GISH Analysis Research Articles

Distribution and evolution of repeated sequences in genomes of Triatominae (Hemiptera-Reduviidae) inferred from genomic in situ hybridization.

The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily.

Fertility of CMS wheat is restored by two Rf loci located on a recombined acrocentric chromosome.

Cytoplasmic male sterility (CMS) results from incompatibility between nuclear and cytoplasmic genomes, and is characterized by the inability to produce viable pollen. The restoration of male fertility generally involves the introgression of nuclear genes, termed restorers of fertility (Rf). CMS has been widely used for hybrid seed production in many crops but not in wheat, partly owing to the complex genetics of fertility restoration. In this study, an acrocentric chromosome that restores pollen fertility of CMS wheat in Hordeum chilense cytoplasm (msH1 system) is studied. The results show that this chromosome, of H. chilense origin and named H(ch)ac, originated from a complex reorganization of the short arm of chromosomes 1H(ch) (1H(ch)S) and 6H(ch) (6H(ch)S). Diversity arrays technology (DArT) markers and cytological analysis indicate that H(ch)ac is a kind of `zebra-like' chromosome composed of chromosome 1H(ch)S and alternate fragments of interstitial and distal regions of chromosome 6H(ch)S. PCR-based markers together with FISH, GISH, and meiotic pairing analysis support this result. A restorer of fertility gene, named Rf6H(ch)S, has been identified on the short arm of chromosome 6H(ch)S. Moreover, restoration by the addition of chromosome 1H(ch)S has been observed at a very low frequency and under certain environmental conditions. Therefore, the results indicate the presence of two Rf genes on the acrocentric chromosome: Rf6H(ch)S and Rf1H(ch)S, the restoration potential of Rf6H(ch)S being greater. The stable and high restoration of pollen fertility in the msH1 system is therefore the result of the interaction between these two restorer genes.

Molecular and cytogenetic characterization of a common wheat-Agropyron cristatum chromosome translocation conferring resistance to leaf rust

Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a perennial species of economic importance as forage that also displays potentially valuable traits for wheat improvement trough intergeneric hybridization. In order to incorporate resistance genes from A. cristatum against wheat leaf rust (Puccinia triticina Erikss.) into common wheat (Triticum aestivum L.) a breeding programme was carried out by crossing and backcrossing the self-fertile amphiploid AABBDDPP (2n = 8x = 56) with T. aestivum (2n = 6x = 42; AABBDD). The AABBDDPP amphiploid was previously obtained by crossing tetraploid wheat (Triticum turgidum L. conv. durum Desf. 2n = 4x = 28; AABB) with a self-fertile allotetraploid (2n = 4x = 28; DDPP) between diploid wheat (Aegilops tauschii Coss.) and crested wheatgrass (A. cristatum). After three backcrosses a fertile stable line (named TH4) was obtained with 42 chromosomes. The fluorescence in situ hybridization and GISH analysis confirmed that TH4 carries a compensating robertsonian translocation involving the long arm of wheat chromosome 1B and the short arm of an unidentified A. cristatum chromosome. Specific molecular markers from A. cristatum also demonstrated the presence of chromatin from this species in the TH4 line. Macroscopic and microscopic observations indicated that the A. cristatum fragment that has been transferred to common wheat contributed a substantial level of partial resistance to leaf rust. The A. cristatum translocation line in bread wheat makes the disease resistance gene(s) from A. cristatum accessible for wheat breeding programmes.

Morphological, Cytological and Biochemical Characterization of wheatAegilops LongissimaDerivatives BC1F6and BC2F4with High Grain Micronutrient

Micronutrient deficiency (Iron and Zinc) is the major problem worldwide mostly in the developing countries with high dependence on staple foods. Biofortification of staple cereal and tuber crops has been considered and taken up as the most effective, feasible and economic approach for alleviating micronutrient deficiency. The present study was the initiative towards biofortification of wheat where the previous work of wide hybridization between HD2687 and Aegilops longissima accession 3506 and subsequent backcrossing with Triticum aestivum cultivar WL711 has been continued. In this study the alien chromosome introgression, chromosomal stability of BC1F6 and BC2F4 wheat-Ae. longissima derivatives has been investigated for their potential as germplasm for future breeding and biofortification program through morphological, cytological and biochemical analysis. We found that the selected derivatives showed stable 42, 44 and 46 chromosomes for most of the plants where single plant reported for each 41, 43 and 45 chromosomes with 19–22 bivalents and few trivalent. The GISH analysis of derivative 79-1-4-8-1-2-2 revealed introgression of two univalent Sl chromosomes. Finally four derivatives were selected from BC1F6 (79-1-4-8-10-2-2, 79-1-4-8-10-2-5, 79-2-4-4-1-1-3 and 79-2-4-4-1-1-5) and three from BC2F4 (HD2687/L3506//WL711-3///WL711-1-2-7-1, HD2687/L3506//WL711-3///WL711-1-2-7-3, HD2687/L3506//WL711-3///WL711-1-2-7-5) as stable biofortified lines for future breeding to alleviate hidden hunger.

Induction of 4VS chromosome recombinants using the CS ph1b mutant and mapping of the wheat yellow mosaic virus resistance gene from Haynaldia villosa

The wheat spindle streak mosaic virus (WSSMV) or wheat yellow mosaic virus (WYMV) resistance gene, Wss1, from Haynaldia villosa, was previously mapped to the chromosome arm 4VS by the development of 4V (4D) substitution and T4DL·4VS translocationlines. For better utilization and more accurate mapping of the Wss1, in this research, the CS ph1b mutant was used to induce new translocations with shortened 4VS chromosome fragments. Thirty-five homozygous translocations with different alien fragment sizes and breakpoints of 4VS were identified by GISH and molecular marker analysis. By field test, it was found that all the identified terminal translocations characterized as having smaller 4VS chromosome segments in the chromosome 4DS were highly resistant to WYMV, while all the interstitial translocations with 4VS inserted into the 4DS were WYMV susceptible. Marker analysis using 32 4VS-specific markers showed that both the terminal and interstitial translocations had different alien fragment sizes. Five specific markers could be detected in the WYMV-resistant terminal translocation line NAU421 with the shortest introduced 4VS fragment, indicating they can be used for marker-assisted selection in wheat breeding. Based on the resistance evaluation, GISH and molecular marker analysis of the available translocations, the gene(s) conferring the WYMV resistance on 4VS could be further cytologically mapped to the distal region of 4VS, immersed in the bin of FL 0.78-1.00. The newly developed small fragment translocations with WYMV resistance and 4VS specific markers have laid solid groundwork for the utilization in wheat breeding for WYMV resistance as well as further cloning of Wss1.

High-throughput genotyping of wheat-barley amphiploids utilising diversity array technology (DArT)

BackgroundHordeum chilense, a native South American diploid wild barley, is one of the species of the genus Hordeum with a high potential for cereal breeding purposes, given its high crossability with other members of the Triticeae tribe. Hexaploid tritordeum (×Tritordeum Ascherson et Graebner, 2n=6×=42, AABBHchHch) is the fertile amphiploid obtained after chromosome doubling of hybrids between Hordeum chilense and durum wheat. Approaches used in the improvement of this crop have included crosses with hexaploid wheat to promote D/Hch chromosome substitutions. While this approach has been successful as was the case with triticale, it has also complicated the genetic composition of the breeding materials. Until now tritordeum lines were analyzed based on molecular cytogenetic techniques and screening with a small set of DNA markers. However, the recent development of DArT markers in H. chilense offers new possibilities to screen large number of accessions more efficiently.ResultsHere, we have applied DArT markers to genotype composition in forty-six accessions of hexaploid tritordeum originating from different stages of tritordeum breeding program and to H. chilense-wheat chromosome addition lines to allow their physical mapping. Diversity analyses were conducted including dendrogram construction, principal component analysis and structure inference. Euploid and substituted tritordeums were clearly discriminated independently of the method used. However, dendrogram and Structure analyses allowed the clearest discrimination among substituted tritordeums. The physically mapped markers allowed identifying these groups as substituted tritordeums carrying the following disomic substitutions (DS): DS1D (1Hch), DS2D (2Hch), DS5D (5Hch), DS6D (6Hch) and the double substitution DS2D (2Hch), DS5D (5Hch). These results were validated using chromosome specific EST and SSR markers and GISH analysis.ConclusionIn conclusion, DArT markers have proved to be very useful to detect chromosome substitutions in the tritordeum breeding program and thus they are expected to be equally useful to detect translocations both in the tritordeum breeding program and in the transference of H. chilense genetic material in wheat breeding programs.

Elucidation of intergenomic recombination and chromosome translocation: meiotic evidence from interspecific hybrids of Lilium through GISH analysis

Intergenomic exchange has been found to be a normal phenomenon in sexual polyploids. In order to distinguish whether such exchanges are derived from intergenomic recombination or translocation, 13 genotypes of an interspecific hybrid, which were previously used as parents to generate sexual polyploids, were selected for a detailed meiosis analysis. In all genotypes under study, variable numbers of bivalents (0–12) resulting from homoeologous pairing were observed. But in two genotypes (006001-6 and 006001-13), a multivalent which was either a quadri- or a trivalent, as well as a bivalent involving two Asiatic chromosomes, was observed. Two Asiatic chromosomes in 006001-6 which were non-homologous were always found to be associated together, and formed trivalent or quadrivalent with chromosomes from homoeologous genome. This indicated that there was a duplication between these two Asiatic chromosomes. Such a duplication might have resulted from the segregation of a chromosomal translocation between two non-homologous chromosomes in the Asiatic parent ‘Connecticut King’ which was transmitted to the progeny (006001-6). With the exception of two genotypes, in 11 genotypes that formed variable frequencies of bivalents, the homoeologous chromosome pairing and chiasma formation were similar to that between homologous chromosomes. After from the analysis of anaphase I stages it was evident that the expected types of chiasma formation involving non-sister chromatids gave rise to two strand single, two strand double, three strand double, four strand double and multiple exchanges. Whereas these events resulted from locus specific homoeologous exchanges, the translocations resulted from an aberrant form of non-homologous chromosomal exchange of segments. Elucidation of such differences is only possible through the analysis of meiosis using GISH.

Molecular cytological characterization of two novel durum -Thinopyrum intermediumpartial amphiploids with resistance to leaf rust, stem rust andFusariumhead blight

Thinopyrum intermedium, a wild relative of wheat, is an excellent source of disease resistance. Two novel partial amphiploids, 08-47-50 and 08-53-55 (2n = 6x = 42), were developed from wide crosses between durum wheat and Th. intermedium. Meiotic analysis showed that pollen mother cells of the two partial amphiploids formed an average 20.49 bivalents for 08-47-50 and 20.67 bivalents for 08-53-55, indicating that they are basically cytologically stable. GISH analysis revealed that the two partial amphiploids carried different chromosome compositions. 08-47-50 had fourteen chromosomes from Th. intermedium and its alien chromosomes included six St-, four E(e) - and four E(e)-St translocated chromosomes, whereas 08-53-55 had four St- and ten E(e)-St translocated chromosomes. Fungal disease evaluation indicated that both partial amphiploids had a high level of resistance to FHB, leaf rust and stem rust race Ug99. These two novel partial amphiploids with multiple disease resistance could be used as a new source of multiple disease resistance in bread wheat and durum wheat breeding programs.

Molecular cytogenetic analysis of wheat – Elymus repens introgression lines with resistance to Fusarium head blight

Elymus repens (L.) Gould (2n = 6x = 42, StStStStHH) is a hexaploid perennial wheatgrass species from the tribe Triticeae, distantly related to bread wheat Triticum aestivum L. (2n = 6x = 42, AABBDD). As a potential source of resistance to Fusarium head blight (FHB), E. repens was crossed to common wheat to transfer resistance genes. The progeny were advanced to homozygosity by single seed descent. A total of eight BC(1)F(9) progeny lines were selected and characterized in this study. The chromosome numbers of these derived lines ranged from 42 to 56, including lines with 44, 52, and 54 chromosomes. All of the lines were cytologically stable in terms of meiotic chromosome behavior. The univalent frequency in the lines varied between 0.34 and 2.36 per cell. Similarly, the multivalent frequency did not exceed 1% in any of the lines. GISH analysis revealed that the number of intact wheat chromosomes in the various lines varied between 40 and 44. Numerous translocated chromosomes were detected in all lines. The translocations involved chromosomal segments from wheat, and the St and H genomes of E. repens. Furthermore, trigenomic translocated chromosomes were detected in some of the lines. The introgression into wheat chromosomes included not only terminal types but also interstitial segments. The Fusarium head blight resistance of the eight lines, following point inoculation, varied from 5.65% infected florets to 11.46% compared with the check cultivars T. aestivum 'Roblin' and T. aestivum 'Crocus' at 100% and 85%, respectively.

A cytogenetic study of the blue-grain line of the common wheat cultivar Saratovskaya 29

The chromosome composition of the blue-grain line i:S29Ba of the cultivar Saratovskaya 29 was identified by cytological, GISH, and microsatellite analyses and C-banding. It was found that common wheat chromosome 4B of the cultivar Saratovskaya 29 was substituted with the Agropyron elongatum Host. chromosome carrying the gene for blue grain (s:S294Ag(4B)) during the construction of this nearly isogenic line. The blue-grain line was tested for productivity. The substitution of total chromosome 4B of the cultivar Saratovskaya 29 by Ag. elongatum chromosome 4 did not significantly affect the spike productivity parameters and grain quality with the exception of spike length (plus effect), spike density, and vitreousness (minus effects). The blue-grain line with s:S294Ag(4B) can be used in further studies associated with chromosome engineering in cereals and wheat breeding.

Molecular cytogenetic characterization and stripe rust response of a trigeneric hybrid involvingTriticum,Psathyrostachys, andThinopyrum

Trigeneric hybrids offer opportunities to transfer alien traits into cultivated wheat. In this study, a new trigeneric hybrid involving species of Triticum, Psathyrostachys, and Thinopyrum was synthesized by crossing Triticum aestivum L. (wheat)--Thinopyrum intermedium (Host) Barkworth & D.R. Dewey amphiploid Zhong 3 with wheat--Psathyrostachys huashanica Keng ex Kuo amphiploid PHW-SA. Crossability of the two amphiploids was 19.74%, and the fertility of the hybrid was 16.20%. The mean meiotic configuration of the trigeneric hybrid (2n=56) was 13.06 I+17.24 IIring+3.73 IIrod+0.28 III+0.04 IV. GISH analysis indicated that the trigeneric F1 had seven P. huashanica chromosomes and seven Th. intermedium chromosomes. The mean chromosome numbers of F2, F3, and F4 progenies were 2n=49.24, 2n=48.13, and 2n=46.78, respectively, a gradual decrease. GISH analysis revealed that most F2 and F3 plants had 2–10 Th. intermedium chromosomes and 0–4 P. huashanica chromosomes. In the F4 progenies, 1–7 Th. intermedium chromosomes were labeled, but no P. huashanica chromosomes were detected. It seems that Th. intermedium chromosomes are more likely than P. huashanica chromosomes to be transmitted to the progenies. The stripe rust response of PHW-SA was expressed in the F1 and some F2 and F3 progenies. The trigeneric hybrid could be a useful bridge for transfering P. huashanica and Th. intermedium chromosomes to common wheat.

First intergeneric hybrids within the tribe Anthemideae Cass. III. Chrysanthemum indicum L. Des Moul. × Opisthopappus taihangensis (Ling) Shih

Ovule rescue was used to obtain the intergeneric hybrid Chrysanthemum indicum L. Des Moul. x Opisthopappus taihangensis (Ling) Shih. The hybridity of the seven independent progeny was confirmed by morphology, chromosome counting and GISH analysis. Plant height, crown width, leaf length, leaf width, epidermal hair height and epidermal hair length of the hybrid were all intermediate between those of the parents. However, their petiole length and epidermal hair density exceeded those of either parent. GISH analysis of the hybrid was able to distinguish between the parental genomes.

Alien genes introgression and development of alien monosomic addition lines from a threatened species, Allium roylei Stearn, to Allium cepa L.

To produce alien monosomic addition lines (AMALs) of Allium cepa (genomes CC, 2n = 2x = 16) carrying extrachromosomes from Allium roylei (RR, 2n = 2x = 16), reciprocal backcrossing of allotriploids (2n = 24, CCR) with diploids (2n = 16, CC) and selfing of a single allotriploid were carried out. The chromosome numbers in the BC(2)F(1) and BC(1)F(2) progenies ranged from 16 to 32. Forty-eight plants were recorded to possess 2n = 17 among a total of 169 plants in observation. Through the analyses of isozymes, expressed sequence tag (EST) markers, and karyotypes, all eight possible types of A. cepa-A. roylei monosomic addition lines (CC+1R-CC+8R) could be identified. Seven types of representative AMALs (without CC+2R) were used for the GISH analysis of somatic chromosomes. Except for CC+6R, all AMALs showed an entire (unrecombined) extrachromosome from A. roylei in the integral diploid background of A. cepa. A single recombination between A. cepa and A. roylei was observed on the extrachromosome in the remaining type. All alloplasmic AMALs possessing A. roylei cytoplasm showed high or complete pollen sterility. Only the autoplasmic CC+4R with A. cepa cytoplasm possessed relatively high pollen fertility. The bulbs of CC+4R displayed the distinct ovoid shape that discriminates them from spherical or oval ones in other AMALs. Downy mildew screening in the field showed higher resistance in A. roylei, a hypo-allotriploid (CCR-nR, 2n = 23), and an allotriploid (CCR, 2n = 24). Meanwhile, no complete resistance was found in some AMALs examined. This was the first trial toward the establishment of a complete set of A. cepa-A. roylei monosomic additions.

The embryo rescue derived intergeneric hybrid between chrysanthemum and Ajania przewalskii shows enhanced cold tolerance

Five intergeneric hybrids between the chrysanthemum cultivar 'Zhongshanjingui' (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While 'Zhongshanjingui' bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids' flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n=45, of which GISH analysis was able to establish that 27 were inherited from 'Zhongshanjingui' and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.

The behavior of Bupleurum scorzonerifolium as a parent of somatic hybrid introgressed lines is associated with UV resistance of its chromosomes

Protoplasts of Bupleurum scorzonerifolium irradiated with 380 μW/cm2 UV for 5 min were fused by the PEG-mediated method with untreated protoplasts of Arabidopsis thaliana. The fusion products were cultured in the P5 liquid medium for single hybrid cell clone formation. As a total, 81 independent putative hybrid clones (cell lines) were obtained, and seventeen of them were identified as somatic hybrids by chromosome counting, GISH, RAPD, and SSR analyses. More than 80 B. scorzonerifolium-like green introgressed plants and leaves were regenerated from 49 somatic hybrid cell lines, which contained chromatin and DNA characteristic of A. thaliana. To assess the UV tolerance of both parents with chromatin exclusion and introgression, their protoplasts were UV-irradiated (380 μW/cm2 for 0 and 5 min), and the protoplasts of A. thaliana were more sensitive to UV than those of B. scorzonerifolium as judged by Single Cell Gel Electrophoresis analysis. The possible relationship between UV resistance of B. scorzonerifolium and A. thaliana chromosome elimination and the formation of somatic introgressed hybrid plants is discussed.

Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization.

BackgroundThe wild herb Swertia mussotii is a source of the anti-hepatitis compounds swertiamarin, mangiferin and gentiopicroside. Its over-exploitation has raised the priority of producing these compounds heterologously. Somatic hybridization represents a novel approach for introgressing Swertia mussotii genes into a less endangered species.ResultsProtoplasts derived from calli of Bupleurum scorzonerifolium and S. mussotii were fused to produce 194 putative hybrid cell lines, of which three (all derived from fusions where the S. mussotii protoplasts were pre-treated for 30 s with UV light) later differentiated into green plants. The hybridity of the calli was confirmed by a combination of isozyme, RAPD and chromosomal analysis. The hybrid calli genomes were predominantly B. scorzonerifolium. GISH analysis of mitotic chromosomes confirmed that the irradiation of donor protoplasts increased the frequency of chromosome elimination and fragmentation. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted and recombined in some hybrid cell lines. Some of the hybrid calli contained SmG10H from donor, and produced swertiamarin, mangiferin and certain volatile compounds characteristic of S. mussotii. The expression of SmG10H (geraniol 10-hydroxylase) was associated with the heterologous accumulation of swertiamarin.ConclusionsSomatic hybrids between B. scorzonerifolium and S. mussotii were obtained, hybrids selected all contained introgressed nuclear and cytoplasmic DNA from S. mussotii; and some produced more mangiferin than the donor itself. The introgression of SmG10H was necessary for the accumulation of swertiamarin.

The differential expression of Rhopalosiphum padi resistance in sibling wheat-rye amphiploids

Thirteen amphiploids were synthesized by chromosome doubling of an F1 plant of a cross between Triticum aestivum Mianyang11 × S. cereale Kustro, which shows resistance to Bird cherry-oat aphid (Rhopalosiphum padi). The amphiploids were confirmed by GISH analysis and tested for their reaction to R. padi. Eleven amphiploids exhibited resistance to R. padiand two of them exhibited high level of susceptibility. The PCR analysis using rye-specific markers provided evidence for the elimination of W-box elements from the susceptible amphiploids. The possible mechanism of loss resistance in some amphiploids was discussed. The resistant amphiploids can be exploited in wheat breeding programs to transfer R. padi resistance to susceptible wheat varieties. Key words: Rhopalosiphum padi, amphiploid, wheat, rye.

A semi-fertile interspecific hybrid of Brassica rapa and B. nigra and the cytogenetic analysis of its progeny

Black rot is a bacterial disease of Brassica rapa caused by Xanthomonas campestris pv. campestris (Xcc.). Sources of resistance to this disease within B. rapa are insufficient and control measures are limited, making the development of resistant breeding lines extremely important. Certain lines of B. nigra exhibit very high resistance to Xcc. For this study, an interspecific cross between Brassica rapa and B. nigra was performed, and a total of 6 F1 hybrids were obtained through ovary culture. Five plants (H1–5) were relatively slow-growing, entirely sterile, and had 18 chromosomes in the majority of pollen mother cells (PMCs). GISH analysis showed that most of the PMCs had 8 B. nigra chromosomes, which indicated the expected AB genomic constitution. The last plant (H6) was partially fertile and the majority of PMCs contained 10 chromosomes of B. rapa and 16 chromosomes of B. nigra, indicating an ABB genomic constitution. Ovary culture techniques were not necessary for the development of the first- or second-backcross generation. Sequence-related amplified polymorphism analysis of F1, BC1, and BC2 plants indicated that some fragments from B. nigra were lost, particularly in the genome of BC1/BC2 in successive generation(s). The BC2 plants expressing good resistance to Xcc. were observed.

Isolation of five new monosomic alien addition lines of Gossypium australe F. Muell in G. hirsutum L. by SSR and GISH analyses

With 2 figures and 3 tables Abstract Gossypium australe F. Muell (2n = 2x = 26) is a wild perennial species possessing agronomic useful traits that would be interesting to introgress into G. hirsutum L. (2n = 4x = 52), the main cultivated cotton species. To isolate monosomic alien addition lines (MAALs) of G. australe in G. hirsutum, the [2(G. hirsutum × G. australe) × G. hirsutum] pentaploid (2n = 5x = 65) was backcrossed as male parent to G. hirsutum. Analysis of 42 BC1 plants and seven alien addition lines, already available, with 150 SSR markers developed from G. hirsutum revealed a cross‐species amplification rate of 100% and a polymorphism rate of 56%. Eighty polymorphic SSR markers generated 87 G. australe‐specific loci that have been assigned by a hierarchical cluster analysis to 13 linkage groups corresponding to the 13 chromosomes of G. australe. Analysis by SSR markers and genomic in situ hybridization of the self‐progeny of disomic alien addition lines, backcross progeny of the pentaploid, allowed the isolation of five new MAALs.

A Tal-PhI wheat genetic stock facilitates efficient alien introgression

The genetic stock Chinese Spring PhI (refer as CS-PhI hereafter) with the Ph I gene (Ph gene inhibitor, introduced from a high-pairing accession of Aegilops speltoides) was crossed as a male parent to a Tai-gu genic male-sterile wheat with the Tal gene and backcrossed once to CS-PhI to develop the Tal-PhI genetic stock. The Tal-PhI stock, CS-PhI as well as Chinese Spring (CS), were test-crossed using Aegilops peregrina (syn. Aegilops variabilis) to test the effectiveness of the Ph I gene inducing homoeologous chromosome pairing in the Tal-PhI stock. At meiotic I(MI) of pollen mother cells (PMCs), the average chromosome pairing of the TC1 testcrosses of TalCSPhIC08–4/Ae. peregrina, TalCSPhIC11–5/Ae. peregrina and CS/Ae. peregrina was 26.81 I + 3.64 II + 0.33 Open image in new window + 0.07 III + 0.01 IV, 24.75 I + 4.58 II + 0.25 Open image in new window + 0.21 III + 0.07 IV and 32.25 I + 1.38 II, respectively. These data indicated that the Ph I gene could promote homoeologous chromosome pairing in the Tai-gu male sterile background. In order to verify the introgression of the chromatin from Ae. speltoides into CS in the three CS-PhI lines, a total of 79 SSR primers located on group-3 chromosomes were used for PCR analysis using the genomic DNA of CS, Ae. speltoides and the three CS-PhI lines. Two markers Xwmc505 -110 and Xwmc674 -160 amplified S genome-specific fragments from Ae. speltoides in lines PhIC04–13 and PhIC11–5, providing molecular evidence for the introgression of Ae. speltoides-specific chromatin in the two lines. Furthermore, some 3D-specifc SSR markers were missing in line PhIC04–13, indicating there might be a deletion of chromosome 3D in this line. In further experiments, a BC1 population was produced from the cross of Tal-PhI/Triticum durum-Dasypyrum villosum amphiploid (AABBVV)//CS-PhI, and the progenies were further screened for chromosome recombination by GISH analysis. A homozygous translocation line T5VS·5VL-5DL was identified from the BC1F2 by genomic in situ hybridization, C-banding and SSR analysis. The results of the present study demonstrate the Tal-PhI is a useful genetic stock for transferring alien genes into common wheat.