Sequential Genomic In Situ Hybridization Research Articles

Development and molecular cytogenetic identification of a new wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust and sharp eyespot.

A wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust, sharp eyespot and high kernel number per spike was developed and characterized by molecular cytogenetic method as novel resistant germplasm. Rye (Secale cereale L.), a close relative of common wheat, is an important and valuable gene donor with multiple disease resistance for wheat improvement. However, resistance genes derived from rye have successively lost resistance to pathogens due to the coevolution of pathogen virulence and host resistance. Development and identification of new effective resistance gene sources from rye therefore are of special importance and urgency. In the present study, a wheat-rye line WR35 was produced through distant hybridization, embryo rescue culture, chromosome doubling and backcrossing. WR35 was then proven to be a new wheat-rye 4R disomic addition line using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization) and ND-FISH (non-denaturing FISH) with multiple probes, mc-GISH (multicolor GISH), rye chromosome arm-specific marker analysis and SLAF-seq (specific-locus amplified fragmentsequencing) analysis. At the adult stage, WR35 exhibited high levels of resistance to the powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and stripe rust (Puccinia striiformis f. sp. tritici, Pst) pathogens prevalent in China, and a highly virulent isolate of Rhizoctonia cerealis, the cause of wheat sharp eyespot. At the seedling stage, it was highly resistant to 22 of 23 Bgt isolates and four Pst races. Based on its disease responses to different pathogen isolates, WR35 may possess resistance gene(s) for powdery mildew, stripe rust and sharp eyespot, which differed from the known resistance genes from rye. In addition, WR35 was cytologically stable and produced high kernel number per spike. Therefore, WR35 with multi-disease resistances and desirable agronomic traits should serve as a promising bridging parent for wheat chromosome engineering breeding.

IDENTIFICATION OF BLUE‑ALEURONE WHEAT DERIVATIVES BY SEQUENTIAL GISH-FISH ASSAY

To select stable inheritable disomic substitution line from the filial generation of blue-grain wheat and common wheat high effectively, sequential GISH (genomic in situ hybridization) and multicolor FISH (fluorescence in situ hybridization) assay was used to investigate the number and characteristics of chromosome 4E in F4 and F6 hybrid offspring. The F4 and F6 plant were selected by the blue aleurone layer future, in which the bluegrain genes from Thinopyrum species were existed. GISH assay showed that the rate of stable inheritable disomic substitution line (blue-grain trait in common wheat) was above 80 percent in F6 plants, but lower in F4 plants. Specially, all of the F6 progenies of ‘qian 09206’, which was derived from crossing parents ‘0147’ and common wheat ’9938’, were homozygous disomic substitution lines. The sequential GISH-FISH assay showed that F4 progeny (B-228) from filial generation of ‘Neimai 10’ and ‘0147’ was a 4D/4E stable inheritable alien disomic substitution line. These results showed that the color based selection was not effective. However, combining color based selection with sequential GISH-FISH assay can improve wheat breeding efficiency.

Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut

Interspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are (TTTAGGG)n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats (ITRs) are also commonly identified. In this study, the telomeric repeat was chromosomally localized in 10 Arachis species through sequential GISH (genomic in situ hybridization) and FISH (fluorescence in situ hybridization) combined with 4’,6-diamidino-2-phenylindole (DAPI) staining. Six ITRs were identified such as in the centromeric region of chromosome Bi5 in Arachis ipaënsis, pericentromeric regions of chromosomes As5 in A. stenosperma, Bho7 in A. hoehnei and Av5 in A. villosa, nucleolar organizer regions of chromosomes As3 in A. stenosperma and Adi3 in A. diogoi, subtelomeric regions of chromosomes Bho9 in A. hoehnei and Adu7 in A. duranensis, and telomeric region of chromosome Es7 in A. stenophylla. The distributions of the telomeric repeat, 5S rDNA, 45S rDNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S rDNA and 45S rDNA sites in this study, along with inversions detected on the long arms of chromosomes Kb10 and Bho10, indicated frequent chromosomal rearrangements during evolution of Arachis species.

Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance.

Rye (Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.

Characterization of a partial wheat-Thinopyrum intermedium amphiploid and its reaction to fungal diseases of wheat

Partial amphiploids between wheat (Triticum aestivum L.) and Thinopyrum species play an important role in the transfer and use of traits from alien species. A wheat-Thinopyrum intermedium partial amphiploid, TAI8335, and its alien parent were characterized by a combination of genomic in situ hybridization (GISH) and cytological observations. Evidence from GISH indicated that the donor parent Th. intermedium possessed seven pairs of S, seven J(s) and 21 J chromosomes. Mitotic observation showed that the majority of TAI8335 plants had 56 chromosomes, but a few had 54 to 55, in some cases with two to three additional telochromosomes. The chromosomes in most pollen mother cells of plants with 2n = 56 formed 28 bivalents, averaging 27.12 in 223 cells, suggesting a basic cytological stability. Sequential GISH patterns using genomic Pseudoroegneria spicata and genomic Th. intermedium DNA as probes revealed that TAI8335 had fourteen chromosomes derived from Th. intermedium and its alien genome consisted of one pair of S-, three pairs of J(s) - and one pair of J-genome chromosomes as well as two translocated chromosome pairs, one being a Robertsonian translocation and another an intercalary translocation, both of which involved J and S genome. Two of the telochromosomes in the aneuploid plants originated from the J genome and one from wheat. Disease screening demonstrated this line was highly resistant to leaf rust, stem rust, stripe rust and powdery mildew. This study showed that the partial amphiploid TAI8335 appears to serve as a novel source for the transfer of resistance genes for multiple fungal pathogens to wheat.

Analysis of introgression of the Tulipa fosteriana genome into Tulipa gesneriana using GISH and FISH

Southern hybridization and genomic in situ hybridization (GISH) have demonstrated that ‘Purissima’ (2n = 2x = 24) is an interspecific hybrid comprised of one genome of Tulipa (T.) gesneriana and one genome of T. fosteriana. Backcrossing T. gesneriana with ‘Purissima’ was partially successful. Simultaneous GISH and fluorescence in situ hybridization (FISH) distinguished chromosomes from both parent genomes, as well as recombinant chromosomes, in interspecific hybrids and their progeny. Chromosome recombination was observed in all cultivars except ‘Purissima’ and ‘Kouki’ (2n = 3x = 36). ‘Kouki’ (2n = 3x = 36) had two genomes of the T. gesneriana and a single genome of the T. fosteriana. The number of nonrecombinant T. fosteriana chromosomes in ‘Judith Leyster’ (2n = 4x = 48) and ‘Purissima’ progeny varied from two in ‘Hatsuzakura’ to six in ‘Kikomachi’ and ‘Momotaro’. The number and type of recombinant chromosomes also differed among cultivars. The total number of translocations ranged from one in ‘Kikomachi’ to six in ‘Hatsuzakura’. Each was a combination of a single T. fosteriana fragment and a single T. gesneriana fragment, indicating that they resulted from a single crossover event. Sequential GISH and FISH analysis with rDNA probes yielded chromosome-specific markers that were used to identify most of the chromosomes in ‘Purissima’ progeny. This is the first report of introgression of T. fosteriana chromatin into the T. gesneriana genome.

Agropyron elongatum chromatin localization on the wheat chromosomes in an introgression line

The introgressed small-chromosome segment of Agropyron elongatum (Host.) Neviski (Thinopyrum ponticum Podp.) in F5 line II-1-3 of somatic hybrid between common wheat (Triticum aestivum L.) and A. elongatum was localized by sequential fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and karyotype data. Karyotype analysis offered basic data of arm ratios and relative lengths of 21 pairs of chromosomes in parent wheat Jinan177 and hybrid II-1-3. Using special high repetitive sequences pSc119.2 and pAs1 for FISH, the entire B- and D-genome chromosomes were detected. The FISH pattern of hybrid II-1-3 was the same as that of parent wheat. GISH using whole genomic DNA from A. elongatum as probe determined the alien chromatin. Sequential GISH and FISH, in combination with some of the karyotype data, localized the small chromosome segments of A. elongatum on the specific sites of wheat chromosomes 2AL, 1BL, 5BS, 1DL, 2DL and 6DS. FISH with probe OPF-03(1296) from randomly amplified polymorphic DNA (RAPD) detected E-genome chromatin of A. elongatum, which existed in all of the small chromosome segments introgressed. Microsatellite primers characteristic for the chromosome arms above were used to check the localization and reveal the genetic identity. These methods are complementary and provide comprehensive information about the genomic constitution of the hybrid. The relationship between hybrid traits and alien chromatin was discussed.

The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers

Genomic in situ hybridization (GISH) is one of the most popular and effective techniques for detecting alien chromatin introgressed into breeding lines; however, GISH analysis alone does not reveal the genetic identity of the alien chromosomes. We previously isolated a set of bacterial artificial chromosomes (BACs) specific to each of the 12 potato chromosomes. These BAC clones can be used as chromosome-specific cytogenetic DNA markers (CSCDMs) for potato chromosome identification. Here we demonstrate that GISH and fluorescence in situ hybridization (FISH), using CSCDMs, can be performed sequentially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows repeated probing up to five times without significant damage to chromosome morphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and also determine whether an alien chromosome has been added to the genetic background of potato or is substituting for a homoeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.

Characterization of euploid backcross progenies derived from interspecific hybrids betweenOryza sativaandO. eichingeriby restriction fragment length polymorphism (RFLP) analysis and genomic in situ hybridization (GISH)

Restriction fragment length polymorphism (RFLP) analysis and GISH (genomic in situ hybridization) were performed on euploid plants derived from crosses between Oryza sativa (2n = 24, AA) and two brown planthopper-resistant accessions of O. eichingeri (2n = 24, CC). After screening with 164 RFLP markers, 60 of the 67 euploid plants were identified as introgression lines, each carrying 1–6 small O. eichingeri segments integrated on chromosomes 1, 2, 6, or 10. In the somatic chromosome preparations of F1hybrid, O. eichingeri chromosomes, fluorescing greenish-yellow in the sequential GISH, appeared to be longer and to contain more heterochromatin than O. sativa ones, and this karyotypic polymorphism can be used to detect some introgressed O. eichingeri segments in euploid plants. In addition, GISH identification presented direct evidence for the transfer of small segments from O. eichingeri to O. sativa chromosome(s) which were subsequently recognized according to their condensation pattern, arm ratio, and chromosome length. The present results would contribute to the molecular mapping and selection of O. eichingeri - derived brown planthopper-resistant gene and positive yield QTLs.Key words: Oryza sativa, Oryza eichingeri, introgression lines, RFLP, genomic in situ hybridization (GISH).

Characterization of euploid backcross progenies derived from interspecific hybrids between Oryza sativa and O. eichingeri by restriction fragment length polymorphism (RFLP) analysis and genomic in situ hybridization (GISH).

Restriction fragment length polymorphism (RFLP) analysis and GISH (genomic in situ hybridization) were performed on euploid plants derived from crosses between Oryza sativa (2n = 24, AA) and two brown planthopper-resistant accessions of O. eichingeri (2n = 24, CC). After screening with 164 RFLP markers, 60 of the 67 euploid plants were identified as introgression lines, each carrying 1-6 small O. eichingeri segments integrated on chromosomes 1, 2, 6, or 10. In the somatic chromosome preparations of F1 hybrid, O. eichingeri chromosomes, fluorescing greenish-yellow in the sequential GISH, appeared to be longer and to contain more heterochromatin than O. sativa ones, and this karyotypic polymorphism can be used to detect some introgressed O. eichingeri segments in euploid plants. In addition, GISH identification presented direct evidence for the transfer of small segments from O. eichingeri to O. sativa chromosome(s) which were subsequently recognized according to their condensation pattern, arm ratio, and chromosome length. The present results would contribute to the molecular mapping and selection of O. eichingeri--derived brown planthopper-resistant gene and positive yield QTLs.

The extent and position of homoeologous recombination in a distant hybrid of Alstroemeria: a molecular cytogenetic assessment of first generation backcross progenies.

To estimate the extent and position of homoeologous recombination during meiosis in an interspecific hybrid between two distantly related Alstroemeria species, the chromosome constitution of six first generation backcross (BC1) plants was analysed using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis. Four different probes were used for the FISH analysis: two species-specific and two rDNA probes. The six BC1 plants were obtained from crosses between the hybrid A. aurea x A. inodora with its parent A. inodora. GISH clearly identified all chromosomes of both parental genomes as well as recombinant chromosomes. The sequential GISH and FISH analysis enabled the accurate identification of all individual chromosomes in the BC1 plants, resulting in the construction of detailed karyotypes of the plants. The identification of the recombinant chromosomes provided evidence which chromosomes of the two species are homoeologous. Two of the BC1 plants were aneuploid (2n=2x+1=17) and four triploid (2n=3x=24), indicating that both n and 2n gametes were functional in the F1 hybrid. Using GISH, it was possible to estimate homeologous recombination in two different types of gametes in the F1 hyrid. The positions of the crossover points ranged from highly proximal to distal and the maximum number of crossover points per chromosome arm was three. Compared with the aneuploid plants, the triploid plants (which received 2n gametes) clearly possessed fewer crossovers per chromosome, indicating reduced chromosome pairing/recombination prior to the formation of the 2n gametes. Besides homeologous recombination, evidence was found for the presence of structural rearrangements (inversion and translocation) between the chromosomes of the parental species. The presence of the ancient translocation was confirmed through FISH analysis of mitotic and meiotic chromosomes.