Monosomic Alien Addition Lines Research Articles

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.

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.

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.

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.

Development of Brassica oleracea-nigra monosomic alien addition lines: genotypic, cytological and morphological analyses.

We report the development and characterization of Brassica oleracea - nigra monosomic alien addition lines (MAALs) to dissect the Brassica B genome. Brassica nigra (2n=16, BB) represents the diploid Brassica B genome which carries many useful genes and traits for breeding but received limited studies. To dissect the B genome from B. nigra, the triploid F1 hybrid (2n=26, CCB) obtained previously from the cross B. oleracea var. alboglabra (2n=18, CC)×B. nigra was used as the maternal parent and backcrossed successively to parental B. oleracea. The progenies in BC1 to BC3 generations were analyzed by the methods of FISH and SSR markers to screen the monosomic alien addition lines (MAALs) with each of eight different B-genome chromosomes added to C genome (2n=19, CC+1B1-8), and seven different MAALs were established, except for the one with chromosome B2 which existed in one triple addition. Most of these MAALs were distinguishable morphologically from each other, as they expressed the characters from B. nigra differently and at variable extents. The alien chromosome remained unpaired as a univalent in 86.24% pollen mother cells at diakinesis or metaphase I, and formed a trivalent with two C-genome chromosomes in 13.76% cells. Transmission frequency of all the added chromosomes was far higher through the ovules (averagely 14.40%) than the pollen (2.64%). The B1, B4 and B5 chromosomes were transmitted by female at much higher rates (22.38-30.00%) than the other four (B3, B6, B7, B8) (5.04-8.42%). The MAALs should be valuable for exploiting the genome structure and evolution of B. nigra.

Characterization of eleven monosomic alien addition lines added from Gossypium anomalum to Gossypium hirsutum using improved GISH and SSR markers.

Background Gossypium anomalum (BB genome) possesses the desirable characteristics of drought tolerance, resistance to diseases and insect pests, and the potential for high quality fibers. However, it is difficult to transfer the genes associated with these desirable traits into cultivated cotton (G. hirsutum, AADD genome). Monosomic alien addition lines (MAALs) can be used as a bridge to transfer desired genes from wild species into G. hirsutum. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, the development of cotton MAALs has lagged far behind many other crops. To date, no set of G. hirsutum-G. anomalum MAALs was reported. Here the amphiploid (AADDBB genome) derived from G. hirsutum × G. anomalum was used to generate a set of G. hirsutum-G. anomalum MAALs through a combination of consecutive backcrossing, genomic in situ hybridization (GISH), morphological survey and microsatellite marker identification.ResultsWe improved the GISH technique used in our previous research by using a mixture of two probes from G. anomalum and G. herbaceum (AA genome). The results indicate that a ratio of 4:3 (G. anomalum : G. herbaceum) is the most suitable for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Using this improved GISH technique, 108 MAAL individuals were isolated. Next, 170 G. hirsutum- and G. anomalum-specific codominant markers were obtained and employed for characterization of these MAAL individuals. Finally, eleven out of 13 MAALs were identified. Unfortunately, we were unable to isolate Chrs. 1Ba and 5Ba due to their very low incidences in backcrossing generation, as these remained in a condition of multiple additions.ConclusionsThe characterized lines can be employed as bridges for the transfer of desired genes from G. anomalum into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, development of chromosome-specific “paints” for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs/genes for a particular G. anomalum chromosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0913-2) contains supplementary material, which is available to authorized users.

Characterization of morphology and resistance to Blumeria graminis of winter triticale monosomic addition lines with chromosome 2D of Aegilops tauschii.

Key messageAllocation of the chromosome 2D ofAe. tauschiiin triticale background resulted in changes of its organization, what is related to varied expression of genes determining agronomically important traits.Monosomic alien addition lines (MAALs) are crucial for transfer of genes from wild relatives into cultivated varieties. This kind of genetic stocks is used for physical mapping of specific chromosomes and analyzing alien genes expression. The main aim of our study is to improve hexaploid triticale by transferring D-genome chromatin from Aegilops tauschii × Secale cereale (2n = 4x = 28, DDRR). In this paper, we demonstrate the molecular cytogenetics analysis and SSR markers screening combined with phenotype analysis and evaluation of powdery mildew infection of triticale monosomic addition lines carrying chromosome 2D of Ae. tauschii. We confirmed the inheritance of chromosome 2D from the BC2F4 to the BC2F6 generation of triticale hybrids. Moreover, we unveiled a high variable region on the short arm of chromosome 2D, where chromosome rearrangements were mapped. These events had direct influence on plant height of hybrids what might be connected with changes at Rht8 loci. We obtained 20 semi-dwarf plants of BC2F6 generation carrying 2D chromosome with the powdery mildew resistance, without changes in spike morphology, which can be used in the triticale breeding programs.

Development of Oryza sativa L. by Oryza punctata Kotschy ex Steud. monosomic addition lines with high value traits by interspecific hybridization.

This paper describes the development of monosomic alien addition and disomic introgression lines through a cross between autotetraploid indica rice and Oryza punctata toward tapping valuable traits for rice improvement. Oryza punctata is a distantly related wild Oryza species having BB genome with untapped genetic resources for rice improvement. Low crossability between the cultivated O. sativa and O. punctata restricts the success of transferring many desirable traits into cultivated rice. Artificially induced autotetraploids of an elite breeding line, IR31917-45-3-2, were produced and crossed with O. punctata. Allotriploid F1 plants were backcrossed to IR31917-45-3-2 and generated progenies with extra chromosomes from O. punctata. Twenty BC1F1 and 59 BC2F1 plants were produced with chromosome numbers ranging from 24 (2n) to 29 (2n+5) and 2n (24) to 26 (2n+2), respectively. Eleven monosomic alien addition lines (MAALs) were characterized morphologically and cytologically and designated as MAAL 1-12. MAALs were genotyped using O. punctata genome-specific molecular markers and detected chromosome segments inherited from O. punctata. O. punctata introgressions across all the chromosomes of O. sativa were identified except for chromosome 8. The most frequent introgressions were observed in chromosomes 4, 6, 10, and 11, which could be the recombination hotspots between A and B genomes. Some of the qualitative traits such as black hull, purple coleoptile base, purple stigma, long awn, and short grain size from O. punctata were inherited in some disomic introgression lines (DILs). Several DILs inherited genes from O. punctata conferring resistance to brown planthopper, green leafhopper, and diseases such as bacterial blight and blast. This is the first report on successful gene transfer from O. punctata into O. sativa.

Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha.

African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75%) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78%) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.

Development of monosomic alien addition lines from the wild rice (Oryza brachyantha A. Chev. et Roehr.) for introgression of yellow stem borer (Scirpophaga incertulas Walker.) resistance into cultivated rice (Oryza sativa L.)

Oryza brachyantha, the African wild rice which is resistant to yellow stem borer (YSB), was exploited as a source for introgression of YSB resistance trait into cultivated rice (O. sativa), through development of monosomic alien addition lines (MAALs). BC2F1 backcross hybrids (O. sativa cv Savitri/O. brachyantha//O. sativa cv Savitri///O. sativa cv Savitri) were produced employing embryo rescue, with a crossability of 0.12 %. Hybrid embryos that were collected at 10 and 12 days after pollination showed highest percentage of survival in culture with a germination percentage of 35.8 %. Cytological analysis of pollen mother cells (PMCs) of the BC2F1 hybrids revealed 15 hybrids with 2n + 1(2n = 25) chromosome number that exhibited typical trisomic chromosomal configurations. These aneuploids were putatively designated as MAALs and characterized morphologically and cytologically. Based on their morphological similarity to primary trisomics of the cultivated rice (O. sativa), 8 MAALs viz., MAALs-4 (Sterile), 5 (Twisted leaf), 7 (Narrow leaf), 8 (Rolled leaf), 9 (Stout), 10 (Short grain), 11 (Pseudonormal) and 12 (Tall) were isolated, each of which exhibited several distinct morphological features. Among the 505 PMCs analysed, 79.80 % had 12II, 5.45 % had 11II and 1.78 % PMCs had 1III configurations. The size of the extra chromosome was found to be smaller than those of O. sativa chromosomes and in 97.25 % of PMCs it remained unpaired and at a distance from the rest of the chromosomes. Of the 8 MAALs screened for YSB resistance, MAAL 11 was found to be moderately resistant. Undesirable morphological traits of O. brachyantha were found to be eliminated in the MAALs.

Generation and characterization of Brassica rapa ssp. pekinensis - B. oleracea var. capitata monosomic and disomic alien addition lines.

Five monosomic alien addition lines (MAALs) of Brassica rapa ssp. pekinensis - B. oleracea var. capitata were obtained by hybridization and backcrossing between B. rapa ssp. pekinensis (female parent) and B. oleracea var. capitata. The alien linkage groups were identified using 42 B. oleracea var. capitata linkage group-specific markers as B. oleracea linkage groups C2, C3, C6, C7 and C8. Based on the chromosomal karyotype of root tip cells, these five MAALs added individual chromosomes from B. oleracea var. capitata: chr 1 (the longest), chr 2 or 3, chr 5 (small locus of 25S rDNA), chr 7 (satellite-carrying) and chr 9 (the shortest). Five disomic alien addition lines were then generated by selfing their corresponding MAALs.