Alien Addition Research Articles

Spontaneous chromosome instability and tissue culture-induced karyotypic alteration in wheat-Thinopyrum intermedium alien addition lines.

Wheat lines harboring wild-relative chromosomes can be karyotypically unstable during long-term maintenance. Tissue culture exacerbates chromosomal instability but appears inefficient to induce somatic homoeologous exchange between alien and wheat chromosomes. We assessed if long-term refrigerator storage with regular renewal via self-fertilization, a widely used practice for crop germplasm maintenance, would ensure genetic fidelity of alien addition lines, and explored the possibility of inducing somatic homoeologues exchange by tissue culture. We cytogenetically characterized sampled stock seeds of originally confirmed 12 distinct wheat-Thinopyrum intermedium alien addition lines (dubbed TAI lines), and subjected immature embryos of the TAI lines to tissue culture. We find eight of the 12 TAI lines were karyotypically departed from their original identity as bona fide disomic alien addition lines due to extensive loss of whole-chromosomes of both Th. intermedium and wheat origins during the ca. 3-decade storage. Rampant numerical chromosome variations (NCVs) involving both alien and wheat chromosomes were detected in regenerated plants of all 12 studied TAI lines, but at variable rates among the wheat sub-genomes and chromosomes. Compared with NCVs, structural chromosome variations (SCVs) occurred at substantially lower rates, and no SCV involving the added alien chromosomes was observed. The NCVs manifested only moderate effects on phenotypes of the regenerated plants under field conditions.

Transcriptional Interactions of Single B-Subgenome Chromosome with C-Subgenome in B. oleracea-nigra Additional Lines.

Serial monosomic alien addition lines (MAALs) provide an ideal system to elucidate the transcriptomic interactions between the alien chromosomes and recipient genome under aneuploidy. Herein, five available Brassica oleracea-nigra MAALs (CCB1, CCB4, CCB5, CCB6, CCB8), their derived B. oleracea plants (non-MAALs), and two parents were analyzed for their gene expressions by using high-throughput technology. Compared to parental B. oleracea, all MAALs showed various numbers of DEGs, but CCB8 gave much higher DEGs; the number of downregulated DEGs was slightly higher than the number of upregulated ones, except for in relation to CCB8. All derived B. oleracea plants also gave certain numbers of DEGs, despite these being much lower than in the respective MAALs. Compared to B. nigra, in all five MAALs more DEGs were downregulated than upregulated. Trans-effects were likely more prevailing than cis-effects, and these DEGs were predominantly associated with material transport by dysregulating the cellular component. Meanwhile, the orthologous genes on alien chromosomes could only play a feeble compensatory role for those gene pairs in C-subgenome, and different levels of the expressed genes had a greater tendency towards downregulation. These results revealed transcriptional aneuploidy response patterns between two genomes and suggested that cis- and trans-mechanisms synergistically regulated alien gene transcriptions after distant hybridization.

Dosage-Dependent Gynoecium Development and Gene Expression in Brassica napus-Orychophragmus violaceus Addition Lines.

Distant hybridization usually leads to female sterility of the hybrid but the mechanism behind this is poorly understood. Complete pistil abortion but normal male fertility was shown by one Brassica napus-Orychophragmus violaceus monosomic alien addition line (MA, AACC + 1 IO, 2n = 39) produced previously. To study the effect of a single O. violaceus chromosome addition on pistil development in different genetic backgrounds, hybrids between the MA and B. carinata (BBCC), B. juncea (AABB), and two synthetic hexaploids (AABBCC) were firstly produced in this study which show complete female sterility. A microspore culture was further performed to produce the haploid monosomic alien addition line (HMA, AC + 1 IO, 2n = 20) and disomic addition line (DA, AACC + 2 IO, 2n = 40) together with haploid (H, AC, 2n = 19) and double haploid (DH, AACC, 2n = 38) plants of B. napus from MA to investigate the dosage effect of the alien O. violaceus chromosome on pistil development and gene expression. Compared to MA, the development of the pistils of DA and HMA was completely or partially recovered, in which the pistils could swell and elongate to a normal shape after open pollination, although no seeds were produced. Comparative RNA-seq analyses revealed that the numbers of the differentially expressed genes (DEGs) were significantly different, dosage-dependent, and consistent with the phenotypic difference in pairwise comparisons of HMA vs. H, DA vs. DH, MA vs. DH, MA vs. DA, and MA vs. HMA. The gene ontology (GO) enrichment analysis of DEGs showed that a number of genes involved in the development of the gynoecium, embryo sac, ovule, and integuments. Particularly, several common DEGs for pistil development shared in HMA vs. H and DA vs. DH showed functions in genotoxic stress response, auxin transport, and signaling and adaxial/abaxial axis specification. The results provided updated information for the molecular mechanisms behind the gynoecium development of B. napus responding to the dosage of alien O. violaceus chromosomes.

Chromosome-scale genome assembly of Cucumis hystrix\u2014a wild species interspecifically cross-compatible with cultivated cucumber

Cucumis hystrix Chakr. (2n = 2x = 24) is a wild species that can hybridize with cultivated cucumber (C. sativus L., 2n = 2x = 14), a globally important vegetable crop. However, cucumber breeding is hindered by its narrow genetic base. Therefore, introgression from C. hystrix has been anticipated to bring a breakthrough in cucumber improvement. Here, we report the chromosome-scale assembly of C. hystrix genome (289 Mb). Scaffold N50 reached 14.1 Mb. Over 90% of the sequences were anchored onto 12 chromosomes. A total of 23,864 genes were annotated using a hybrid method. Further, we conducted a comprehensive comparative genomic analysis of cucumber, C. hystrix, and melon (C. melo L., 2n = 2x = 24). Whole-genome comparisons revealed that C. hystrix is phylogenetically closer to cucumber than to melon, providing a molecular basis for the success of its hybridization with cucumber. Moreover, expanded gene families of C. hystrix were significantly enriched in “defense response,” and C. hystrix harbored 104 nucleotide-binding site–encoding disease resistance gene analogs. Furthermore, 121 genes were positively selected, and 12 (9.9%) of these were involved in responses to biotic stimuli, which might explain the high disease resistance of C. hystrix. The alignment of whole C. hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C. hystrix chromosomes. Finally, we developed four cucumber–C. hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods. The assembled C. hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.

Alien introgression and morpho-agronomic characterization of diploid progenies of Solanum lycopersicoides monosomic alien addition lines (MAALs) toward pre-breeding applications in tomato (S. lycopersicum)

Key messageAlien introgressions that were captured in the genome of diploid plants segregating from progenies of monosomic alien addition lines of S. lycopersicoides confer novel phenotypes with commercial and agronomic value in tomato breeding.Solanum lycopersicoides is a wild relative of tomato with a natural adaptation to a wide array of biotic and abiotic challenges. In this study, we identified and characterized diploid plants segregating from the progenies of monosomic alien addition lines (MAALs) of S. lycopersicoides to establish their potential as donors in breeding for target trait improvement in tomato. Molecular genotyping identified 28 of 38 MAAL progenies having the complete chromosome complement of the cultivated tomato parent and limited chromosome introgressions from the wild S. lycopersicoides parent. Analysis of SSR and indel marker profiles identified 34 unique alien introgressions in the 28 MAAL-derived introgression lines (MDILs) in the genetic background of tomato. Conserved patterns of alien introgressions were detected among sibs of MDILs 2, 3, 4 and 8. Across MDILs, a degree of preferential transmission of specific chromosome segments was also observed. Morphologically, the MDILs closely resembled the cultivated tomato more than S. lycopersicoides. The appearance of novel phenotypes in the MDILs that are lacking in the cultivated parent or the source MAALs indicates the capture of novel genetic variation by the diploid introgression lines that can add commercial and agronomic value to tomato. In particular, screening of representative MDILs for drought tolerance at the vegetative stage identified MDIL 2 and MDIL 11III as drought tolerant based on visual scoring. A regulated increase in stomatal conductance of MDIL 2 under drought stress indicates better water use efficiency that allowed it to survive for 7 days under 0% moisture level.

Development and Cytomolecular Identification of Monosomic Alien Addition and Substitution Lines of Triticale (×Triticosecale Wittmack) With 2Sk Chromosome Conferring Leaf Rust Resistance Derived From Aegilops kotschyi Boiss.

Alien chromosome introgression has become a valuable tool to broaden the genetic variability of crop plants via chromosome engineering. This study details the procedure to obtain monosomic addition and monosomic substitution lines of the triticale carrying 2Sk chromosome from Aegilops kotchyi Boiss., which harbors Lr54 + Yr37 leaf and stripe rust-resistant gene loci, respectively. Initially, A. kotschyi × Secale cereale artificial amphiploids (2n = 6x = 42 chromosomes, UUSSRR) were crossed with triticale cv. “Sekundo” (2n = 6x = 42, AABBRR) in order to obtain fertile offspring. Cyto-molecular analyses of five subsequent backcrossing generations revealed that 2Sk chromosome was preferentially transmitted. This allowed for the selection of monosomic 2Sk addition (MA2Sk) lines of triticale. Finally, the 2Sk(2R) substitution plants were obtained by crossing MA2Sk with the nullisomic (N2R) plants of triticale. The presence of 2Sk chromosome in subsequent generations of plants was evaluated using SSR markers linked to Lr54 + Yr37 loci. Disease evaluation of the monosomic 2Sk(2R) substitution plants for the reaction to leaf and stripe rust infection were carried out under controlled conditions in a growth chamber. The results showed significant improvement of leaf rust resistance severity of monosomic substitution plants compared with control (“Sekundo”). In contrast, the introgression of the Lr54 + Yr37 loci did not lead to improvement of stripe rust resistance. In summary, the creation of monosomic addition and monosomic substitution lines of triticale is the starting point for the precise and guided transfer of Lr54 + Yr37 loci. The results showed that the developed materials could be exploited for the development of triticale varieties with resistance to leaf rust.

An allotriploid hybrid developed from a female 2n gamete was obtained from Eriobotrya japonica (Thunb.) Lindl × E. deflexa (Hemsl.) Nakai

A triploid was obtained from loquat (Eriobotrya japonica cv. ‘Ninghaibai’) (2n = 2x = 34) × bronze loquat (Eriobotrya deflexa, 2n = 2x = 34). The leaves of the triploid had no petioles, similar to the female parent, and its stipules were obvious and dark green, similar to those of the male parent. There were signals of male parent DNA on ten chromosomes of the triploid according to genomic in situ hybridization (GISH) when DNA of bronze loquat was used as probe; the diploid hybrid also had ten chromosomes with signals of male parent DNA. All five selected simple sequence repeats (SSR) primers amplified specific loci of both parents. In particular, primer CH04c06 amplified both specific loci of the female parent but only amplified one specific locus of the male parent. The results of GISH and SSR marker analysis indicated that the 2n gamete which formed the triploid was from first-division restitution or pre-meiotic doubling of the female parent cv. ‘Ninghaibai’. The allotriploid can be used to breed seedless loquat germplasm with different flowering times from loquat directly or to breed new germplasm containing a few genomic components from bronze loquat, such as monosomic alien addition lines, monosomic alien substitution lines, and introgression lines. This is the first report of allotriploid in the genus Eriobotrya and it will provide a basis for breeding new loquat germplasms by distant hybridization and provide an ideal material for breeding new loquat varieties with different flowering time or time to maturity.

Development of alien addition lines from Cucumis hystrix in Cucumis sativus: cytological and molecular marker analyses.

Transferring desired genes from wild species to cultivars through alien addition lines (AALs) has been shown to be an effective method for genetic improvement. Cucumis hystrix Chakr. (HH, 2n = 24) is a wild species of Cucumis that possesses many resistant genes. A synthetic allotetraploid species, C. hytivus (HHCC, 2n = 38), was obtained from the cross between cultivated cucumber, C. sativus (CC, 2n = 14), and C. hystrix followed by chromosome doubling. Cucumis sativus - C. hystrix AALs were developed by continuous backcrossing to the cultivated cucumbers. In this study, 10 different types of AALs (CC-H01, CC-H06, CC-H08, CC-H10, CC-H12, CC-H06+H09, CC-H06+H10, CC-H06+H12, CC-H08+H10, CC-H01+H06+H10) were identified based on the analysis of fluorescence in situ hybridization (FISH) and molecular markers specific to C. hystrix chromosomes. And the behavior of the alien chromosomes in three AALs (CC-H01, CC-H06+H10, CC-H01+H06+H10) at meiosis was investigated. The results showed that alien chromosomes paired with C. sativus chromosome in few pollen mother cells (PMCs). Further, disomic alien addition lines (DAALs) carrying a pair of C. hystrix chromosome H10 were screened from the selfed progenies of CC-H10. Chromosome pairing between genomes provides cytological evidence for the possible introgression of alien chromosome segments. The development of AALs could serve as a key step for exploiting and utilizing valuable genes from C. hystrix.

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.

A novel locus from the wild allotetraploid rice species Oryza latifolia Desv. confers bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice (O. sativa).

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major limiting factor to rice productivity worldwide. Genetic control through the identification of novel sources of bacterial blight resistance and their utilization in resistance breeding remains the most effective and economical strategy to manage the disease. Here we report the identification of a novel locus from the wild Oryza species, Oryza latifolia, conferring a race-specific resistance to Philippine Xoo race 9A (PXO339). The locus was identified from two introgression lines i.e. WH12-2252 and WH12-2256 that segregated from O. latifolia monosomic alien addition lines (MAALs). The discrete segregation ratio of susceptible and resistant phenotypes in the F2 (χ2[3:1] = 0.22 at p>0.05) and F3 (χ2[3:1] = 0.36 at p>0.05) populations indicates that PXO339 resistance in the MAAL-derived introgression lines (MDILs) is controlled by a single, recessive gene. Genotyping of a total of 216 F2, 1130 F3 and 288 F4 plants derived from crossing either of the MDILs with the recurrent parent used to generate the MAALs narrowed the candidate region to a 1,817 kb locus that extends from 10,425 to 12,266 kb in chromosome 12. Putative candidate genes that were identified by data mining and comparative sequence analysis can provide targets for further studies on mapping and cloning of the causal gene for PXO339 resistance in the MDILs. To our knowledge, this is the first report of a genetic locus from the allotetraploid wild rice, O. latifolia conferring race-specific resistance to bacterial blight.

A complete set of monosomic alien addition lines developed from Gossypium anomalum in a Gossypium hirsutum background: genotypic and phenotypic characterization.

Gossypium anomalum (B1B1) is a valuable wild resource for the genetic improvement of G. hirsutum (A1A1D1D1) in terms of fiber quality and disease and pest resistance, but the inherent difficulties in distant hybridization hinder its utilization in breeding programs. Monosomic alien addition lines (MAALs) are powerful tools for interspecific gene transfer. First, to access useful genes from G. anomalum, a fertile hexaploid from G. hirsutum × G. anomalum was obtained and then additional chromosomes were selected using SSR markers in successive backcrosses and self-crossing from BC2F1 to BC4F4. Finally, a complete set of 13 MAALs were developed. All the MAALs were confirmed by chromosome-specific anchored SSRs and genome-wide resequencing. The MAALs demonstrated abundant variation in morphological, agronomic, yield-related, and fiber quality traits. MAAL_3B had excellent fiber strength and fineness, indicating that the transmitted chromosome may carry desirable genes for the observed phenotypes. This complete set of MAALs will provide important genetic bridge material for the identification and introgression of favorable genes from G. anomalum and lay an important foundation for the genetic improvement of cotton.

Resistance of Nicotiana tabacum to Phytophthora parasitica var. nicotianae Race 0 Is Enhanced by the Addition of N. plumbaginifolia Chromosome 9 with a Slight Effect on Host Genomic Expression

ABSTRACTBlack shank is a devastating disease in tobacco production worldwide, and resistance to this disease in Nicotiana plumbaginifolia Viv. tobacco is controlled by the Php gene. The position of the Php gene on chromosomes of N. plumbaginifolia remains unclear. The tobacco line TP‐1, which was derived from N. tabacum L. cv. Yunyan87 and N. plumbaginifolia, was used in the present study to help locate the Php gene on the N. plumbaginifolia chromosomes and understand the effects of the N. plumbaginifolia chromosome addition on the genomic expression of N. tabacum. An in vitro infection test verified that TP‐1 was as resistant to Phytophthora parasitica var. nicotianae race 0 as N. plumbaginifolia and more resistant than Yunyan87. According to cytogenetic analysis, TP‐1 was identified as a monosomic alien addition line and the alien chromosome was recognized as the ninth chromosome of N. plumbaginifolia through. Thus, the P. parasitica var. nicotianae race 0 resistance gene might be Php on chromosome 9 of N. plumbaginifolia. A superficial transcriptomic analysis showed that addition of the alien chromosome slightly affected the genomic expression of the host, with only 3.32% of differentially expressed genes (DEGs) identified between TP‐1 and Yunyan87. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, most (9/13) genes classified as involved in plant–pathogen reactions, particularly five serine/threonine‐protein kinase genes, were upregulated in TP‐1. It is worth noting that flavonoid biosynthesis may be greatly downregulated in TP‐1: two genes were both downregulated in TP‐1 according to gene ontology analysis, and all 20 DEGs in the flavonoid biosynthesis pathway were downregulated in TP‐1 according to KEGG analysis.

Assignment of unanchored scaffolds in genome of Brassica napus by RNA-seq analysis in a complete set of Brassica rapa-Brassica oleracea monosomic addition lines

The economically valuable oil crop Brassica napus (AACC, 2n=38), which arose from interspecific hybridization between the diploid ancestors Brassica rapa (AA, 2n=20) and Brassica oleracea (CC, 2n=18), has a complex genome. More than 10% of the assembled sequences, most of which belong to the C subgenome, have not been anchored to the corresponding chromosome. Previously, a complete set of monosomic alien addition lines (MAALs, C1–C9) with each of the nine C-subgenome chromosomes added to the extracted A subgenome was obtained from the allotetraploid B. napus donor Oro, after the ancestral B. rapa (RBR Oro) genome was restored. These MAALs effectively reduced the complexity of the B. napus genome. Here, we determined the expression values of genes on unanchored scaffolds in the MAALs and RBR Oro. Then, multiple comparisons of these gene expression values were used to determine the affiliations of the non-anchored scaffolds on which the genes were located. In total, 54.68% (44.11 Mb) of the 80.67 Mb of non-anchored scaffolds belonging to the C subgenome were assigned to corresponding C chromosomes. This work highlights the potential value of these MAALs in improving the genome quality of B. napus.

Development of a Fertility Restorer for inap CMS (Isatis indigotica) Brassica napus Through Genetic Introgression of One Alien Addition.

Novel Brassica napus cytoplasmic male sterility (CMS) with carpelloid stamens (inap CMS) was produced by intertribal somatic hybridization with Isatis indigotica (Chinese woad), but its RF (restorer of fertility) gene(s) existed in one particular woad chromosome that was carried by one fertile monosomic alien addition line (MAAL) of rapeseed. Herein, the selfed progenies of this MAAL were extensively selected and analyzed to screen the rapeseed-type plants (2n = 38) with good male fertility and to produce their doubled haploid (DH) lines by microspore culture. From the investigation of fertility restoration in the F1 hybrids with inap CMS, one DH line (RF 39) was identified to adequately restore male fertility and likely carried one dominant RF gene. Specifically, this restorer produced brown pollen grains, similar to the woad and the MAAL, suggesting that this trait is closely linked with the RF gene(s) and serves as one phenotypic marker for the restorer. This restorer contained 38 chromosomes of rapeseed and no intact chromosomes of woad, but some DNA fragments of woad origin were detected at low frequency. This restorer was much improved for pollen and seed fertility and for low glucosinolate content. The successful breeding of the restorer for inap CMS rendered this new pollination control system feasible for rapeseed hybrid production.

Transcriptional Aneuploidy Responses of Brassica rapa-oleracea Monosomic Alien Addition Lines (MAALs) Derived From Natural Allopolyploid B. napus.

Establishing the whole set of aneuploids, for one naturally evolved allopolyploid species, provides a unique opportunity to elucidate the transcriptomic response of the constituent subgenomes to serial aneuploidy. Previously, the whole set of monosomic alien addition lines (MAALs, C1-C9) with each of the nine C subgenome chromosomes, added to the extracted A subgenome, was developed in the context of the allotetraploid Brassica napus donor “Oro,” after the restitution of the ancestral B. rapa (RBR Oro) was realized. Herein, transcriptomic analysis using high-throughput technology was conducted to detect gene expression alterations in these MAALs and RBR. Compared to diploid RBR, the genes of all of the MAALs showed various degrees of dysregulated expressions that resulted from cis effects and more prevailing trans effects. In addition, the trans-effect on gene expression in MAALs increased with higher levels of homology between the recipient A subgenome and additional C subgenome chromosomes, instead of gene numbers of extra chromosomes. A total of 10 trans-effect dysregulated genes, among all pairwise comparisons, were mainly involved in the function of transporter activity. Furthermore, highly expressed genes were more prone to downregulation and vice-versa, suggesting a common trend for transcriptional pattern responses to aneuploidy. These results provided a comprehensive insight of the impact of gene expression of individual chromosomes, in one subgenome, on another intact subgenome for one allopolyploid with a long evolutionary history.

Monosomic alien addition lines (MAALs) of Oryza rhizomatis in Oryza sativa: production, cytology, alien trait introgression, molecular analysis and breeding application.

Key message Development of MAALs and disomic introgression lines derived from the cross between O. sativa and O. rhizomatis to exploit and utilize the valuable traits for rice improvement. The CC genome wild species, Oryza rhizomatis, possesses valuable traits for rice improvement. Unlike other CC genome wild rice, O. rhizomatis is less studied and none of the research has focused on the utilization of this resource in rice breeding. The transfer of novel genes governing the valuable traits from O. rhizomatis is difficult due to high genome incompatibility with O. sativa. Here we report the development of backcross progenies and complete sets of monosomic alien addition lines (MAALs) for the first time from O. rhizomatis in O. sativa line IR31917-45-3-2. Autotetraploid IR31917-45-3-2 (4x = AAAA) was used to generate allotriploid F1, and the F1 plant was backcrossed to IR31917-45-3-2 (2x). Forty-seven BC1F1 and 73 BC2F1 plants were produced with chromosome numbers ranging from 24 to 33 (2x + 9) and 24 to 27 (2x + 3), respectively. A complete set of MAALs were identified by morphological, cytological and marker-based analysis. A total of 116 CC genome-specific InDel markers across the 12 chromosome of rice were used to detect O. rhizomatis chromosome segments in F1, BC1F1, BC2F2, MAALs and disomic introgression lines (DILs). Expressions of major phenotypic traits inherited from O. rhizomatis were observed in MAAL-derived DILs. Small chromosomal segments of O. rhizomatis for chromosomes 1, 2, 4, 5, 6, 7, 10 and 12 were detected in DILs, and some of the introgression lines showed insect resistance against brown planthopper and green leafhopper. These newly developed MAALs and DILs will be useful for gene mining and more precise faster transfer of favorable genes to improve rice cultivars.

Two Orthodox Churches (the Old and the New) of the Holy Apostles Peter and Paul in Kalisz

The Kalisz Orthodox church from the 1870s (Fig. 1) was demolished in the interwar period and quickly replaced by a “new” Orthodox church by the same name (Fig. 6). The surviving official correspondence reveals a specific set of arguments for the dismantling of the “old” church, e.g. that it was becoming dilapidated, was a threat to public safety and constituted an alien addition to the architectural landscape of the city. The demolition of the Orthodox church was to provide jobs for the unemployed and to open up the possibility of erecting a post office in that spot. The municipal authorities convinced the Ministry of Culture and Art that the local Orthodox parish was not interested in reclaiming the church for their own needs, even though this was not the case. The community ultimately conceded to having the church dismantled but demanded that a new temple be erected as compensation. The example of Kalisz aptly illustrates the attitude the authorities of the Second Republic of Poland had towards Russian Orthodox churches that had been erected in the partition period. The situation mirrored the controversies around the fate of the Orthodox church in Saski Square in Warsaw, if in a more provincial environment. The architectural style of the “new” Orthodox church in Kalisz puzzles many authors – the building, clearly representative of Russian historicism, is associated with Rundbogenstil and Latin- and Occidental-style Orthodox churches, which were spared by the interwar Polish authorities who wished to convert Orthodox citizens to Catholicism within the framework of the so-called neo-Union.

Development and characterization of a complete set of Triticum aestivum-Roegneria ciliaris disomic addition lines.

A complete set wheat-R. ciliaris disomic addition lines (DALs) were characterized and the homoeologous groups and genome affinities of R. ciliaris chromosomes were determined. Wild relatives are rich gene resources for cultivated wheat. The development of alien addition chromosome lines not only greatly broadens the genetic diversity, but also provides genetic stocks for comparative genomics studies. Roegneria ciliaris (genome ScScYcYc), a tetraploid wild relative of wheat, is tolerant or resistant to many abiotic and biotic stresses. To develop a complete set of wheat-R. ciliaris disomic addition lines (DALs), we undertook a euplasmic backcrossing program to overcome allocytoplasmic effects and preferential chromosome transmission. To improve the efficiency of identifying chromosomes from Sc and Yc, we established techniques including sequential genomic in situ hybridization/fluorescence in situ hybridization (FISH) and molecular marker analysis. Fourteen DALs of wheat, each containing one pair of R. ciliaris chromosomes pairs, were characterized by FISH using four repetitive sequences [pTa794, pTa71, RcAfa and (GAA)10] as probes. One hundred and sixty-two R. ciliaris-specific markers were developed. FISH and marker analysis enabled us to assign the homoeologous groups and genome affinities of R. ciliaris chromosomes. FHB resistance evaluation in successive five growth seasons showed that the amphiploid, DA2Yc, DA5Yc and DA6Sc had improved FHB resistance, indicating their potential value in wheat improvement. The 14 DALs are likely new gene resources and will be phenotyped for more agronomic performances traits.

Ten alien chromosome additions of Gossypium hirsutum–Gossypium bickii developed by integrative uses of GISH and species-specific SSR markers

Gossypium bickii: (2n = 26, G1G1), a wild diploid cotton, carries many favourable traits. However, these favourable traits cannot be directly transferred into G. hirsutum (2n = 52, AADD) cultivars due to the differences in genomes. Monosomic alien addition lines (MAALs) are considered an invaluable tool for the introgression of genes of interest from wild relatives into cultivated crops. In this study, the G. hirsutum-G. bickii amphidiploid (2n = 78, AADDG1G1) was backcrossed with G. hirsutum to develop alien additions containing individual G. bickii chromosomes in a G. hirsutum background. Genomic in situ hybridization was employed to detect the number of alien chromosomes added to the backcross progenies. A total of 183 G. bickii-specific DNA markers were developed to discriminate the identities of the G. bickii chromosomes added to G. hirsutum and assess the alien chromosome transmissibility. Chromosomes 4Gb and 13Gb showed the highest transmissibility, while chromosomes 1Gb, 7Gb and 11Gb showed the lowest. Ten of the 13 possible G. hirsutum-G. bickii MAALs were isolated and characterized, which will lay the foundation for transferring resistance genes of G. bickii into G. hirsutum, as well as for gene assignment, physical mapping, and selective isolation and mapping of cDNAs for particular G. bickii chromosomes. The strategies of how to use MAALs to develop varieties with the trait of interest from wild species (such as glanded plant-glandless seed) were proposed and discussed.

Genome-Wide Gene Expression Disturbance by Single A1/C1 Chromosome Substitution in Brassica rapa Restituted From Natural B. napus.

Alien chromosome substitution (CS) lines are treated as vital germplasms for breeding and genetic mapping. Previously, a whole set of nine Brassica rapa-oleracea monosonic alien addition lines (MAALs, C1-C9) was established in the background of natural B. napus genotype “Oro,” after the restituted B. rapa (RBR) for Oro was realized. Herein, a monosomic substitution line with one alien C1 chromosome (Cs1) in the RBR complement was selected in the progenies of MAAL C1 and RBR, by the PCR amplification of specific gene markers and fluorescence in situ hybridization. Cs1 exhibited the whole plant morphology similar to RBR except for the defective stamens without fertile pollen grains, but it produced some seeds and progeny plants carrying the C1 chromosome at high rate besides those without the alien chromosome after pollinated by RBR. The viability of the substitution and its progeny for the RBR diploid further elucidated the functional compensation between the chromosome pairs with high homoeology. To reveal the impact of such aneuploidy on genome-wide gene expression, the transcriptomes of MAAL C1, Cs1 and euploid RBR were analyzed. Compared to RBR, Cs1 had sharply reduced gene expression level across chromosome A1, demonstrating the loss of one copy of A1 chromosome. Both additional chromosome C1 in MAAL and substitutional chromosome C1 in Cs1 caused not only cis-effect but also prevalent trans-effect differentially expressed genes. A dominant gene dosage effects prevailed among low expressed genes across chromosome A1 in Cs1, and moreover, dosage effects for some genes potentially contributed to the phenotype deviations. Our results provided novel insights into the transcriptomic perturbation and gene dosage effects on phenotype in CS related to one naturally evolved allopolyploid.