Alien Chromatin Research Articles

Phenotyping in Rye Landrace and Regeneration Frequency Study in Wheat × Rye F1 Hybrid Seeds

The present research aimed to study the phenotyping characterization of a high-altitude Himalayan rye landrace and its crossability with wheat genotypes and investigate the regeneration frequency of wheat × rye F1 hybrid seeds. The study was conducted during the rabi season of 2022-2023 at the Agriculture Research Farm, Department of Plant Breeding and Genetics, Lovely Professional University, Phagwara, Punjab. The phenotypic characterization of the Himalayan rye landrace revealed several adaptive traits that were found suitable for cultivation in the northwest Indo-Gangetic plains. Furthermore, the study investigated the crossability of the Himalayan rye landrace with various wheat genotypes, including 4 genotypes of bread wheat HD2967, UNNAT 343, WH1105, and PBW502 and 4 genotypes of durum wheat PDW291, PDW274, PBW34, and WHD943. Among the germination percentage of all 8 genotypes, 4 in Bread wheat and 4 in Durum wheat was observed. Due to its self-incompatibility, Rye cannot be used to develop inbred lines directly. However, the doubled haploid approach can produce inbred lines in the rye, facilitating the extension of genetic bases and introducing alien chromatin to identify stress tolerances.

Triticum durum Desf. is a valuable source for spring bread wheat genetic diversity enhancement

Background. Enhancement of the Triticum aestivum L. genetic diversity by means of alien chromatin, including that from Triticum durum Desf., is an important element in the breeding of bread wheat cultivars that meet modern requirements.Materials and methods. The study included the L153, L154 and L155 introgressive spring bread wheat lines produced from crosses between the spring bread wheat cultivars ‘L503’ and ‘L505’ and spring durum wheat cultivars ‘Yazi10’ and ‘Tarro’ developed in Mexico. Evaluation of these introgressive lines included phytopathological, phenological, genetic, and breadmaking analyses carried out with conventional methods both under open-air and laboratory conditions. Seventeen DNA markers were used to identify genes for resistance to Puccinia triticina Erikss. f. sp. tritici in the lines. The obtained data were statistically analyzed using the Agros-2.10 package of breeding and genetic programs.Results. Studying introgressive spring bread wheat lines L153, L154 and L155, developed with the participation of durum wheat cvs. ‘Yazi10’ and ‘Tarro’, showed that leaf rust resistance of these lines was induced by the Lr genes from durum wheat cultivars. The effect of the gene donor and the recipient cultivars on a number of useful agronomic traits was identified. The recipient cv. ‘L505’ was noted for its good combining abilities.Conclusion. Introgression of chromatin from durum wheat cultivars into the spring bread wheat gene pool made it possible to produce lines resistant to the local P. triticina population, as well as to some test clones of this pathogen. Such lines can be used as donors of the Lr genes. In addition, the L154 introgressive line combined good grain productivity and baking quality.

Systemic development of wheat-Thinopyrum elongatum translocation lines and their deployment in wheat breeding for Fusarium head blight resistance.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum) around the world. FHB causes significant yield losses and reduces grain quality. The lack of resistance resources is a major bottleneck for wheat FHB resistance breeding. As a wheat relative, Thinopyrum elongatum contains many genes that can be used for wheat improvement. Although the novel gene Fhb-7EL was mapped on chromosome 7EL of Th. elongatum, successful transfer of the FHB resistance gene into commercial wheat varieties has not been reported. In this study, we developed 836 wheat-Th. elongatum translocation lines of various types by irradiating the pollen of the wheat-Th.elongatum addition line CS-7EL at the flowering stage, among which 81 were identified as resistant to FHB. By backcrossing the FHB-resistant lines with the main cultivar Jimai 22, three wheat-Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, were successfully applied in wheat breeding without yield penalty. Combining karyotype and phenotype analyses, we mapped the Fhb-7EL gene to the distal end of chromosome 7EL. Five molecular markers linked with the FHB resistance interval were developed, which facilitates molecular marker-assisted breeding. Altogether, we successfully applied alien chromatin with FHB resistance from Th. elongatum in wheat breeding without yield penalty. These newly developed FHB-resistant wheat-Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, can be used as novel resistance resources for wheat breeding.

Development and cytological characterization of wheat-Thinopyrum intermedium translocation lines with novel stripe rust resistance gene.

Wheat stripe rust is a destructive disease in many cool and temperate regions around the world. Exploiting novel sources of resistance can provide wheat cultivars with robust and durable resistance to stripe rust. The wheat-Thinopyrum intermedium addition line TAI-14 was proven to carry a stripe rust resistance gene (named as YrT14) on the alien Th. intermedium chromosome. In order to transfer the resistance gene to wheat, wheat-Th. intermedium translocation lines were created by irradiating the pollen of the line TAI-14. We totally obtained 153 wheat-Th. intermedium translocation lines, among which the long alien segmental translocation line Zhongke 78 and the intercalary translocation line Zhongke 15 not only showed good integrated agronomic traits but also were identified as highly resistant to stripe rust in both seedling and adult plant stages. The alien chromatin in Zhongke 15 was identified as an insertion into the satellite of chromosome 6B, a type of translocation never reported before in chromosome engineering. By screening Simple Sequence Repeat (SSR) and Expressed Sequence Tag (EST) markers as well as the markers developed from RNA-sequencing (RNA-Seq) data, 14 markers were identified specific for the alien chromosome and a physical map was constructed. Both Zhongke 78 and Zhongke 15 could be used as a novel source of stripe rust resistance for wheat breeding, and the linked marker T14K50 can be used for molecular marker-assisted breeding. Finally, based on the karyotype, reaction to stripe rust, and genome resequencing data of different wheat-Th. intermedium translocation lines, the stripe rust resistance gene YrT14 was located to an 88.1 Mb interval from 636.7 to 724.8 Mb on Th. intermedium chromosome 19 corresponding to 7J or 7Js.

Significance of introgression hybridization for extension of genetic variability in recipient genome

Alien chromatin (introgressions) can be incorporated into the wheat genome either through recombination or through translocation. Lines of introgressive origin may not contain identified alien chromatin, although they appear to be changed relative to the original wheat sample by some characteristics that are registered at different levels of phenotype assessment. It has been shown numerous facts of the variability of the recipient genome of hybrid origin. They can be caused by irregular behavior of chromosomes in mitoses of somatic cells with hybrid genome, and DNA changes. Wide phenotypic variability was observed among the progeny of hybrid genomes up to appearance of phenotypic novelties. The study of DNA profiles using several methods revealed the presence of numerous genetic and epigenetic changes, including deletions, changes in gene expression, changes in the methylation pattern, and activation of retrotransposons in the recipient genome. When these changes were not directly related to alien fragments, they are considered as possible mechanisms of extension of genetic variability intra genome of hybrid origin. Transcriptomic analysis of lines of hybrid origin and parental plants using the RNA-seq analyses showed that the change of expression affected a different number of genes. The change in the expression of alien genes occurred in the direction of down-regulation, upregulation was typical for wheat genes. Phenotypic variability during introgressive hybridization can be accompanied by attenuation of the expression of an alien gene when it enters the recipient genome. That may be a consequence of the interaction between the alien gene and the genetic environment of the host genome. Today there is a lot of evidence that these mechanisms are epigenetic, which include DNA methylation, histone modification, the involvement of small RNAs in the regulation of gene expression, and the involvement of transposons. Moreover, none of these mechanisms act independently, but are a component of events, the results of which can be a change in gene expression and the appearance of a new phenotype that is not related to the expression of an alien gene.

Identification of Alien Chromosome/Chromatin Introgressions in Triticale × Wheat Derived Stable Lines Through Molecular Cytogenetic Analysis

Molecular cytogenetic study was conducted for identifying rye chromosome/chromatin translocation, substitution and addition in 43 triticale x wheat derived stable lines (BC1F7 and F7 generations) six different crosses using FISH (Fluorescence in situ hybridization) and GISH (Genomic in situ hybridization) analyses with different probes. Rye chromosomes/chromatin was identified in 17 BC1F7 and 12 F7 lines by these two analyses. The GISH and FISH techniques worked satisfactorily for identifying the presence of rye chromatin at metaphase. All the lines were associated with alien chromosome translocation, addition or substitution except a few lines. Most of the recombinants present in TW 1, TW 2 and TW 6 crosses were observed to be associated with important alien chromatin translocation like 1BL/1RS, substitutions 1R (1D), 5R (5D) and combination of both i.e., 1BL/1RS + 5R (5D) and in some cases presence of more than 4 rye chromosomes. The crosses TW 3 and TW 5 possessed 8 to 14 rye chromosomes in their chromosome constitution. So, their morphological expression was similar to triticale. These reconstituted lines will be of immense application for the future wheat breeding programmes.

Aegilops Species for the Improvement of the Leaf and Stripe Rust Resistance in Cultivated Triticale (×Triticosecale Wittmack)

Hexaploid triticale (×Triticosecale Wittmack, 2n = 6x = 42 chromosomes, AABBRR) is a cultivated hybrid, which combines wheat (Triticum aestivum L.) and rye (Secale cereale L.) properties. It has a better ability to be grown on poor soils, compared to wheat. Mainly, triticale is produced for forage feed and bioethanol. Considering the limited diversity of this human-made crop, there is a need to widen its genetic variability, especially to introduce new genes, responsible for agronomic traits, such as resistance to biotic stresses. Leaf rust caused by Puccinia triticina Eriks. and stripe rust caused by Puccinia striiformis Westend are the most destructive foliar diseases of triticale and related cereals. Developing resistant triticale varieties is an important strategy for the control of these diseases. A number of leaf and stripe rust resistance genes have been already introduced into bread wheat from related species using chromosome manipulations. Exploitation of related species conferring desirable loci is the most effective non-GMO way of improving the rust resistance of triticale. The procedure encompasses chromosome doubling of obtained hybrids followed by a number of backcrosses to eliminate unnecessary alien chromatin and to reduce the linkage drag. In this review, we show the recent status of pre-breeding studies, which are focused on transfer of leaf and stripe rust resistance genes from Aegilops species into cultivated triticale using distant crossing and chromosome engineering.

Characterization of Resistance to Cereal Cyst Nematode, Agronomic Performance, and End-Use Quality Parameters in Four Perennial Wheat-Thinopyrum intermedium Lines.

Perennial wheat is considered to be a practical way to increase the flexibility and profitability of sustainable agricultural system, as it can be either a forage grass or a grain crop. Four perennial wheat lines SX12-480, SX12-787, SX12-1150, and SX12-1269 were developed from a series of interspecific crosses between common wheat (Triticum aestivum, 2n = 42) or durum wheat (T. turgidum var. durum, 2n = 28) and the intermediate wheatgrass (Thinopyrum intermedium, 2n = 42). These lines were characterized by the vigorous regrowth for at least 3 years. The one- and 2-year-old plants had higher grain yield potential than the 3-year-old perennial plants. The decline of grain yield was associated with plant age-related effects on yield components. The perennial wheat lines were all resistant to both Heterodera avenae and H. filipjevi, the two distinct cereal cyst nematode species that occur in China, except that line SX12-787 exhibited moderate resistance only to H. avenae. The dual-purpose perennial wheat lines were evaluated for quality values of both defoliated grass and harvested grains in the form of amino acid profile, mineral concentration, and contents of protein and fiber. Difference in the quality profile was observed between the perennial lines. These perennial lines had an overall improved quality levels over those of the perennial wheat control Montana-2 (T. turgidum × Th. intermedium) and the annual wheat cultivar Jinchun 9. The amplification profiles of the molecular markers provided molecular evidence for the introgression of alien chromatin. Genomic in situ hybridization detected 16, 14, 14, and 12 Th. intermedium chromosomes in lines SX12-480 (2n = 48), SX12-787 (2n = 56), SX12-1150 (2n = 56), and SX12-1269 (2n = 54), respectively, in addition to either 32 or the complete set of wheat chromosomes. The four perennial wheat-Th. intermedium lines described here provide valuable sources of perennial wheat for the dual-purpose application of both grain and forage.

Development of RNA-seq-based molecular markers for characterizing Thinopyrum bessarabicum and Secale introgressions in wheat.

Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of Thinopyrum bessarabicum- and Secale anatolicum-specific EST-STS markers and their application in tracing respective alien chromatin introgressions in wheat. The parental lines, Chinese Spring (CS), ISR991.1 (CS/Th. bessarabicum amphidiploid), and ISR1049.2 (CS/Secale anatolicum amphidiploid), were used as core experimental materials. Using comparative analysis of RNA-Seq data, 10 903 and 10 660 candidate sequences specific to Th. bessarabicum and S. anatolicum, respectively, were assembled and identified. To validate the genome specificity of these candidate sequences, 68 and 64 EST-STS markers were developed from randomly selected candidate sequences of Th. bessarabicum and S. anatolicum, respectively, and tested on sets of alien addition lines. Fifty-five and 53 markers for Th. bessarabicum and S. anatolicum chromatin, respectively, were assigned to chromosomal location(s), covering all seven chromosomes. Approximately 83% of S. anatolicum-specific markers were transferable to S. cereale. The genome-specific candidate sequences identified and the EST-STS markers developed will be valuable resources for exploitation of Th. bessarabicum and Secale species diversity in wheat and triticale breeding.

Reducing the size of an alien segment carrying leaf rust and stripe rust resistance in wheat

BackgroundLeaf and stripe rusts are two major wheat diseases, causing significant yield losses. The preferred way for protecting wheat from rust pathogens is by introgression of rust resistance traits from wheat-related wild species. To avoid genetic drag due to replacement of large wheat chromosomal segments by the alien chromatin, it is necessary to shorten the alien chromosome segment in primary recombinants.ResultsHere we report on shortening of an alien chromosome segment in wheat that carries leaf and stripe rust resistance from Sharon goatgrass (Aegilops sharonensis). Rust resistant wheat introgression lines were selected and the alien region was mapped using genotyping by sequencing. Single polymorphic nucleotides (SNP) were identified and used to generate diagnostic PCR markers. Shortening of the alien fragment was achieved by induced homoeologous pairing and lines with shortened alien chromosome were identified using the PCR markers. Further reduction of the segment was achieved in tertiary recombinants without losing the rust resistance.ConclusionsAlien chromatin in wheat with novel rust resistance genes was characterized by SNP markers and shortened by homoeologous recombination to avoid deleterious traits. The resulting wheat lines are resistant to highly virulent races of leaf and stripe rust pathogens and can be used as both resistant wheat in the field and source for gene transfer to other wheat lines/species.

Genome-wide impacts of alien chromatin introgression on wheat gene transcriptions

Agronomic characteristics and tolerance to biotic and abiotic stresses in hexaploid wheat can be drastically improved through wheat-alien introgression. However, the transcriptional level interactions of introduced alien genes in the wheat genetic background is rarely investigated. In this study, we report the genome-wide impacts of introgressed chromosomes derived from Ae. longissima on gene transcriptions of the wheat landrace Chinese Spring. RNA-seq analyses demonstrated 5.37% and 4.30% of the genes were significantly differentially expressed (DEGs) in CS-Ae. longissima disomic 3Sl#2(3B) substitution line TA3575 and disomic 6Sl#3 addition line TA7548, respectively when compared to CS. In addition, 561 DEGs, including 413 up-regulated and 148 down-regulated or not transcribed genes, were simultaneously impacted by introgressed chromosomes 3Sl#2 and 6Sl#3, which accounts for 41.25% of the DEGs in TA3575 and 38.79% in TA7548. Seventeen DEGs, annotated as R genes, were shared by both introgression lines carrying chromosomes 3Sl#2 and 6Sl#3, which confer resistance to powdery mildew. This study will benefit the understanding of the wheat gene responses as result of alien gene(s) or chromosome intogression and the plant defense response initiated by powdery mildew resistance genes in chromosomes 3Sl#2 and 6Sl#3.

Chromosome specific microsatellite analysis of common wheat lines resistant to powdery mildew

Aim. Determine the chromosome localization of introgressions containing alien genes for resistance to powdery mildew transferred to the wheat genome from three Aegilops species: Ae. sharonensis, Ae. speltoides, and Ae. umbellulata. Methods. Comparative microsatellite analysis of amplicons’ spectra, derived from PCR with primers to SSR loci localized on seven chromosomes of wheat D subgenome. Results. Spectra of amplification of products with DNA of Aurora cultivar, genome substitution amphidiploids Aurosis, Aurodes and Aurolata, using primers to 44 microsatellite loci localized on seven chromosomes of common wheat D subgenome have been obtained. Absence of genome specificity has been determined for most of the analyzed loci. All the studied introgressive lines have been characterized by allele polymorphism for different number of microsatellite loci from all seven chromosomes of D subgenome. This polymorphism included new components in amplicons spectra, which have been different from components of Aurora and genome substituted amphidiploids. Heterozygous genotypes have been also observed. A part of polymorphic loci has been uninformative because the chromosome of genome substituted amphidiploid had null-allele for such loci. For specification of the information about homeological identity of alien chromatin in resistant lines, genes of morphological traits with known chromosome localization have been used. Conclusions. Screening of resistant to powdery mildew wheat introgressive lines for microsatellite loci does not allow an unambiguous identification of chromosome localization of resistance gene because of high polymorphism of microsatellite loci in the genomes of introgressive lines, and because many loci are non-informative. Assumptions have been made about possible connections of resistance genes with chromosomes of homeological groups 3 and 6 in lines derived from Aurodes; 1, 2, 3, and 6 – in lines derived from Aurolata; and homeological group 2 in lines derived from Aurosis.

Inheritance of powdery mildew resistance in Aurotica’s introgressive lines

Aim. Frequency of alien chromatin inheritance in hybrids from crosses of common wheat and introgressive lines determines whether transfer of alien chromatin controlling desirable trait into common wheat genome would be successful. The paper presents results of introgressive lines assessment for frequency of transfer of alien dominant trait of powdery mildew resistance to their progeny in the crossing with recurrent genotype Aurora, which was a recipient cultivar in the development of these lines. Methods. Artificial hybridization of introgressive lines with common wheat, visual assessment of cross components and hybrids F1 and F2 for powdery mildew resistance. Determination of chromosome numbers and chromosome configurations in meiosis M1 of F1 plants. Results. Among F2 plants, from the crossing of resistant to powdery mildew introgressive lines of Aurotica and susceptible cultivar Aurora excess of susceptible plants was observed. In order to explain distortion of ratio susceptible : resistant plants in comparison with expected for monogenic segregation, we propose the determined fact of absence in meiosis M1 of MPC of bivalent chromosome conjugation, which results in formation of aneuploid gametes and zygotes with reduced viability. As the third parameter, the one that can influence the mentioned ratio, we propose d parameter, which quantitatively expresses excess of gametes without alien chromatin controlling resistance to powdery mildew. Conclusion. Model of inheritance, which best explains experimental data of F2 plants segregation for resistant and susceptible if the trait is controlled by one dominant gene, takes into account the following conditions for calculation of probabilities of resistant or susceptible plant formation: all female and only euploid male gametes function, nullisomic zygotes are not viable, during meiosis elimination of meiosis products with alien chromatin occurs with parameter d = 0,11–0,12.Received 11.06.2018

Yield of chromosomally engineered durum wheat-Thinopyrum ponticum recombinant lines in a range of contrasting rain-fed environments

Introgressions of Thinopyrum ponticum 7AgL chromosome segments, spanning 23%, 28% and 40% of the distal end of durum wheat 7AL arm, were previously shown to contain multiple beneficial gene(s)/QTL for yield-related traits, in addition to effective disease resistance (Lr19, Sr25) and quality (Yp) genes. In the present study, durum wheat near isogenic recombinant lines (NIRLs), harbouring each of the three introgressions, were included for the first time in multi-location field trials, to evaluate general and environment-specific effects of the alien chromatin on 26 yield-related traits. Results from nine different trials across contrasting environments of Italy, Morocco and South Australia over four years revealed that the overall impact of 7AgL introgressions into the tetraploid wheat background did not incur, except in one environment, a major yield penalty. The effect of the three 7AgL segments on individual yield-contributing traits resulted in significant increases of biomass m−2 (+9%), spike number m−2 (+13%), grain number m−2 (+11%) and spikelet-1 (+8%), as well as a significant decrease in grain weight (−8%). When the separate NIRLs were analysed, each of the three 7AgL segments were associated with specific yield component variation. The effects of the 40%-long segment proved to be the most stably expressed across environments and involved significant increases of spike and grain number m−2 (13% and 15%, respectively), grain number spike-1 (10%) and spike fertility index (46%), though accompanied by a significant decrease in thousand grain weight (−23%). In spite of this trade-off between grain number and grain weight, their interplay was such that in four trials, including drier environments, a grain yield advantage was observed. This evidence, and comparison with the two other NIRLs, substantiates the hypothesized existence of major gene(s)/QTL for grain number in the most proximal 28–40% 7AgL region, exclusive to the 40%-long 7AgL introgression. The present study represents an important validation of the use of chromosomally engineered genetic stocks for durum wheat improvement, targeting not only disease resistance and quality traits but also relevant yield components.

Development of EST-PCR markers specific to the long arm of chromosome 6V of Dasypyrum villosum

Expressed sequence tags-derived polymerase chain reaction (EST-PCR) molecular markers specific for alien chromosomes can be used to not only monitor the introgressed alien chromatin in wheat background, but also provide the evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers specific for chromosome 6VL of Dasypyrum villosum, 297 primer pairs were designed based on the expressed sequence tags (EST) sequences, which were previously mapped in different bins of the long arms of wheat homoeologous 6AL, 6BL, and 6DL. By using the Triticum aestivum, D. villosum, T. durum-D. villosum amphiploid, and T. aestivum-D. villosum alien chromosome lines involving chromosome 6V, it was found that 32 (10.77%) primers could amplify specific bands for chromosome 6V, and 31 could be allocated to chromosome arm 6VL. These 6VL specific markers provided efficient tools for the characterization of structural variation involving the chromosome 6VL in common wheat background as well as for the selection of useful genes located on 6VL in breeding programs.

Development of Thinopyrum ponticum-specific molecular markers and FISH probes based on SLAF-seq technology.

Based on SLAF-seq, 67 Thinopyrum ponticum-specific markers and eight Th. ponticum-specific FISH probes were developed, and these markers and probes could be used for detection of alien chromatin in a wheat background. Decaploid Thinopyrum ponticum (2n=10x=70) is a valuable gene reservoir for wheat improvement. Identification of Th. ponticum introgression would facilitate its transfer into diverse wheat genetic backgrounds and its practical utilization in wheat improvement. Based on specific-locus-amplified fragment sequencing (SLAF-seq) technology, 67 new Th. ponticum-specific molecular markers and eight Th. ponticum-specific fluorescence in situ hybridization (FISH) probes have been developed from a tiny wheat-Th. ponticum translocation line. These newly developed molecular markers allowed the detection of Th. ponticum DNA in a variety of materials specifically and steadily at high throughput. According to the hybridization signal pattern, the eight Th. ponticum-specific probes could be divided into two groups. The first group including five dispersed repetitive sequence probes could identify Th. ponticum chromatin more sensitively and accurately than genomic in situ hybridization (GISH). Whereas the second group having three tandem repetitive sequence probes enabled the discrimination of Th. ponticum chromosomes together with another clone pAs1 in wheat-Th. ponticum partial amphiploid Xiaoyan 68.

Genome Targeted Introgression of Resistance to African Stem Rust from Aegilops sharonensis into Bread Wheat.

Many accessions of the wheat wild relative Sharon goatgrass ( Eig., ) are resistant to African races of the stem rust pathogen (i.e., Ug99 group races), which currently threaten wheat production worldwide. A procedure was designed to introgress the respective resistances to specific bread wheat genomes by producing plants homozygous for the A and B genomes and hemizygous for the D and S genomes or homozygous for the A and D genomes and hemizygous for the B and S genomes. In these genotypes, which lack the allele, homeologous pairing was expected mainly between chromosomes of the D and S genomes or B and S genomes, respectively. An antigametocidal (AG) wheat mutant () was used to overcome gametocidal effects. Wheat lines initially found resistant at the seedling stage were also highly resistant at the adult plant stage in rust nurseries established in the field. DNA of 41 selected homozygous resistant lines, analyzed by the Axiom wheat 820K SNP array, showed alien chromatin mainly in wheat chromosomes 1B, 1D, and 5B. This work suggests that, in most cases, it is possible to target introgressions into the homeologous chromosome of a selected genome of bread wheat.

Whole genome development of intron targeting (IT) markers specific for Dasypyrum villosum chromosomes based on next-generation sequencing technology

Dasypyrum villosum (Dv), a wild relative of wheat, is an important and useful gene resource for wheat improvement. A large number of wheat-Dv aneuploid lines harboring whole or fragments of Dv chromosomes have been developed. However, the lack of sufficient molecular markers hindered accurate identification of Dv chromatin, especially when the introgressed fragments are small. Development of molecular markers covering the whole Dv genome and evenly distributed on different chromosome regions is not only useful for the detection of the introgressed alien chromatin in wheat background, but also provides evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers on individual Dv chromosomes, genomic DNA of Dv leaves was sequenced and assembled. Sequence assemblies of all wheat chromosomes were first used to identify exon–exon junctions and localize introns in Dv. Intron length polymorphisms suitable for designing Dv primers flanking introns were evaluated, and a total of 1624 intron targeting (IT) markers was designed. By using the Chinese Spring, the Triticum durum-Dv amphiploid and the Dv sequenced DNA libraries, 841 IT molecular markers specific for Dv chromosomes were developed, with maximum efficiency up to 51.79%. We assigned the 841 IT markers to seven Dv chromosomes (1V–7V) using seven wheat–Dv chromosome addition and substitution lines: 135 to 1V, 175 to 2V, 120 to 3V, 89 to 4V, 140 to 5V, 71 to 6V, and 111 to 7V, respectively. Using T. aestivum-Dv telosomic and whole arm translocation lines, they were further located on the short or long chromosome arms. These specific markers for individual chromosomes of Dv provided efficient tools for the characterization of structural variation involving the individual chromosome of Dv, as well as for the selection of useful genes located on individual Dv chromosome in breeding programs.

Heterosis, transmission genetics, and selection for increased growth rate in a N. tabacum × synthetic tobacco cross

Cultivated tobacco (Nicotiana tabacum L.) is a classic amphidiploid, and hybrids between this cultivated species and closely related diploid Nicotiana relatives often exhibit heterotic effects for growth rate and yield. Crosses between N. tabacum and synthetic tobaccos, 4x(Nicotiana sylvestris × Nicotiana otophora) or 4x(N. sylvestris × Nicotiana tomentosiformis), may provide superior routes for genome-wide introgression from diploid relatives and allow increased potential to capitalize on heterotic effects in tobacco. Significant levels of mid-parent heterosis were observed for yield and growth rate in F1 hybrids between synthetic tobaccos and a standard tobacco cultivar. Microsatellite marker genotyping of an F2 population derived from a K326 × [4x(N. sylvestris × N. otophora)] cross was carried out to preliminarily investigate the relative importance of different types of gene action on observed heterosis in the original interspecific cross. Results suggested a role for both partial dominance and overdominance. Marker genotyping also indicated an overall reduced level of recombination in the N. tabacum × synthetic tobacco cross relative to a N. tabacum × N. tabacum cross but no evidence of genomic regions with severely restricted levels of recombination. Results suggest that populations derived from N. tabacum × synthetic tobacco crosses may be more efficient for introgressing germplasm from diploid relatives, as compared to populations derived from crosses between N. tabacum and diploid forms where preferential pairing between N. tabacum homologues can reduce the potential for introgression of alien chromatin. Such materials may be useful as sources of favorable alleles influencing quantitative characters in tobacco.

Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing

BackgroundHaynaldia villosa (L.) Schur (syn. Dasypyrum villosum L. Candargy, 2n = 14, genome VV) is the tertiary gene pool of wheat, and thus a potential resource of genes for wheat improvement. Among other, wheat yellow mosaic (WYM) resistance gene Wss1 and a take-all resistance gene were identified on the short arm of chromosome 4 V (4VS) of H. villosa. We had obtained introgressions on 4VS chromosome arm, with the objective of utilizing the target genes. However, monitoring these introgressions has been a daunting task because of inadequate knowledge as to H.villosa genome, as reflected by the lack of specific markers.ResultsThis study aims to develop 4VS-specific markers by combination of chromosome sorting and next-generation sequencing. The short arm of chromosome 4VS of H.villosa was flow-sorted using a FACSVantage SE flow cytometer and sorter, and then sequenced by Illumina sequencing. The sequence of H. villosa 4VS was assembled by the software Hecate, and then was compared with the sequence assemblies of wheat chromosome arms 4AL, 4BS and 4DS and Ae. tauschii 4DS, with the objectives of identifying exon-exon junctions and localizing introns on chromosome 4VS of H. villosa. The intron length polymorphisms suitable for designing H. villosa primers were evaluated with criteria. Consequently, we designed a total of 359 intron targeting (IT) markers, among which 232 (64.62%) markers were specific for tracing the 4VS chromatin in the wheat background.ConclusionThe combination of chromosome sorting and next-generation sequencing to develop specific IT markers for 4VS of H. villosa has high success rate and specificity, thus being applicable for the development of chromosome-specific markers for alien chromatin in wheat breeding.