Study of mitochondrial genomes of allopllasmic recombinant wheat lines constructed on the basis of barley-wheat hybrids Hordeum geniculatum all. (= H. marinum subsp. gussoneanum) (2n = 28) x x Triticum aestivum L. (2n = 42) with using of RELP and PCR analyses

Using RELP analysis with three probes homologous to specific regions of mitochondrial DNA genes and PCR analysis of the mitochondrial recombining-repeat-sequence 18S/5S region of cereals, five alloplasmic wheat lines of different origin and fertility expression were studied. These lines are self-pollinated progeny of BC1-BC4 generations of barley-wheat hybrids Hordeum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42). It was found that recombinant alloplasmic lines characterized by partial fertility contain either maternal (barley) DNA fragments or maternal and paternal (wheat) DNA fragments simultaneously (heteroplasmy). In lines with stable expression of self-fertility, fragments of only paternal mitochondrial DNA were detected. It is assumed that in alloplasmic lines, there is the interrelation between the presence of definite fragments of the mitochondrial genome belonging to either parental type and fertility expression.     

Molecular study and C-banding of chromosomes in common wheat alloplasmic lines obtained from the backcross progeny of barley–wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42) and differing in fertility

We studied common wheat alloplasmic lines differing in fertility traits, which had been obtained from the backcross progeny of barley—wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42), using molecular analysis and chromosome C-banding. It was found that the nuclei of all alloplasmic lines studied, regardless of their fertility traits, contained only the common wheat chromosomes (2n = 42). The formation of line L-79(10)(3)F₆, stable for self-fertility, from line L-79(10)F₃ was accompanied by changes of the proportions of simple sequence repeats of the parental common wheat varieties in the nuclear genome. The presence of barley genome fragments in line accessions with incomplete self-fertility was shown by RAPD. Heteroplasmy for mitochondrial genome loci was detected in these lines with the use of primers specific to the tMet-18S-5S repeat of mitochondrial ribosomal genes.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1668–1677.Original Russian Text Copyright © 2004 by Bildanova, Badaeva, Pershina, Salina.     

Expression of Fertility during Morphogenesis in Self-Pollinated Backcrossed Progenies of Barley—Wheat Amphiploids [Hordeum geniculatum All. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70)

The fertility characteristics expressed during morphogenesis in first-generation self-pollinated backcrossed progenies (BC₁) obtained from amphiploid barley–wheat hybrids [Hordeum geniculatum All. (2n= 28) ×Triticum aestivum L. (2n= 42)] (2n = 70) backcrossed with common wheat were studied. It was found that, in the case of self-pollination of BC₁ plants, karyotype stabilization leads to the formation of alloplasmic euploid (2n = 42), telocentric substitution (2n = 40 + 2t), and telocentric addition (2n = 42 + t), (2n = 42 + 2t) plant forms, which may serve as the sources of the respective alloplasmic lines of common wheat. That the expression of fertility characters in BC₁F₈plants was shown to depend on growth conditions. The main mechanism of hybrid incompatibility of BC₁F₁–BC₁F₈plants was expressed as grass-clump dwarfism.     

RAPD-Based Analysis of Introgression of Barley Genetic Material into the Genome of Alloplasmic Wheat Lines (Hordeum geniculatumAll./Triticum aestivum L.)

Genomes of three alloplasmic wheat lines obtained on the basis of barley–wheat hybrid Hordeum geniculatumAll. (2n = 28) ×Triticum aestivumL. (2n = 42)(Pyrotrix 28) were examined using random amplified polymorphic DNA (RAPD) analysis. Line L-29 was obtained after first backcross of the initial hybrid with the wheat variety Pyrotrix 28 and ten subsequent self-pollinating generations. This line was represented by euploid plants with typical to the common wheat chromosome number (2n = 42), as well as by aneuploids, which contained an additional telocentric chromosome in the main karyotype (2n = 42 + t). Lines L-26 and L-27 were obtained by two backcrosses of one BC₁ plant with the wheat variety Novosibirskaya 67 and one subsequent self-polination of one BC₃ plant. Chromosome number in all these plants corresponded to 2n = 40 + 4t. RAPD analysis was carried out using seven primers, which were previously proved to be effective for identification of the barley genome fragments within hybrid genomes of alloplasmic lines. The presence of barley genome fragments in line L-29 was revealed by use of five primers, while in lines L-26 and L-27 these fragments were detected by use of one primer. The significant difference in the number of barley RAPD fragments in the genomes of alloplasmic lines obtained at different backcrossing stages suggests more intense displacement of barley genome during backcrossing compared to self-pollination in BC₁ plants.     

Production of alloplasmic and euplasmic wheat-barley ditelosomic substitution lines 7H<Superscript>1</Superscript>L<Superscript>mar</Superscript>(7D) and analysis of the 18S/5S mitochondrial repeat in these lines

Alloplasmic lines of common wheat with disomic substitution of chromosome 7D for telocentric chromosome 7H¹L^mar of barley H. marinum subsp. gussoneanum Hudson were isolated from the plants of generation BC₃, produced as a result of backcrossing of barley-wheat hybrids H. marinum subsp. gussoneanum (2n = 28) × T. aestivum (2n = 42), Pyrotrix, cultivar, with 28 common wheat cultivars Pyrotrix 28 and Novosibirskaya 67. Chromosome substitution pattern was determined using SSR analysis and C-banding. In preliminary genomic in situ hybridization experiments, telocentric chromosomes were assigned to wild barley was established. In the BC₃F₈ generations of three alloplasmic lines with the 7H¹L^mar(7D) substitution type the differences in fertility manifestation were observed: most of the L-32(1) plants were sterile, in line L-32(2) only sporadic plants were sterile, and line L-32(3) was fertile. Simultaneously with these experiments, using selfpollinated progeny of the hybrids obtained in crosses of common wheat cultivar Saratovskaya 29 (2n = 41), monosomic for chromosome 7D, with common wheat cultivar Pyrotrix 28 with addition of pair of telocentric chromosomes 7H¹L^mar (7D) of barley H. marinum subsp. gussoneanum, euplasmic wheat-barley ditelosomic substitution 7H¹L^mar (7D) lines were isolated. The lines obtained had normal fertility. PCR analysis of the 18S/5S mitochondrial repeat (hereafter, mtDNA sequence) in alloplasmic and euplasmic ditelosomic substitution lines 7H¹L^mar(7D) was performed. In the plants from alloplasmic sterile line L-32(1), the sequences only of the barley (maternal) type were revealed, while the plants from alloplasmic fertile lines L-32(2) and L-32(3) demonstrated heteroplasmy (the presence of barley- and wheat-like sequences within one individual). In euplasmic ditelosomic substitution lines the presence of only wheat-like 18S/5S mitochondrial repeat sequences was observed. The results indicate that the presence of barley-like mtDNA sequences in alloplasmic substitution lines was not associated with the presence of barley chromosomes in their nuclear genomes.     

Specific Features of the Nuclear Genome Recombination in Backcross Progenies of Barley–Wheat Hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42)

The backcross progenies of the barley–wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n= 42) and two alloplasmic lines derived from them were studied using microsatellite markers of barley and wheat. The F₁ hybrids and first backcross plants BC₁ contained the genetic material of both cultivated barley and the cultivars of common wheat involved in developing of these hybrid genotypes. The genomes of BC₃, BC₄, and alloplasmic lines contained no microsatellite markers of the cultivated barley, whereas chromosomes of each homeologous group of common wheat were identified. In chromosomes of backcross progenies BC₃, BC₄, and alloplasmic lines yielded by backcrosses of hybrids and various common wheat cultivars, microsatellite markers of the parental wheat cultivars were shown to undergo recombination.     

Investigation of the Backcross Progeny of Barley–Wheat Hybrids by the RAPD and RAMPO Methods

The genomes of alloplasmic wheat lines were analyzed by PCR-based methods: random amplified polymorphic DNA (RAPD) and random amplified microsatellite polymorphism (RAMPO). Lines L-16(1) and L-17(2) were obtained by three backcrosses and line L-79(10), by four backcrosses of the barley–wheat hybrid Hordeum vulgare (2n = 14) (variety Nepolegayushchii) × Triticum aestivum (2n = 42) (variety Saratovskaya 29) with different common wheat varieties. These lines proved to be euploid (2n= 42). The aneuploid line L-9 (2n = 43 +t) was obtained after a second backcross of the hybridH. geniculatum All. (2n = 28) × T. aestivum (2n = 42) (Pyrotrix 28) with the variety Pyrotrix 28. The RAPD patterns of L-16(1) and L-17(1) contained fragments present only in the patterns of the parental wheat varieties and, in addition, fragments absent from the latter. This fragment from the pattern of L-16(1) was cloned. Analysis of its primary structure showed that the difference between L-16(1) and the parental wheat genotypes may be related to a mutation that had occurred during the development of the alloplasmic line at the binding site of an arbitrary primer. The genomes of plants of the lines L-79(10) and L-9 contain, in addition to the RAPD fragments of wheat, those characteristic of barley. RAMPO revealed higher polymorphism level among wheat varieties than that detected by RAPD. The hybridization patterns of the lines L-16(1), L-17(1), and L-79(10) contained fragments specific for wheat, and the patterns of L-9 contained both wheat and barley fragments.     

Production and molecular and cytogenetic analyses of euploid (2n = 42) and telocentric addition (2n = 42 + 2t) alloplasmic lines (Hordeum marinum subsp. gussoneanum)-Triticum aestivum

Individual plants from the BC₁F₆ and BC₁F₈ backcross progenies of barley-wheat [H. marinum subsp. gussoneanum Hudson (=H. geniculatum All.) (2n = 28) × T. aestivum L. (2n = 42)] and the BC₁F₆ progeny of their amphiploids were used to obtain alloplasmic euploid (2n = 42) lines L-28, L-29, and L-49 and alloplasmic telocentric addition (2n = 42 + 2t) lines L-37, L-38, and L-50. The lines were examined by genomic in situ hybridization (GISH), microsatellite analysis, chromosome C-banding, and PCR analysis of the mitochondrial 18S/5S repeat. Lines L-29 and L-49 were characterized by substitution of wild barley chromosome 7H¹ for common wheat chromosome 7D. In line L-49, common wheat chromosomes 1B, 5D, and 7D were substituted with homeologous barley chromosomes. Lines L-37, L-38, and L-50 each contained a pair of telocentric chromosomes, which corresponded to barley chromosome arm 7H¹L. All lines displayed heteroplasmy for the mitochondrial 18S/5S locus; i.e., both barley and wheat sequences were found. Original Russian Text ? N.V. Trubacheeva, E.D. Badaeva, I.G. Adonina, L.I. Belova, E.P. Devyatkina, L.A. Pershina, 2008, published in Genetika, 2008, Vol. 44, No. 1, pp. 81–89.     

Alloplasmic male sterility in AD allotetraploid Gossypium hirsutum upon replacement of its resident A cytoplasm with that of D species G. harknessii

Summary Alloplasmic male sterile (cms) and restoration-of-fertility (Rf) lines of the AD allotetraploid Gossypium hirsutum were earlier derived from the presumed introgression of the cytoplasm of the D species G. harknessii. To confirm that this happened and address its significance, cytoplasms of the maternal progenitor, backcross intermediates, derived breeding lines, related A, D, and F species, and a synthetic AD tetraploid were examined by agarose and polyacrylamide gel electrophoresis of 140 restriction enzyme fragments of chloroplast DNA. Length mutations of 10–50 nucleotides predominate over site loss/gain mutations. Chloroplast DNA is maternally inherited and that of G. harknessii has been maintained in the cms lines for at least 13 successive generations without detectable alteration. Chloroplast DNA divergence is consistent with current nuclear genome classification and shows that the A progenitor was the maternal parent of the AD tetraploids. As predicted from incompatability models of cms, the degree of male sterility in alloplasmic Gossypium tetraploids is correlated with the extent of evolutionary divergence of their cytoplasms. It is suggested that the A genome in the AD tetraploids dominates those nuclear-cytoplasm interactions reflected by male fertility.     

Construction of Rye-Wheat Amphidiploids with the Cytoplasm of Rye—Secalotriticum (RRAABB, 2<Emphasis Type="Italic">n</Emphasis> = 42): Meiosis Characteristics in Rye-Triticale F<Subscript>1</Subscript> Hybrids (RRABR, 5<Emphasis Type="Italic">x</Emphasis> = 35)

Results of cytogenetic analysis of microsporogenesis in rye-triticale F₁ hybrids (RRABR, 5x = 35) are presented. Meiosis of pentaploids is described as the key stage in the synthesis of secalotriticum, lines with intergenomic substitutions of chromosomes, or alloplasmic rye and wheat lines. Properties of meiotic processes and cytological mechanisms in formation of functional gametes and plants of various genomic and chromosomal composition are discussed.__________Translated from Genetika, Vol. 41, No. 7, 2005, pp. 902–909.Original Russian Text Copyright © 2005 by Lyusikov, Bel’ko, Shchet’ko, Gordei.     

Barley Chromosome Identification Using Genomic in Situ Hybridization in the Genome of Backcrossed Progeny of Barley–Wheat Amphiploids [Hordeum geniculatum all. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70)

Genomic in situ hybridization (GISH) has been used to study characteristics of the formation of alloplasmic lines detected among self-pollinated backcrossed progeny (BC₁F₅–BC₁F₈) of barley–wheat amphiploids [Hordeum geniculatum All. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70). The chromosome material of the wild barley H. geniculatum has been shown to contribute to these lines. For example, fifth-generation plants (BC₁F₅) had genotypes (2n= 42w + 2g), (2n = 42w + 1g + 1tg), and (2n = 41w + 1g), where w is common wheat chromosomes, g is barley (H. geniculatum) chromosomes, and tg is the telocentric chromosome of wild barley. Beginning from theBC₁F₆ generation, alloplasmic telocentric addition lines (2n= 42 + 2tg) and (2n = 42 + 1tg) appear. This lines has been found cytogenetically unstable. The progeny of each of these cytological types include not only the (2n= 42 + 2tg) and (2n = 42 + 1tg) addition plants, but also plants with the monosomic (2n = 41 + 1tg) and the disomic (2n = 40 + 2tg) substitutions, as well as the (2n = 41 + 2tg) plants, which lack one wheat chromosome and have two telocentric barley chromosomes. It has been demonstrated that the selection for well-filled grains favors the segregation of telocentric addition lines (2n = 42 + 2tg) and (2n = 42 + 1tg).     

Search for Protein Markers for the Genetic CMS–Rf System in Sunflower

Pollen and anthers of the F₁ hybrid with the restored fertility and the parental forms, a maternal male-sterile line harboring the mitochondrial orfH522 gene and a paternal male-fertile line harboring dominant alleles of the nuclear Rf gene, were studied. In order to find protein markers for the genetic CMS–Rf system in sunflower, we analyzed cytoplasmic proteins by disc electrophoresis under denaturing conditions. As a result, a protein fraction with a mol wt of 43 kD specific for cytoplasmic proteins of the paternal anthers, and a protein fraction with a mol wt of 16 kD specific for cytoplasmic proteins of the maternal anthers, were detected. The electrophoretic patterns of water-soluble proteins from the hybrid and paternal lines did not differ.     

Heteroplasmy and paternally oriented shift of the organellar DNA composition in barley-wheat hybrids during backcrosses with wheat parents.

Mitochondrial (mt) and chloroplast (ct) genome inheritance was studied in barley-wheat hybrids, as were their progenies obtained from backcrosses with different common wheat cultivars, by monitoring the composition of 4 mtDNA (coxI, a 5'-flanking region of cob, nad3-orf156, and 5'-upstream region of 18S/5S) and 2 ctDNA (simple-sequence repeat locus downstream of trnS and a 3'-flanking region of rbcL) loci. In male sterile F1 and BC1 plants, maternal barley mtDNA fragments were mainly detected and very low levels of paternal wheat fragments were occasionally detected by PCR in coxI, a 5'-flanking region of cob and nad3-orf156, whereas a 5'-upstream region of 18S/5S showed clear heteroplasmy, containing both maternal and paternal copies, with maternal copies prevailing. Plants showing such heteroplasmic mtDNA composition remained either semisterile or became completely sterile in the later backcross generations. Only maternal ctDNA copies were detected in these plants. In 3 stable, self-fertile, and vigourous lines obtained in the advanced backcross generations and possessing recombinant wheat nuclear genome, however, only mt- and ctDNA copies of wheat parents were detected; thus, the original alloplasmic condition appeared to be lost. Our results suggest that transmission followed by selective replication of the paternal wheat organellar DNA leads to a paternally oriented shift of the organellar DNA composition in barley-wheat hybrids, which correlates with the restoration of fertility and plant vigour. These 2 processes seem to be related to nucleocytoplasmic compatibility and to be under the control of the nuclear genome composition.     

Characterization of fertility restoration in alloplasmic lines derived from hybridization of self-fertilized offspring of barley-wheat (Hordeum vulgare L. × Triticum aestivum L.) amphiploid with common wheat varieties Saratovskaya 29 and Pyrotrix 28

The problems of fertility restoration in the progeny of barley-wheat hybrids (H. vulgare × T. aestivum) are explained by incompatibility between the cytoplasm of cultivated barley and the nuclear genome of common wheat. Appropriate models for studying these problems are alloplasmic lines that combine the cytoplasm of barley and the nuclear genome of wheat. In this work, the differences of fertility restoration in alloplasmic common wheat lines (H. vulgare)-T. aestivum were studied depending on the influence of wheat varieties Saratovskaya 29 (Sar29) and Pyrotrix 28 (Pyr28) used to produce these lines. The alloplasmic lines were created using hybrids between the 48-chromosome offspring (Amph₁) of the barley-wheat amphiploid H. vulgare (ya-319) × T. aestivum (Sar29) and these wheat varieties. Backcrossing of the Amph₁ (2n = 48) × Sar29 hybrid with the wheat variety Sar29 resulted in the complete sterility in the (H. vulgare)-Sar29 line, which suggests the incompatibility of the nuclear genome of the common wheat variety Sar29 with the cytoplasm of H. vulgare. Crossing of Amph₁ (2n = 48) with Pyr28 resulted in the restoration of self-fertility in the hybrid with 2n = 44. In the alloplasmic lines (2n = 42) formed based on plants of the self-fertilized generations of this hybrid, the barley chromosomes were eliminated, and recombination between the nuclear genomes of the parental wheat varieties Sar29 and Pyr28 took place. Alloplasmic recombinant lines (H. vulgare)-T. aestivum with different levels of fertility were isolated. As was shown by the SSR analysis, differences in the fertility between these lines are determined by differences in the content of the genetic material from the wheat varieties Sar29 and Pyr28. The complete restoration of fertility in these alloplasmic recombinant lines is accompanied by the formation of a nuclear genome in which the genetic material of Pyr28 significantly prevails. The conclusion is made that the common wheat variety Pyrotrix 28 is a carrier of a gene (or genes), which determines the restoration of common wheat fertility on the cytoplasm of cultivated barley.     

Molecular study and C-banding of chromosomes in common wheat alloplasmic lines obtained from the backcross progeny of barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42) and differing in fertility

We studied common wheat alloplasmic lines differing in fertility traits, which had been obtained from the backcross progeny of barley-wheat hybrids Hordeum vulgare L. (2n = 14) x Triticum aestivum L. (2n = 42), using molecular analysis and chromosome C-banding. It was found that the nuclei of all alloplasmic lines studied, regardless of their fertility traits, contained only the common wheat chromosomes (2n = 42). The formation of line L-79(10)(3)F6, stable for self-fertility, from line L-79(10)(3)F6 was accompanied by changes of the proportions of simple sequence repeats of the parental common wheat varieties in the nuclear genome. The presence of barley genome fragments in line accessions with incomplete self-fertility was shown by RAPD. Heteroplasmy for mitochondrial genome loci was detected in these lines with the use of primers specific to the tMet-18S-5S repeat of mitochondrial ribosomal genes.     

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 mol·mg^–1·min^–1, 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.Abbreviation RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Cytogenetic analysis of alloplasmic recombinant lines (H. vulgare)—T. aestivum unstable in fertility and viability

Comparative cytogenetic analysis was performed with four alloplasmic recombinant (Hordeum vulgare)—Triticum aestivum lines differing in morphological traits, number of seeds per spike, and seed plumpness. None of the lines displayed introgression of the barley genetic material: the karyotypes included only common wheat chromosomes. Two lines, 79(B) and 79(D), were cytogenetically stable. Plants of lines 79(A) and 79(C) displayed a high frequency of unbalanced chromosome aberrations, including dicentric and polycentric chromosomes, terminal deletions varying in size, acentric fragments, and multiple unidentifiable translocations. Previous studies of the mitochondrial (mt) genome showed that the two cytologically unstable lines 79(C) and 79(A), which were also unstable in fertility and viability, are characterized by heteroplasmy at the mitochondrial 18S-5S locus (simultaneous presence of barley and wheat mt-fragments). Stable lines 79(B) and 79(D) with normal fertility contained only wheat mitochondrial markers. It was assumed that the substantial instability of the nuclear genome in lines 79(C) and 79(A) was a result of nuclear-cytoplasmic incompatibility and was associated with heteroplasmy, while elimination or considerable reduction of barley material in the mitochondrial genome stabilized the nuclear genome of lines 79(B) and 79(D). In turn, the instability of the nuclear genome was responsible for a decrease in viability and fertility of plants.