 4, depending on the marker (2.5 on average). InT. timopheevii, amplification fragments are produced by 80, 55, and 30% of primers specific to the A, B, and D common wheat genomes, respectively. Microsatellite analysis revealed two major areas of introgression of the T. timopheevii genome: chromosomes of homoeological groups 2 and 5. Translocations were detected in the 2A and 2B chromosomes simultaneously in 11 lines of 24. The length of the translocated fragment in the 2B chromosome was virtually identical in all hybrid lines and did not depend on the parental wheat variety. In 15 lines developed on the basis of the Saratovskaya-29, Irtyshanka, and Tselinnaya-20, changes occurred in the telomeric region of the long arm of the 5A chromosome. Analysis with markers specific to the D genome suggested that introgressions of the T. timopheevii genome occurred in chromosomes of the D genome. However, the location of these markers on T. timopheevii chromosomes is unknown. Our data suggest that the genes for leaf rust resistance transferred from T. timopheevii to T. aestivum are located on chromosomes of homoeological group 2.     

Wheat Microsatellites: The Prospects of Application for Gene Mapping and Analysis of the Reconstructed Genomes

Microsatellite markers Xgwmand Xgdmwere used to map the S1, S2, and S3genes of the induced sphaerococcoid mutants of Triticum aestivumL. and to analyze the introgressive lines of common wheat, obtained by crossing several common wheat cultivars to T. timopheeviiZhuk.; these lines carry the Lrgenes of resistance to leaf rust. All sphaerococcoid genes were linked to centromeric markers of the short and long arms of chromosomes of homoeologous group 3 of T. aestivum: the S1locus was located between the markers Xgdm72and Xgwm456; the S2gene, betweenXgwm845and Xgwm566; and the S3was located between Xgwm2and Xgwm720. The introgressive lines of common wheat carry the following substitutions from T. timopheevii, most of 2A and 2B and telomeric region of the 5AL chromosome in the line 821, the same introgression and also the completely substituted chromosome 4B in line 837, and the partially substituted chromosomes 2A and 2B in line 842. The introgression of the genomic material fromT. timopheeviiinto the chromosomes of homoeologous group 2 was the common trait of all three lines resistant to leaf rust. The authors discuss the feasibility of using microsatellite-derived data for analyzing nonmapped wheat species, linking new genes to wheat molecular genetic maps, and analyzing wheat genomes of diverse hybrid origins.     

Chromosome painting in meiosis reveals pairing of specific chromosomes in polyploid <Emphasis Type="Italic">Solanum</Emphasis> species

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n?=?4x?=?48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66–78% of the meiotic cells at the pachytene stage. Solanum demissum (2n?=?6x?=?72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83–98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.     

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.     

Structural chromosome differentiation between Triticum timopheevii and T. turgidum and T. aestivum

 Chromosome pairing at metaphase-I was analyzed in F₁ hybrids among T. turgidum (AABB), T. aestivum (AABBDD), and T. timopheevii (A^tA^tGG) to study the chromosome structure of T. timopheevii relative to durum (T. turgidum) and bread (T. aestivum) wheats. Individual chromosomes and their arms were identified by means of C-banding. Homologous pairing between the A-genome chromosomes was similar in the three hybrid types AA^tBG, AA^tBGD, and AABBD. However, associations of B-G were less frequent than B-B. Homoeologous associations were also observed, especially in the AA^tBGD hybrids. T. timopheevii chromosomes 1A^t, 2A^t, 5A^t, 7A^t, 2G, 3G, 5G, and 6G do not differ structurally from their counterpart in the A and B genomes. Thus, these three polyploid species inherited translocation 5AL/4AL from the diploid A-genome donor. Chromosome rearrangements that occurred at the tetraploid level were different in T. turgidum and T. timopheevii. Translocation 4AL/7BS and a pericentric inversion of chromosome 4A originated only in the T. turgidum lineage. The two lines of T. timophevii studied carry four different translocations, 6A^tS/1GS, 1GS/4GS, 4GS/4A^tL, and 4A^tL/3A^tL, which most likely arose in that sequence. These structural differences support a diphyletic origin of polyploid wheats. Received: 15 June 1998 / Accepted: 19 August 1998     

The pattern of polytene chromosome conjugation and crossing-over in interspecific hybrids of Drosophila

Summary The pairing of polytene chromosomes was investigated in the hybrids between three closely related species of Drosophila belonging to the virilis species group. It was found that within the same hybrid different chromosome bands lost the ability to pair by differing degrees. Furthermore, the same chromosome sections paired with different frequencies depending on the hybrid involved. This study revealed that poor polytene chromosome pairing in the hybrids is not due to specific genetic interaction in the hybrids, but depends solely on the properties of the homologous loci themselves. It was also of interest to find whether the pattern of polytene chromosome somatic pairing resembled in some way the picture of chromosome synapsis during meiosis. To obtain evidence for this, crossing-over in the hybrid 5th chromosome was analyzed both genetically and cytologically (from salivary gland chromosome observations). It was found that the sections of the fifth chromosome which were characterized by a high frequency of conjugation in the salivary glands of hybrids also exhibited a high frequency of crossing-over in hybrid females. It may be concluded that sections of the polytene chromosome characterized by a low frequency of conjugation behave in the same manner in meiosis, and thus rarely take part in genetic recombination.     

A study of the storage proteins in the introgression lines of common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L. × <Emphasis Type="Italic">T. timopheevii</Emphasis> Zhuk.) Resistant to brown leaf rust

Storage proteins, prolamins, were studied in ten introgression lines of common wheat bred with involvement of Triticum timopheevii (Tt) Zhuk. and five commercial hexaploid wheat cultivars. The lines are resistant to leaf rust. A comparative analysis of the storage proteins in the Triticum aestivum L. (Ta) introgression lines and the parental forms allowed us to (1) detect the active genes of prolamins on the chromosomes homeologous groups 1 and 6 in the introgression lines of T. aestivum and T. timopheevii; (2) clarify their origin; (3) identify the chromosome attribution of the products; (4) estimate the degree of introgression and postulate the introgression mechanisms; and (5) predict the bread-making quality of these introgression lines.     

Characterization of an Alien Chromosome of <Emphasis Type="Italic">Oryza officinalis</Emphasis> Transferred the Genomic and Cytological Environment of <Emphasis Type="Italic">Oryza sativa</Emphasis>

Distant hybridization between rice species and their genome interactions have become the hot points of genetic research. The new formed hybrid was unstable and many of the alien chromosomes would be excluded out in the meiosis procedure. In this study, we investigated the phenomenon that the monosomic alien addition lines (MAALs) of Oryza officinalis (CC)–Oryza sativa (AA) inherited in the genomic and cytoplasmic environments of cultivated rice. In a series of backcross programs, every time the MAALs were backcrossed with the recurrent parent Hejiang19, we get a portion of the backcross progenies that were similar to the female parent MAALs in morphological characters (with rolled leaves) and chromosome constitutions (2n + 1). The monosomic alien chromosome exhibited stable inheritance in the genomic and cytoplasmic environments of cultivated rice. Molecular biology and fluorescence in situ hybridization (FISH) analyses on mitotic cells showed that the alien chromosomes in MAALs were inclined to be transferred integrated into the backcross progenies with rolled leaves and no big fragments of alien chromosomes introgressing into the chromosomes of cultivated rice. By FISH analysis on the meiotic cells, we proved that the single alien chromosome performing nonstandard meiosis and behaving like a dispensable genetic factor, which would be the cytological reasons for the stable inheritance of alien chromosomes.     

Chromosomal Behavior during Meiosis in the Progeny of Triticum timopheevii × Hexaploid Wild Oat

The meiotic behavior of pollen mother cells (PMCs) of the F₂ and F₃ progeny from Triticum timopheevii × hexaploid wild oat was investigated by cytological analysis and sequential C-banding-genomic in situ hybridization (GISH) in the present study. A cytological analysis showed that the chromosome numbers of the F₂ and F₃ progeny ranged from 28 to 41. A large number of univalents, lagging chromosomes, chromosome bridges and micronuclei were found at the metaphase I, anaphase I, anaphase II and tetrad stages in the F₂ and F₃ progeny. The averages of univalents were 3.50 and 2.73 per cell, and those of lagging chromosomes were 3.37 and 1.87 in the F₂ and F₃ progeny, respectively. The PMC meiotic indices of the F₂ and F₃ progeny were 12.22 and 20.34, respectively, indicating considerable genetic instability. A sequential C-banding-GISH analysis revealed that some chromosomes and fragments from the hexaploid wild oat were detected at metaphase I and anaphase I in the progeny, showing that the progeny were of true intergeneric hybrid origin. The alien chromosomes 6A, 7A, 3C and 2D were lost during transmission from F₂ to F₃. In addition, partial T. timopheevii chromosomes appeared in the form of univalents or lagging chromosomes, which might result from large genome differences between the parents, and the wild oat chromosome introgression interfered with the wheat homologues’ normally pairing.     

Chromosome synteny of the a genome of two evolutionary wheat lines

In order to estimate synteny between A^t and A polyploid wheat genomes belonging to different evolutionary lines (Timopheevi and Emmer), saturation of chromosome maps of Triticum timopheevii A^t genome by molecular markers has been conducted. Totally, 179 EST-SSR and 48 genomic SSR-markers have been used with the following integration of 13 and 7 markers correspondingly into chromosome maps of A^t genome. ESTSSR showed higher transferability and lower polymorphism than genomic SSR markers. The chromosome maps designed were compared to maps of homoeologous chromosome group of the T. aestivum A genome. No disturbances of colinearity, i.e., of the order of markers within the chromosome segments on which they had been previously mapped, were observed. According to the quantity assessment of markers amplifying in homoeologous chromosomes, the maximum divergence was detected in two groups (4A^t/4A and 3A^t/3A) among the seven chromosomes examined in the A ^t and A genomes. Comparison of molecular genetic mapping results with the published results of studying meiosis of F₁ hybrids and the frequency of chromosomes substitution in introgressive T. aestivum × T. timopheevii lines suggest that individual chromosomes of the A^t and A genomes evolve differently. Translocations were shown to introduce the major impact on the divergence of 4A^t/4A and 6A^t/6A chromosomes, while mutations of the primary DNA structure, on the divergence of homoeologous group 3 chromosomes. The level of reorganization of other chromosomes during the evolution in the A^t and A genomes was significantly lower.     

应用原位杂交技术对小麦—黑麦代换系间杂交的细胞遗传研究

应用原位杂交技术结合染色体组型分析方法,对两个小麦-黑麦异源双代换系5R/5A和6R/6A杂交后代的遗传进行了研究,探讨同祖染色体配对的可能性并获得小麦-黑麦易位系。实验中对杂种F1代植株减数分裂各时期的花粉母细胞染色体行为进行分析,结果发现有22.91%的花粉母细胞中黑麦染色体与小麦染色体发生同祖配对。F2代通过C-分带、原位杂交鉴定,在45株中检测到9株易位,易位频率为20%,是目前报道易位频率最高的。染色体易位有的来源于同祖配对交换,有的来源于单价体错分裂或断裂的重建。     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Chromosome substitutions of Triticum timopheevii in common wheat and some observations on the evolution of polyploid wheat species

Whether the two tetraploid wheat species, the well known Triticum turgidum L. (macaroni wheat, AABB genomes) and the obscure T. timopheevii Zhuk. (A^tA^tGG), have monophyletic or diphyletic origin from the same or different diploid species presents an interesting evolutionary problem. Moreover, T. timopheevii and its wild form T. araraticum are an important genetic resource for macaroni and bread-wheat improvement. To study these objectives, the substitution and genetic compensation abilities of individual T. timopheevii chromosomes for missing chromosomes of T. aestivum Chinese Spring (AABBDD) were analyzed. Chinese Spring aneuploids (nullisomic-tetrasomics) were crossed with a T. timopheevii x Aegilops tauschii amphiploid to isolate T. timopheevii chromosomes in a monosomic condition. The F₁ hybrids were backcrossed one to four times to Chinese Spring aneuploids without selection for the T. timopheevii chromosome of interest. While spontaneous substitutions involving all A^t- and G-genome chromosomes were identified, the targeted T. timopheevii chromosome was not always recovered. Lines with spontaneous substitutions from T. timopheevii were chosen for further backcrossing. Six T. timopheevii chromosome substitutions were isolated: 6A^t (6A), 2G (2B), 3G (3B), 4G (4B), 5G (5B) and 6G (6B). The substitution lines had normal morphology and fertility. The 6A^t of T. timopheevii was involved in a translocation with chromosome 1G, resulting in the transfer of the group-1 gliadin locus to 6A^t. Chromosome 2G substituted for 2B at a frequency higher than expected and may carry putative homoeoalleles of gametocidal genes present on group-2 chromosomes of several alien species. Our data indicate a common origin for tetraploid wheat species, but from separate hybridization events because of the presence of a different spectrum of intergenomic translocations.     

白菜型油菜(B. Chinensis)与海甘蓝(Crambe abyssinica)属间杂种的细胞学观察

白菜型油菜与海甘蓝属间杂种F₁代染色体数目早期为2n=55。经多代组培无性繁殖后,杂种染色体大量减少,数目变化于25～28条间,平均为26条。杂种PMC减数分裂中,平均配对构型为0.06Ⅲ+11.26Ⅱ+3.80Ⅰ,二价体数目变化于8～13之间,以10Ⅱ、11Ⅱ、12Ⅱ的细胞出现频率较高,分别为26.60%,23.91%和30.98%,单价体数目变化于0～8个之间。杂种染色体数目减少,二价体个数较高,这说明海甘蓝染色体在大量丢失的同时,白菜型油菜的染色体组发生了自然加倍。在后期Ⅱ观察到三分体、染色体落后、染色体桥等异常现象。      

Characterization and mapping of cryptic alien introgression from <Emphasis Type="Italic">Aegilops geniculata</Emphasis> with new leaf rust and stripe rust resistance genes <Emphasis Type="Italic">Lr57</Emphasis> and <Emphasis Type="Italic">Yr40</Emphasis> in wheat

Leaf rust and stripe rust are important foliar diseases of wheat worldwide. Leaf rust and stripe rust resistant introgression lines were developed by induced homoeologous chromosome pairing between wheat chromosome 5D and 5M^g of Aegilops geniculata (U^gM^g). Characterization of rust resistant BC₂F₅ and BC₃F₆ homozygous progenies using genomic in situ hybridization with Aegilops comosa (M) DNA as probe identified three different types of introgressions; two cytologically visible and one invisible (termed cryptic alien introgression). All three types of introgression lines showed similar and complete resistance to the most prevalent pathotypes of leaf rust and stripe rust in Kansas (USA) and Punjab (India). Diagnostic polymorphisms between the alien segment and recipient parent were identified using physically mapped RFLP probes. Molecular mapping revealed that cryptic alien introgression conferring resistance to leaf rust and stripe rust comprised less than 5% of the 5DS arm and was designated T5DL·5DS-5M^gS(0.95). Genetic mapping with an F₂ population of Wichita × T5DL·5DS-5M^gS(0.95) demonstrated the monogenic and dominant inheritance of resistance to both diseases. Two diagnostic RFLP markers, previously mapped on chromosome arm 5DS, co-segregated with the rust resistance in the F₂ population. The unique map location of the resistant introgression on chromosome T5DL·5DS-5M^gS(0.95) suggested that the leaf rust and stripe rust resistance genes were new and were designated Lr57 and Yr40. This is the first documentation of a successful transfer and characterization of cryptic alien introgression from Ae. geniculata conferring resistance to both leaf rust and stripe rust in wheat.     

B chromosomes in Stylidium crossocephalum (Angiospermae:Stylidiaceae)

Populations of Stylidium crossocephalum contain two common types of B chromosomes, macro B chromosomes and micro B chromosomes. The macro B chromosomes are telocentric, slightly smaller than the smallest A chromosomes and mitotically unstable. They have so far been found associated with 6 of the 16 stable genomes known in S. crossocephalum, occurring in populations covering a substantial portion of the species range. Micro B chromosomes are about one third the length of the smallest A chromosome, acrocentric and show some mitotic instability. They occur associated with 3 stable genomes in populations found in the more medial regions of the species range. Both types of B chromosomes generally show regular behaviour during meiosis, although when two or more are present their pairing efficiency is reduced when compared to the A chromosomes. In addition a single very large mega chromosome was found in a single cell of one heterokaryotypic plant. Its origin although conjectural at this stage may be of relevance in understanding the origin of macro and micro B chromosomes in this species.     

The genetics of meiotic chromosome pairing in Lolium temulentum x Lolium perenne tetraploids

Summary The degree of preferential pairing of homologous chromosomes was estimated in a series of tetraploid hybrids of Lolium temulentum x Lolium perenne by means of cytological and genetic analyses. The correlations between the frequency of bivalents at first metaphase of meiosis in the hybrid tetraploids and the degree of preferential pairing calculated from the segregation pattern of isozyme alleles in a test cross was extremely high. The results showed clearly that suppression of heterogenetic pairing in these Lolium tetraploids is achieved by a genetic system involving the A chromosomes as well as the B chromosome system which has been known for some time. Certain similarities with the genetic system controlling pairing in polyploid wheats are discussed.     

Marker-assisted development and characterization of a set of Triticum aestivum lines carrying different introgressions from the T. timopheevii genome

A set of common wheat introgression lines carrying one or two introgressions from Triticum timopheevii was produced by means of marker-assisted backcross selection. The starting material consisted of two BC1F20 (T. aestivum*2/T. timopheevii) lines with resistance to leaf rust, stem rust, powdery mildew, spot blotch, and loose smut and containing multiple 1A^t, 2A^t, 2G, 3A^tL, 3GL, 4GL, 5A^tL, 5GL, and 6G T. timopheevii chromosome fragments. The two lines were crossed with, and backcrossed three times to common wheat cultivar Saratovskaya 29. In total, 275 BC2F1 and BC3F2 plants were characterized by microsatellite markers and in situ hybridization. Molecular and cytological analyses revealed 38 plants with a single introgression from chromosomes 2G, 5GL, or 6G of T. timopheevii and 72 plants, each with two introgressions, among them three plants carrying a T. timopheevii translocation involving the D genome (2DS.2GL). It was observed that the lengths of fragments introgressed from the A^t genome were more than halved in the BC2 generation, while the lengths of 2G and 5GL introgressed fragments were only slightly reduced after the third backcross. The introgression lines were tested for resistance to the native Puccinia triticina population of the Western Siberian region of Russia. Lines with a single introgressed 5GL region carrying the major leaf rust resistance locus, QLr.icg-5B, were completely resistant. The presence of two minor resistance loci, QLr.icg-2A and QLr.icg-1A, suppressed disease development and reduced the number of urediniospores by up to 25 % but did not lead to a hypersensitive response. The introgression lines therefore constitute promising sources of new resistance to Puccinia triticina.     

Genome relationships between Hordeum vulgare L. and H. bulbosum L.

Interrelationships between H. vulgare (2x=14) and H. bulbosum (2x=14; 4x=28) were estimated on the basis of the karyotypes and the pairing behaviour of the chromosomes in diploid, triploid and tetraploid hybrids obtained with the aid of embryo culture. — A comparison of the karyotypes of the two species revealed similarities as well as differences. It was concluded that at least 4 or more of the chromosomes were similar in morphology and probably closely related. — Diploid and tetraploid hybrids are rarely obtained and their chromosome numbers tend to be unstable whereas triploid hybrids (1 vulgare + 2 bulbosum genomes) were stable and relatively easy to produce. In the diploid hybrid only 40% of the meiotic cells contained 14 chromosomes while the numbers ranged from 7 to 16 in other cells. All hybrids exhibited pairing between the chromosomes of the two species. Diploid hybrids had a mean of 5.0 and a maximum of 7 bivalents per cell in those cells having 14 chromosomes. Triploid hybrids from crosses between 2x H. vulgare and 4x H. bulbosum exhibited a mean of 1.5 and a maximum of 5 trivalents per cell. In a hexaploid sector found following colchicine treatment of a triploid the mean frequencies of chromosome associations per cell were: 5.5_I+8.0_II+0.7_III+3.7_IV+0.3_V+0.4_VI. One unstable 27 chromosome hybrid obtained from crosses between the autotetraploid forms had a mean of 1.1 and a maximum of 4 quadrivalents per cell. The chromosome associations observed in these hybrids are consistent and are taken as evidence of homoeologous pairing between the chromosomes of the two species. Interspecific hybridization between these two species also reveals that chromosome stable hybrids are only obtained when the genomes are present in a ratio of 1 vulgare2 bulbosum. Based upon the results obtained, the possibility of transferring genetic characters from H. bulbosum into cultivated barley is discussed.     

Inducing chromosome pairing through premature condensation: analysis of wheat interspecific hybrids

At the onset of meiosis, chromosomes first decondense and then condense as the process of recognition and intimate pairing occurs between homologous chromosomes. We show here that okadaic acid, a drug known to induce chromosome condensation, can be introduced into wheat interspecific hybrids prior to meiosis to induce chromosome pairing. This pairing occurs in the presence of the Ph1 locus, which usually suppresses pairing of related chromosomes and which we show here delays condensation. Thus the timing of chromosome condensation during the onset of meiosis is an important factor in controlling chromosome pairing.