Genomic In Situ Hybridization Research Articles

Rapid and cost-effective molecular karyotyping in wheat, barley, and their cross-progeny by chromosome-specific multiplex PCR

BackgroundInterspecific hybridisation is a powerful tool for increasing genetic diversity in plant breeding programmes. Hexaploid wheat (Triticum aestivum, 2n = 42) × barley (Hordeum vulgare, 2n = 14) intergeneric hybrids can contribute to the transfer of agronomically useful traits by creating chromosome addition or translocation lines as well as full hybrids. Information on the karyotype of hybrid progenies possessing various combinations of wheat and barley chromosomes is thus essential for the subsequent breeding steps. Since the standard technique of chromosome in situ hybridisation is labour-intensive and requires specific skills. a routine, cost-efficient, and technically less demanding approach is beneficial both for research and breeding.ResultsWe developed a Multiplex Polymerase Chain Reaction (MPCR) method to identify individual wheat and barley chromosomes. Chromosome-specific primer pairs were designed based on the whole genome sequences of ‘Chinese Spring’ wheat and ‘Golden Promise’ barley as reference cultivars. A pool of potential primers was generated by applying a 20-nucleotide sliding window with consecutive one-nucleotide shifts on the reference genomes. After filtering for optimal primer properties and defined amplicon sizes to produce an ordered ladder-like pattern, the primer pool was manually curated and sorted into four MPCR primer sets for the wheat A, B, and D sub-genomes, and for the barley genome. The designed MPCR primer sets showed high chromosome specificity in silico for the genome sequences of all 18 wheat and barley cultivars tested. The MPCR primers proved experimentally also chromosome-specific for the reference cultivars as well as for 13 additional wheat and four barley genotypes. Analyses of 16 wheat × barley F1 hybrid plants demonstrated that the MPCR primer sets enable the fast and one-step detection of all wheat and barley chromosomes. Finally, the established genotyping system was fully corroborated with the standard genomic in situ hybridisation (GISH) technique.ConclusionsWheat and barley chromosome-specific MPCR offers a fast, labour-friendly, and versatile alternative to molecular cytogenetic detection of individual chromosomes. This method is also suitable for the high-throughput analysis of distinct (sub)genomes, and, in contrast to GISH, can be performed with any tissue type. The designed primer sets proved to be highly chromosome-specific over a wide range of wheat and barley genotypes as well as in wheat × barley hybrids. The described primer design strategy can be extended to many species with precise genome sequence information.

Identification and transfer of resistance to Fusarium head blight from Elymus repens chromosome arm 7StL into wheat

Fusarium head blight (FHB), mainly caused by Fusarium graminearum (Fg), is one of the most devastating fungal diseases in wheat production worldwide. Elymus repens (2n=6x=42, StStStStHH) is a wild relative of wheat with many biotic and abiotic stress resistance traits. To transfer and apply the wild germplasm's resistance gene (s) for wheat breeding, we identified a new translocation line K140-7 with high resistance to FHB, developed from the derivative progenies of E. repens crossed with common wheat cultivars. Cytogenetic analyses based on genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), oligonucleotide-FISH painting (Oligo-FISH painting), and single-gene FISH revealed that K140-7 had 40 wheat chromosomes and two 7DS·7StL translocated chromosomes. Wheat 55K SNP array analysis confirmed that the translocated breakpoint (340.8~342.5 Mb) was close to the centromere region of chromosome 7D (336.3~341.7 Mb), supporting the 7DS·7StL translocation event. Based on the diploid reference St genome of Pseudoroegneria libanotica, we developed 21 simple sequence repeats (SSR) markers, specific for chromosome arm 7StL. Genotyping and phenotyping analysis of the 7DS·7StL translocation in different wheat backgrounds demonstrated that the chromosome arm 7StL confers FHB resistance and possesses the dominant FHB resistance locus (s) named QFhb.Er-7StL. We further transferred QFhb.Er-7StL into three different wheat cultivars, their second 7DS·7StL translocation line-generations showed improved agronomic traits, representing new germplasms that could be used in wheat FHB-resistant breeding programs.

Introgression of an all-stage and broad-spectrum powdery mildew resistance gene Pm3VS from Dasypyrum villosum chromosome 3V into wheat.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious disease tothat threatens wheat production globally. It is imperative to explore novel resistance genes in order to control this disease throughby developing and planting resistant varieties. Here, we identified a wheat-Dasypyrum villosum 3V (3D) disomic substitution line, NAU3815 (2n=42), with a high level of powdery mildew resistance at both the seedling and adult-plant stages. Subsequently, NAU3815 was used to generate recombination between chromosomes 3V and 3D. Through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH)GISH/FISH and 3VS, 3VL-specific markers analysis, four introgression lines were developed from the selfing progenies of 3V and 3D double monosomic line NAU3816, which was derived from the F1 hybrids of NAU3815/NAU0686. There were t3VS (3D) ditelosomic substitution line NAU3817, t3VL (3D) ditelosomic substitution line NAU3818, homozygous T3DL·3VS translocation line NAU3819, and homozygous T3DS·3VL translocation line NAU3820. Powdery mildew tests of these lines confirmed the presence of an all-stage and broad-spectrum powdery mildew resistance gene, Pm3VS, located on chromosome arm 3VS. When compared with the recurrent parent NAU0686 plants, the T3DL·3VS translocation line NAU3819 showed no obvious negative effect on yield-related traits. However, the introduction of the T3DL·3VS translocated chromosome had a strong effect on reducing the flag-leaf length. Consequently, the T3DL·3VS translocation line NAU3819 provides a new germplasm in breeding for both resistance and plant architecture.

Cytogenetic and genomic characterization of a novel wheat-tetraploid Thinopyrum elongatum 1BS·1EL translocation line with stripe rust resistance.

Stripe rust, caused by Puccinia striiformis f. sp tritici (Pst), is a destructive wheat disease pathogen. Thinopyrum elongatum is a valuable germplasm including diploid, tetraploid, and decaploid with plenty of biotic and abiotic resistance. In a previous study, we generated a stripe rust resistance wheat-tetraploid Th. elongatum 1E/1D substitution line K17-841-1. To further apply the wild germplasm for wheat breeding, we selected and obtained a new homozygous wheat-tetraploid Th. elongatum translocation line T1BS·1EL using genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), oligo-FISH-Painting, and the wheat 55K single nucleotide polymorphisms (SNPs) genotyping array. The T1BS·1EL is highly resistant to stripe rust at the seedling and adult stage. Pedigree and molecular marker analyses revealed that the resistance gene was located on chromosome arm 1EL of tetraploid Th. elongatum, tentatively named Yr1EL. Besides, we developed and validated 32 Simple Sequence Repeats (SSR) markers and two kompititive allele specific PCR (KASP) assays which were specific to tetraploid Th. elongatum chromosome arm 1EL to facilitate marker-assisted selection for alien 1EL stripe rust resistance breeding. This will help us explore and locate the stripe rust resistance gene mapping on the 1E chromosome and deploy it in the wheat breeding program.

Analysis of the genetic characteristics and variations in disease-resistant grass carp based on whole-genome resequencing and transcriptome sequencing

Disease-resistant grass carp (DR-GGC) was obtained by backcrossing gynogenetic grass carp (GGC, 2n = 48, female) with common grass carp (Ctenopharyngodon idellus [GC], 2n = 48, male). The genetic constitution and the potential mutation associated with growth dominance in DR-GGC were analyzed by genomic in situ hybridization (GISH), whole-genome resequencing (WGRS) and RNA-sequencing (RNA-seq). The GISH results showed partial translocations and deletions in the chromosomes of DR-GGC compared with GC. The WGRS results demonstrated that there were numerous genetic variations between DR-GGC and its parents, among which DR-GGC had the highest genomic heterozygosity and GGC had the highest genomic purity. The RNA-seq results revealed that the differentially expressed genes (DEGs) were significantly enriched in cellular processes and immune pathways in DR-GGC and GGC compared with GC. Meanwhile, the combined analysis of WGRS and RNA-seq identified DEGs with overlapping mutation loci in DR-GGC and GGC compared with GC. These DEGs were significantly enriched in the pathways of growth, metabolism, and immunity, including cellular metabolism, biosynthesis, glycolysis/gluconeogenesis, and extra cellular matrix (ECM) receptor interactions. These results provide a genetic basis for exploring the better growth characteristics and disease resistance of DR-GGC than GC, valuable information on the genetic breeding for grass carp, and a further understanding of the genetic characteristics of gynogenetic individuals.

Development, identification, and utilization of wheat-tetraploid Thinopyrum elongatum 4EL translocation lines resistant to stripe rust.

The new stripe rust resistance gene Yr4EL in tetraploid Th. elongatum was identified and transferred into common wheat via 4EL translocation lines. Tetraploid Thinopyrum elongatum is a valuable genetic resource for improving the resistance of wheat to diseases such as stripe rust, powdery mildew, and Fusarium head blight. We previously reported that chromosome 4E of the 4E (4D) substitution line carries all-stage stripe rust resistance genes. To optimize the utility of these genes in wheat breeding programs, we developed translocation lines by inducing chromosomal structural changes through 60Co-γ irradiation and developing monosomic substitution lines. In total, 53 plants with different 4E chromosomal structural changes were identified. Three homozygous translocation lines (T4DS·4EL, T5AL·4EL, and T3BL·4EL) and an addition translocation line (T5DS·4EL) were confirmed by the genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), FISH-painting, and wheat 55K SNP array analyses. These four translocation lines, which contained chromosome arm 4EL, exhibited high stripe rust resistance. Thus, a resistance gene (tentatively named Yr4EL) was localized to the chromosome arm 4EL of tetraploid Th. elongatum. For the application of marker-assisted selection (MAS), 32 simple sequence repeat (SSR) markers were developed, showing specific amplification on the chromosome arm 4EL and co-segregation with Yr4EL. Furthermore, the 4DS·4EL line could be selected as a good pre-breeding line that better agronomic traits than other translocation lines. We transferred Yr4EL into three wheat cultivars SM482, CM42, and SM51, and their progenies were all resistant to stripe rust, which can be used in future wheat resistance breeding programs.

Molecular cytogenetic identification and nutritional composition evaluation of newly synthesized Triticum turgidum-Triticum boeoticum amphiploids (AABBAbAb).

Triticum boeoticum Boiss. (AbAb, 2n = 2x = 14) is a wheat-related species with the blue aleurone trait. In this study, 18 synthetic Triticum turgidum-Triticum boeoticum amphiploids were identified, which were derived from crosses between T. boeoticum and T. turgidum. Three probes (Oligo-pTa535, Oligo-pSc119.2, and Oligo-pTa713) for multicolor fluorescence in situ hybridization (mc-FISH) were combined with genomic in situ hybridization (GISH) to identify chromosomal composition. Seven nutritional indices (anthocyanins, protein, total essential amino acids TEAA, Fe, Zn, Mn and Cu) were measured, and the nutritional components of 18 synthetic amphiploids were comprehensively ranked by principal component analysis (PCA). The results showed that all three synthetic amphiploids used for cytological identification contained 42 chromosomes, including 14A, 14 B, and 14 Ab chromosomes. The average anthocyanin content was 82.830 μg/g to 207.606 μg/g in the whole meal of the 17 blue-grained lines (Syn-ABAb-1 to Syn-ABAb-17), which was obviously higher than that in the yellow-grained line Syn-ABAb-18 (6.346 μg/g). The crude protein content was between 154.406 and 180.517 g/kg, and the EAA content was 40.193-63.558 mg/g. The Fe, Zn, Mn and Cu levels in the 17 blue-grained lines were 60.55 to 97.41 mg/kg, 60.55-97.41 mg/kg, 35.11 to 65.20 mg/kg and 5.74 to 7.22 mg/kg, respectively, which were higher than those in the yellow-grained line. The contribution of the first three principal components reached 84%. The first principal component was mainly anthocyanins, Fe, Zn and Mn. The second principal component contained protein and amino acids, and the third component contained only Cu. The top 5 Triticum turgidum-Triticum boeoticum amphiploids were Syn-ABAb-11, Syn-ABAb-17, Syn-ABAb-5, Syn-ABAb-8 and Syn-ABAb-4. These amphidiploids exhibited the potential to serve as candidates for hybridization with common wheat, as indicated by comprehensive score rankings, toward enhancing the nutritional quality of wheat.

Development and characterization of a line with substitution of chromosome 4B of wheat Triticum aestivum L. on chromosome 4Hmar of wild barley Hordeum marinum ssp. gussoneanum (4x).

Introgressive hybridization is the main method of broadening the genetic diversity of bread wheat. Wild barley Hordeum marinum ssp. gussoneanum Hudson (2n = 4x = 28) has useful agronomical traits, such as high resistance to stress factors, that could be a potential source of new genes for bread wheat improvement. This study aimed to evaluate the possibility of introgression of H. marinum chromosomes into the genome of bread wheat using an incomplete amphiploid H. marinum ssp. gussoneanum (4x)-T. aestivum (Pyrotrix 28) (2n = 54) carrying the cytoplasm of wild barley. For this purpose, we crossed the line of bread wheat variety Pyrotrix 28 with an incomplete amphiploid, and then selected cytogenetically stable 42-chromosome plants with a high level of fertility in hybrid progeny. Genomic in situ hybridization (GISH) revealed a pair of H. marinum chromosomes in the genome of these plants. C- banding analysis confirmed that bread wheat chromosome 4B was replaced by wild barley chromosome 4Hmar. SSR markers Xgwm368 and Xgwm6 confirmed the absence of chromosome 4B, and EST markers BAWU808 and BAW112 identified chromosome 4Hmar in the genome of the isolated disomic wheat-barley substitution line. The study of this line showed that the substitution of chromosome 4B with chromosome 4Hmar resulted in a change of some morphological traits. It included intense anthocyanin coleoptile coloration, specific for H. marinum, as well as a lack of purple coloration of the ears in the leaf sheath, specific for Pyrotrix 28. Line 4Hmar(4B) showed increased performance for several traits, including plant height, number of spikes and tillers per plant, spikelet and grain number in the main spike, grain number per plant, but it had decreased values of 1000-grain weight compared to wheat. Cytogenetic stability and fertility of line 4Hmar(4B) indicated a high compensation ability of barley 4Hmar for wheat chromosome 4B and confirmed their homeology.

Cytological origination of the first found allotriploid tree peony, Paeonia × lemoinei ‘Oukan’ (AAB), reveled by molecular karyotype comparation

Polyploid breeding is one of the most important approaches to improve the traits of cultivated plants, but there has been no feasible method to get polyploid tree peony. In this study, we revealed a sexual triploidization approach from a diploid intersubsectional hybrid tree peony, Paeonia × lemoinei ‘High Noon’, to an allotriploid one, P. × lemoinei ‘Oukan’, by accurate karyotyping based on genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). The ancestor genomes from P. suffruticosa and P. delavayi, which were defined as A-genome and B-genome respectively, were clearly distinguished by GISH in ‘High Noon’ and ‘Oukan’. Nonhomologous chromosomes were successfully recognized by means of 5S and 45S rDNA FISH. Karyotype comparation based on GISH and FISH indicated that the B-genome and one of the two A-genomes in ‘Oukan’ (2n=3x=15, AAB) were highly consistent to the corresponding genomes in ‘High Noon’ (2n=2x=10, AB), implying the triploid origination via 2n gametes produced by first division restitution (FDR). In addition, it was demonstrated for the first time that the dual in series sites of 5S rDNA located on the short arm of chromosome 3 in both A and B-genomes. Besides the familiar strong 45S rDNA signals observed at terminal region of short arms, single weak 45S rDNA signal was newly detected at terminal region in the short arm of certain chromosome 1 in A-genome, and at interstitial-distal region in the long arm of chromosome 5 in both genomes. These findings provide a proof and theoretical support for tree peony polyploid breeding, and an accurate karyotyping method for genetic research of plants in Paeonia.

Development and molecular cytogenetic identification of a new wheat-rye 6RL ditelosomic addition and 1R (1B) substitution line with powdery mildew resistance

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious disease that critically threatens the yield and quality of wheat (Triticum aestivum L.). Using effective powdery mildew resistance genes is considered to be the optimal choice for controlling this disease. Against the background of high homogeneity among the modern commercial cultivars that are mainly derived from conventional interbreeding, the resistance genes from wheat relatives have especially prominent advantages. Octoploid triticale, produced by common wheat and rye (Secale cereale L.) through distant hybridization, is a new synthetic species and valuable gene donor for wheat improvement. In this study, we developed a wheat-rye line YT5 through the hybridization of octoploid triticale and two wheat lines. YT5 was confirmed to be a 6RL ditelosomic addition and 1R (1B) substitution line using genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), multicolor GISH (mc-GISH) and molecular marker analysis. Genetic analysis showed that the powdery mildew resistance in YT5 was derived from the rye chromosome arm 6RL. After inoculation with different Bgt isolates at the seedling stage, YT5 had compound reaction patterns with both obvious spores and hypersensitivity and gradually became highly resistant until the adult-plant stage, thus showing a resistance response significantly different from the reported Pm genes from rye chromosome 6RL. Meanwhile, YT5 showed promising agronomic performance and hence is expected to be an elite resistance donor for wheat improvement. To promote the transfer of the chromosome arm 6RL of YT5 in marker-assisted selection breeding, we selected and verified two 6RL-specific kompetitive allele-specific PCR markers that can be applied to efficiently detect this chromosome arm in different wheat backgrounds.

Revealing the abnormal meiosis and the variation of the functional female gametes of aneuploid lily (Lilium) using genomic in situ hybridization (GISH)

Revealing the abnormal meiosis and the variation of the functional female gametes of aneuploid lily (Lilium) using genomic in situ hybridization (GISH)

Chromosome Rearrangement in Elymus dahuricus Revealed by ND-FISH and Oligo-FISH Painting.

As a perennial herb in Triticeae, Elymus dahuricus is widely distributed in Qinghai-Tibetan Plateau and Central Asia. It has been used as high-quality fodders for improving degraded grassland. The genomic constitution of E. dahuricus (2n = 6x = 42) has been revealed as StStHHYY by cytological approaches. However, the universal karyotyping nomenclature system of E. dahuricus is not fully established by traditional fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH). In this study, the non-denaturing fluorescent in situ hybridization (ND-FISH) using 14 tandem-repeat oligos could effectively distinguish the entire E. dahuricus chromosomes pairs, while Oligo-FISH painting by bulked oligo pools based on wheat-barley collinear regions combined with GISH analysis, is able to precisely determine the linkage group and sub-genomes of the individual E. dahuricus chromosomes. We subsequently established the 42-chromosome karyotype of E. dahuricus with distinctive chromosomal FISH signals, and characterized a new type of intergenomic rearrangement between 2H and 5Y. Furthermore, the comparative chromosomal localization of the centromeric tandem repeats and immunostaining by anti-CENH3 between cultivated barley (Hordeum vulgare L.) and E. dahuricus suggests that centromere-associated sequences in H subgenomes were continuously changing during the process of polyploidization. The precise karyotyping system based on ND-FISH and Oligo-FISH painting methods will be efficient for describing chromosomal rearrangements and evolutionary networks for polyploid Elymus and their related species.

Autotetraploid Origin of Chinese Cherry Revealed by Chromosomal Karyotype and In Situ Hybridization of Seedling Progenies.

Polyploidy is considered a driving force in plant evolution and diversification. Chinese cherry [Cerasus pseudocerasus (Lindl.) G.Don], an economically important fruit crop native to China, has evolved at the tetraploid level, with a few pentaploid and hexaploid populations. However, its auto- or allo-polyploid origin remains unclear. To address this issue, we analyzed the ploidy levels and rDNA chromosomal distribution in self- and open-pollinated seedling progenies of tetraploid and hexaploid Chinese cherry. Genomic in situ hybridization (GISH) analysis was conducted to reveal the genomic relationships between Chinese cherry and diploid relatives from the genus Cerasus. Both self- and open-pollinated progenies of tetraploid Chinese cherry exhibited tetraploids, pentaploids, and hexaploids, with tetraploids being the most predominant. In the seedling progenies of hexaploid Chinese cherry, the majority of hexaploids and a few pentaploids were observed. A small number of aneuploids were also observed in the seedling progenies. Chromosome 1, characterized by distinct length characteristics, could be considered the representative chromosome of Chinese cherry. The basic Chinese cherry genome carried two 5S rDNA signals with similar intensity, and polyploids had the expected multiples of this copy number. The 5S rDNA sites were located at the per-centromeric regions of the short arm on chromosomes 4 and 5. Three 45S rDNA sites were detected on chr. 3, 4 and 7 in the haploid complement of Chinese cherry. Tetraploids exhibited 12 signals, while pentaploids and hexaploids showed fewer numbers than expected multiples. Based on the GISH signals, Chinese cherry demonstrated relatively close relationships with C. campanulata and C. conradinae, while being distantly related to another fruiting cherry, C. avium. In combination with the above results, our findings suggested that Chinese cherry likely originated from autotetraploidy.

Development and Identification of a Wheat-Rye Breeding Line for Harmonious Improvement Between Powdery Mildew Resistance and High Yield Potential.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease that seriously threatens wheat yield and quality. To control this disease, host resistance is the preferred measure. However, wheat breeding is a complex process with elusive exchange and recombination of the traits from their parents. Increased resistance often leads to a decline in other key traits, such as yield and quality. Developing breakthrough germplasms with harmonious powdery mildew resistance and other key breeding traits is attractive in wheat breeding. In this study, we developed an ideal wheat breeding line AL46 that pyramided its hexaploid triticale parent-derived desirable yield traits and its wheat parent-derived powdery mildew resistance gene Pm2. Sequential genomic in situ hybridization (GISH), multicolor GISH, multicolor fluorescence in situ hybridization, and molecular marker analyses revealed that AL46 was a wheat-rye T1RS·1BL translocation line. Genetic analysis combined with function marker detection and sequence alignment were used to confirm that AL46 carried the Pm2 gene. Then, we evaluated the powdery mildew resistance and comprehensive traits of AL46, and just as we designed, AL46 showed harmonious powdery mildew resistance with some key breeding traits. This study not only developed an ideal wheat germplasm resource but also provided a successful example for pyramiding breeding, which could be a promising direction for wheat improvement in the future.

Homoeologous Chromosome Pairing and Alien Introgression in Backcrossing Progenies Derived from Hybrids Solanum tuberosum (+) Mexican 2x (1 EBN) B-Genome Potato Species

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC1–BC5), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC1 progeny (AAAAB), whereas in the subsequent BC2–BC4 generations, the partial loss of alien chromosomes was observed and almost all the BC5 genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed.

Establishment of transformation system in wheat close relatives under assistance of TaWOX5

Wheat relative species are important for agriculture production, functional genomics study and wheat improvement as useful genetic resources. In this study, a regeneration related wheat gene TaWOX5 was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) region of the chromosomes in transgenic T. monococcum and hexaploid triticale plants; in the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB genome and RR genome. Furthermore, the transgene was proved to be almost stably inherited in the next generation by Mendel’s law. The findings in this study will promote genetic improvement for grain or forage production of the three plant species and for functional genomics study of cereal species including wheat.

The Control of the Crossover Localization in Allium.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F1 diploid (2n = 2x = 8C + 8F) and F1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

Relocation of Sr48 to Chromosome 2D Using an Alternative Mapping Population and Development of a Closely Linked Marker Using Diverse Molecular Technologies.

The Ug99-effective stem rust resistance gene Sr48 was mapped to chromosome 2A based on its repulsion linkage with Yr1 in an Arina/Forno recombinant inbred line (RIL) population. Attempts to identify markers closely linked to Sr48 using available genomic resources were futile. This study used an Arina/Cezanne F5:7 RIL population to identify markers closely linked with Sr48. Using the Arina/Cezanne DArTseq map, Sr48 was mapped on the short arm of chromosome 2D and it co-segregated with 12 markers. These DArTseq marker sequences were used for BlastN search to identify corresponding wheat chromosome survey sequence (CSS) contigs, and PCR-based markers were developed. Two simple sequence repeat (SSR) markers, sun590 and sun592, and two Kompetitive Allele-Specific PCR (KASP) markers were derived from the contig 2DS_5324961 that mapped distal to Sr48. Molecular cytogenetic analysis using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) identified a terminal translocation of chromosome 2A in chromosome 2DL of Forno. This translocation would have led to the formation of a quadrivalent involving chromosomes 2A and 2D in the Arina/Forno population, which would have exhibited pseudo-linkage between Sr48 and Yr1 in chromosome 2AL. Polymorphism of the closet marker sunKASP_239 among a set of 178 wheat genotypes suggested that this marker can be used for marker-assisted selection of Sr48.

Multispecies polyploidization, chromosome shuffling, and genome extraction in Zea/Tripsacum hybrids.

By hybridization and special sexual reproduction, we sequentially aggregated Zea mays, Zea perennis, and Tripsacum dactyloides in an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize and Z. perennis by natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenome stability, chromosome pairings and rearrangements, and their impacts on an organism's fitness were investigated by fertility phenotyping and molecular cytogenetic techniques genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n = 35-84) with varying proportions of subgenomic chromosomes, of which one individual (2n = 54, MMMPT) overcame self-incompatibility barriers and produced a self-fertile nascent near-allotetraploid by preferentially eliminating Tripsacum chromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, the mean chromosome number preferably maintained at the near-tetraploid level (2n = 40) with full integrity of 45S rDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 for maize, Z. perennis, and T. dactyloides chromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

Cytogenetic Characterization and Metabolomic Differences of Full-Sib Progenies of Saccharum spp.

Sugarcane smut is a worldwide fungal disease. Disease resistance breeding is the most economical and effective measure to prevent and control sugarcane smut. The cytogenetic characteristics and metabolomic differences of sugarcane F1s are closely related to disease resistance. Zhongzhe 1 and G160 sugarcane from the same parents (ROC25 and Yunzhe89-7) were used; the plants were grown in accordance with the barrel method. When the seedlings had 4-5 leaves, genomic in situ hybridization (GISH) was performed; digoxigenin (DIG)-labeled female parental (ROC25)DNA and biotin-labeled male parental (Yunzhe89-7) DNA were used as probes, and the karyotypes of two hybrids were analyzed. The new sugarcane smut-resistant variety (Zhongzhe 1) and the susceptible variety (G160) derived from the same parent were analyzed via gas chromatography-mass spectrometry technology (GC-MS) to compare the metabolomic differences between them. GISH analysis revealed that the chromosome ploidy number of Zhongzhe 1 sugarcane and G160 sugarcane were 114 and 110, respectively. However, the two contain different numbers of chromosomes from the female (ROC25) and male (Yunzhe89-7) parents. Moreover, 258 significantly changed metabolites were identified in smut-resistant Zhongzhe 1, as compared with the smut-susceptible G160 sugarcane: 56 flavonoids, 52 phenolic acids, 30 lipids, 26 organic acids, 26 amino acids and derivatives, 19 nucleotides and derivatives, 5 alkaloids, 9 terpenoids, and 35 others. Multivariate statistical analysis revealed a distinct difference in metabolic pathways between Zhongzhe 1 sugarcane and G160, and both of these varieties had unique functional metabolites. Differences in chromosome composition may constitute the genetic basis for the difference in resistance to smut disease between Zhongzhe 1 sugarcane and G160 sugarcane, and a high accumulation of flavonoids, lipids, terpenoids and tannins may constitute the basis of resistance to smut disease for the Zhongzhe 1 variety.