Genomic In Situ Hybridization Analysis Research Articles

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.

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.

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.

Genomic relationships of the polyploid rhizoma peanut (Arachis glabrata Benth.) inferred by genomic in situ hybridization (GISH)

The rhizoma peanut (Arachis glabrata Benth., section Rhizomatosae) is a tetraploid perennial legume. Although several A. glabrata cultivars have been developed as forage and ornamental turf, the origin and genomic constitution of this species are still unknown. In this study, we evaluated the affinity between the genomes of A. glabrata and the probable diploid donors of the sections Rhizomatosae, Arachis, Erectoides and Procumbentes by genomic in situ hybridization (GISH). Single GISH analyses detected that species of the sections Erectoides (E2 subgenome) and Procumbentes (E3 subgenome) were the diploid species with the highest degree of genomic affinity with A. glabrata. Based on single GISH experiments and DNA sequence similarity, three species -A. duranensis, A. paraguariensis subsp. capibarensis, and A. rigonii-, which showed the most uniform and brightest hybridization patterns and lowest genetic distance, were selected as probes for double GISH experiments. Double GISH experiments showed that A. glabrata is constituted by four identical or very similar chromosome complements. In these assays, A. paraguariensis subsp. capibarensis showed the highest brightness onto A. glabrata chromosomes. Thus, our results support the autopolyploid origin of A. glabrata and show that the species with E2 subgenome are the most probable ancestors of this polyploid legume forage.

GISH Analysis of the Introgression of the B Subgenome Genetic Material of Wild Allotetraploid Species Solanum stoloniferum into Backcrossing Progenies with Potato

Wild relatives of cultivated potato are used in breeding to increase the genetic diversity of Solanum tuberosum (AAAA genome) varieties. Wild Mexican allotetraploid species Solanum stoloniferum (AABB genome) was used in breeding for extreme resistance to viruses and late blight. In this study, genomic in situ hybridization (GISH) was used for visualization of introgression of genetic material of the B subgenome of S. stoloniferum into the genome of backcross hybrids. The fertile hexaploid hybrid had 48 chromosomes of the A genome and 24 chromosomes of the B subgenome. Plants of the BC1 generation were pentaploid having the AAAAB genome constitution and three selected BC2 hybrids were aneuploid, containing one to six chromosomes of the B subgenome and 48 chromosomes of the A genome. The B subgenome of S. stoloniferum was inherited in the backcross generations as single chromosomes and in rare cases as recombinant chromosomes. GISH showed that chromosome pairing in the backcross hybrids was predominantly intragenomic. Most chromosomes of the B subgenome remained as univalents in backcross hybrids. Rare homeologous A/B chromosome pairing was detected in all analyzed hybrids. The obtained data indicate that the B subgenome of S. stoloniferum was able to recombine with the A genome.

Identification of Parental Genome Construction and Inherited Morphological Characteristics in Triploid and AneuploidIntergeneric Hybrids from a Diploid−Diploid Cross between Citrus and Fortunella

We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst; H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E; Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidization processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even number) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.

Identification of Alien Chromosome/Chromatin Introgressions in Triticale × Wheat Derived Stable Lines Through Molecular Cytogenetic Analysis

Molecular cytogenetic study was conducted for identifying rye chromosome/chromatin translocation, substitution and addition in 43 triticale x wheat derived stable lines (BC1F7 and F7 generations) six different crosses using FISH (Fluorescence in situ hybridization) and GISH (Genomic in situ hybridization) analyses with different probes. Rye chromosomes/chromatin was identified in 17 BC1F7 and 12 F7 lines by these two analyses. The GISH and FISH techniques worked satisfactorily for identifying the presence of rye chromatin at metaphase. All the lines were associated with alien chromosome translocation, addition or substitution except a few lines. Most of the recombinants present in TW 1, TW 2 and TW 6 crosses were observed to be associated with important alien chromatin translocation like 1BL/1RS, substitutions 1R (1D), 5R (5D) and combination of both i.e., 1BL/1RS + 5R (5D) and in some cases presence of more than 4 rye chromosomes. The crosses TW 3 and TW 5 possessed 8 to 14 rye chromosomes in their chromosome constitution. So, their morphological expression was similar to triticale. These reconstituted lines will be of immense application for the future wheat breeding programmes.

Chromosomal composition analysis and molecular marker development for the novel Ug99-resistant wheat-Thinopyrum ponticum translocation line WTT34.

A novel Ug99-resistant wheat-Thinopyrum ponticum translocation line was produced, its chromosomal composition was analyzed and specific markers were developed. Stem rust caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn (Pgt) has seriously threatened global wheat production since Ug99 race TTKSK was first detected in Uganda in 1998. Thinopyrum ponticum is near immune to Ug99 races and may be useful for enhancing wheat disease resistance. Therefore, developing new wheat-Th. ponticum translocation lines that are resistant to Ug99 is crucial. In this study, a novel wheat-Th. ponticum translocation line, WTT34, was produced. Seedling and field evaluation revealed that WTT34 is resistant to Ug99 race PTKST. The resistance was derived from the alien parent Th. ponticum. Screening WTT34 with markers linked to Sr24, Sr25, Sr26, Sr43, and SrB resulted in the amplification of different DNA fragments from Th. ponticum, implying WTT34 carries at least one novel stem rust resistance gene. Genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), and multi-color GISH (mc-GISH) analyses indicated that WTT34 carries a T5DS·5DL-Th translocation, which was consistent with wheat660K single-nucleotide polymorphism (SNP) array results. The SNP array also uncovered a deletion event in the terminal region of chromosome 1D. Additionally, the homeology between alien segments and the wheat chromosomes 2A and 5D was confirmed. Furthermore, 51 PCR-based markers derived from the alien segments of WTT34 were developed based on specific-locus amplified fragment sequencing (SLAF-seq). These markers may enable wheat breeders to rapidly trace Th. ponticum chromosomal segments carrying Ug99 resistance gene(s).

Characterization of chromosome constitution in three wheat - Thinopyrum intermedium amphiploids revealed frequent rearrangement of alien and wheat chromosomes

BackgroundThinopyrum intermedium (2n = 6x = 42) is an important wild perennial Triticeae species exhibiting many potentially favorable traits for wheat improvement. Wheat-Th. intermedium partial amphiploids serve as a bridge to transfer desirable genes from Th. intermedium into common wheat.ResultsThree octoploid Trititrigia accessions (TE261–1, TE266–1, and TE346–1) with good resistances to stripe rust, powdery mildew and aphids were selected from hybrid progenies between Th. intermedium and the common wheat variety ‘Yannong 15’ (YN15). Genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and multicolor GISH (McGISH) analyses demonstrated that the three octoploid Trititrigia possess 42 wheat chromosomes and 14 Th. intermedium chromosomes. The 14 alien (Th. intermedium) chromosomes belong to a mixed genome consisting of J-, JS- and St-genome chromosomes rather than a single J, JS or St genome. Different types of chromosomal structural variation were also detected in the 1A, 6A, 6B, 2D and 7D chromosomes via FISH, McGISH and molecular marker analysis. The identity of the alien chromosomes and the variationes in the wheat chromosomes in the three Trititrigia octoploids were also different.ConclusionsThe wheat-Th. intermedium partial amphiploids possess 14 alien chromosomes which belong to a mixed genome consisting of J-, JS- and St- chromosomes, and 42 wheat chromosomes with different structural variations. These accessions could be used as genetic resources in wheat breeding for the transfer of disease and pest resistance genes from Th. intermedium to common wheat.

Visualizing Meiotic Chromosome Pairing and Segregation in Interspecific Hybrids of Rice by Genomic in situ Hybridization

Meiotic disturbances in F1 hybrids and their progenies are still major problems in wide hybridization. To investigate the genome affinity reflected in chromosome pairing and segregation, we studied chromosome behaviors during meiosis in two interspecific F1 hybrids [O. minuta×O. australiensis (Om×Oa, BCE genome) and Oa×O. ridleyi (Or, EHJ genome)] by using both traditional staining methods and genomic in situ hybridization (GISH). GISH analysis has been successfully performed on mitotic chromosomes to distinguish different Oryza genomes, but relatively fewer systematic analyses of meiotic chromosomes of interspecific hybrids have been reported. In the hybrids, highly irregular chromosome behaviors through meiosis resulted in producing microspores with unbalanced genome. At diakinesis of these two hybrids, most chromosomes present as univalent, with low frequency as bivalents and occasionally as trivalents. In a pollen mother cell, 2 to 8 bivalents and 0 to 4 trivalents were observed in the hybrid Oa×Or, and 1 to 8 bivalents and 0 to 5 trivalents were observed in the hybrid Om×Oa. GISH results indicated that 51.52% bivalents in Oa×Or and 79.65% bivalents in Om×Oa involved allosyndetic association, which indicates that recombination and introgression should be possible if viable backcrosses can be recovered even from triploid hybrids. In this study, we revealed that the meiotic disturbance due to low affinities between parental genomes is the major reason for the sterility of these two triploid interspecific hybrids. The two hybrids showing vigor in reproductive growth are potential genetic resources in future breeding programs. A better understanding of genomic affinities between these distant Oryza species can facilitate planning an effective breeding program by using wide hybridization, and efficient and routine GISH analysis is helpful to monitor alien introgression in the process.

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.

Dissection and physical mapping of wheat chromosome 7B by inducing meiotic recombination with its homoeologues in Aegilops speltoides and Thinopyrum elongatum.

We constructed a homoeologous recombination-based bin map of wheat chromosome 7B, providing a unique physical framework for further study of chromosome 7B and its homoeologues in wheat and its relatives. Homoeologous recombination leads to the dissection and diversification of the wheat genome. Advances in genome sequencing and genotyping have dramatically improved the efficacy and throughput of homoeologous recombination-based genome studies and alien introgression in wheat and its relatives. In this study, we aimed to physically dissect and map wheat chromosome 7B by inducing meiotic recombination of chromosome 7B with its homoeologues 7E in Thinopyrum elongatum and 7S in Aegilops speltoides. The special genotypes, which were double monosomic for chromosomes 7B' + 7E' or 7B' + 7S' and homozygous for the ph1b mutant, were produced to enhance 7B - 7E and 7B - 7S recombination. Chromosome-specific DNA markers were developed and used to pre-screen the large recombination populations for 7B - 7E and 7B - 7S recombinants. The DNA marker-mediated preselections were verified by fluorescent genomic in situ hybridization (GISH). In total, 29 7B - 7E and 61 7B - 7S recombinants and multiple chromosome aberrations were recovered and delineated by GISH and the wheat 90K SNP assay. Integrated GISH and SNP analysis of the recombinants physically mapped the recombination breakpoints and partitioned wheat chromosome 7B into 44 bins with 523 SNPs assigned within. A composite bin map was constructed for chromosome 7B, showing the bin size and physical distribution of SNPs. This provides a unique physical framework for further study of chromosome 7B and its homoeologues. In addition, the 7B - 7E and 7B - 7S recombinants extend the genetic variability of wheat chromosome 7B and represent useful germplasm for wheat breeding. Thereby, this genomics-enabled chromosome engineering approach facilitates wheat genome study and enriches the gene pool of wheat improvement.

Identification of an Elite Wheat-Rye T1RS·1BL Translocation Line Conferring High Resistance to Powdery Mildew and Stripe Rust.

Wheat-rye T1RS·1BL translocations have been widely used worldwide in wheat production for multiple disease resistance and superior yield traits. However, many T1RS·1BL translocations have successively lost their resistance to pathogens due to the coevolution of pathogen virulence with host resistance. Because of the extensive variation in rye (Secale cereale L.) as a naturally cross-pollinating relative of wheat, it still has promise to widen the variation of 1RS and to fully realize its application value in wheat improvement. In the present study, the wheat-rye breeding line R2207 was characterized by comprehensive analyses using genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization with multiple probes, multicolor GISH, and molecular marker analysis, and then was proven to be a cytogenetically stable wheat-rye T1RS·1BL translocation line. Based on the disease responses to different isolates of powdery mildew and genetic analysis, R2207 appears to possess a novel variation for resistance, which was confirmed to be located on the rye chromosome arm 1RS. Line R2207 also exhibited high levels of resistance to stripe rust at both seedling and adult stages, as well as enhanced agronomic performance, so it has been transferred into a large number of commercial cultivars using an efficient 1RS-specific kompetitive allele specific PCR marker for marker-assisted selection.

Characterization and Evaluation of Resistance to Powdery Mildew of Wheat-Aegilops geniculata Roth 7Mg (7A) Alien Disomic Substitution Line W16998.

Aegilops geniculata Roth has been used as a donor of disease-resistance genes, to enrich the gene pool for wheat (Triticum aestivum) improvement through distant hybridization. In this study, the wheat–Ae. geniculata alien disomic substitution line W16998 was obtained from the BC1F8 progeny of a cross between the common wheat ‘Chinese Spring’ (CS) and Ae. geniculata Roth (serial number: SY159//CS). This line was identified using cytogenetic techniques, analysis of genomic in situ hybridization (GISH), functional molecular markers (Expressed sequence tag-sequence-tagged site (EST–STS) and PCR-based landmark unique gene (PLUG), fluorescence in situ hybridization (FISH), sequential fluorescence in situ hybridization–genomic in situ hybridization (sequential FISH–GISH), and assessment of agronomic traits and powdery mildew resistance. During the anaphase of meiosis, these were evenly distributed on both sides of the equatorial plate, and they exhibited high cytological stability during the meiotic metaphase and anaphase. GISH analysis indicated that W16998 contained a pair of Ae. geniculata alien chromosomes and 40 common wheat chromosomes. One EST–STS marker and seven PLUG marker results showed that the introduced chromosomes of Ae. geniculata belonged to homoeologous group 7. Nullisomic–tetrasomic analyses suggested that the common wheat chromosome, 7A, was absent in W16998. FISH and sequential FISH–GISH analyses confirmed that the introduced Ae. geniculata chromosome was 7Mg. Therefore, W16998 was a wheat–Ae. geniculata 7Mg (7A) alien disomic substitution line. Inoculation of isolate E09 (Blumeria graminis f. sp. tritici) in the seedling stage showed that SY159 and W16998 were resistant to powdery mildew, indeed nearly immune, whereas CS was highly susceptible. Compared to CS, W16998 exhibited increased grain weight and more spikelets, and a greater number of superior agronomic traits. Consequently, W16998 was potentially useful. Germplasms transfer new disease-resistance genes and prominent agronomic traits into common wheat, giving the latter some fine properties for breeding.

A 1Ns Disomic Addition from Psathyrostachys Huashanica Keng Confers Resistance to Powdery Mildew in Wheat

Powdery mildew is a fungal disease that threatens wheat production throughout the world. Breeding resistant cultivars is an effective way to reduce harm caused by powdery mildew. In this study, 35 wheat-Psathyrostachys huashanica-derived lines were developed by crossing common wheat and P. huashanica Keng (2n = 2x = 14, NsNs) using embryo culture. Resistance to powdery mildew in the derived lines was identified at the seedling and adult stages. Line H5-5-4-2 was selected with immunity to powdery mildew at both growth stages. The chromosome structure of this line was characterized by cytology, genomic in situ hybridization (GISH), and expressed sequence tag-sequence-tagged site (EST-STS) analysis. The chromosome configuration was 2n = 44 = 22II. Two P. huashanica chromosomes with strong hybridization signals were detected by GISH analysis. Among 83 EST-STS markers that covered all seven homologous groups in wheat, three pairs of STS markers, BE497584, BF202643, and BG262410, located in wheat homologous group 1 amplified clear specific bands related to P. huashanica. The results indicated that resistant line H5-5-4-2 was a wheat-P. huashanica 1Ns disomic addition line.

GISH-based comparative genomic analysis in Urochloa P. Beauv.

The genus Urochloa P. Beauv. [syn. Brachiaria (Trin.) Griseb.] comprises species of great economic relevance as forages. The genomic constitution for the allotetraploid species Urochloa brizantha (cv. Marandu) and Urochloa decumbens (cv. Basilisk) and the diploid Urochloa ruziziensis was previously proposed as BBB1B1, B1B1B2B2 and B2B2, respectively. Evidence indicates U. ruziziensis as the ancestral donor of genome B2 in U. decumbens allotetraploidy, but the origin of the genomes B and B1 is still unknown. There are diploid genotypes of U. brizantha and U. decumbens that may be potential ancestors of the tetraploids. The aim of this study was to determine the genomic constitution and relationships between genotypes of U. brizantha (2x and 4x), U. decumbens (2x and 4x) and U. ruziziensis (2x) via genomic in situ hybridization (GISH). Additionally, chromosome number and genome size were verified for the diploid genotypes. The diploids U. brizantha and U. decumbens presented 2n = 2x = 18 chromosomes and DNA content of 1.79 and 1.44pg, respectively. The GISH analysis revealed high homology between the diploids U. brizantha and U. decumbens, which suggests relatively short divergence time. The GISH using genomic probes from the diploid accessions on the tetraploid accessions' chromosomes presented similar patterns, highlighting the genome B1 present in both of the tetraploids. Based on GISH results, the genomic constitution was proposed for the diploid genotypes of U. brizantha (B1B1) and U. decumbens (B1'B1') and both were pointed as donors of genome B1 (or B1'), present in the allotetraploid genotypes.

Identification of a conserved ph1b-mediated 5DS–5BS crossing over site in soft-kernel durum wheat (Triticum turgidum subsp. durum) lines

Genetic recombination is the major mechanism and basis of genetic diversity and crop improvement. Recombination typically occurs between homologous chromosomes. In wheat, however, it can also occur between homoeologous chromosomes if a functional Ph1 (Pairing homoeologous-1) locus is absent (ph1b). Recently, the genes for soft kernel trait on the distal 5DS chromosome of common wheat were transferred into chromosome 5BS of durum wheat through ph1b-mediated recombination. Several independent 5DS–5BS recombination lines carrying the 5DS translocation were obtained. However, the specific size of the translocation and region where the translocation breakpoint occurred was not determined for any of the lines. In the present study, four independent 5DS–5BS recombination lines were investigated and the translocation breakpoints were fine-mapped and sequenced. All the analyzed lines lost a 5BS fragment of ~ 20,742,425 bp and gained a 5DS fragment of ~ 28,163,252 bp. The recombination breakpoint in each line was located within a predicted gene in a conserved 39 bp region, suggesting the presence of a recombination hotspot. The information obtained was then used to develop a KASP marker diagnostic for the 5DS–5BS translocation. Finally, a subset of the soft durum wheat lines exhibiting varying degrees of kernel hardness was analyzed through genomic in situ hybridization (GISH) and scanning electron microscopy (SEM). From both the GISH and SEM analyses no major differences were detected among the lines, indicating that factors other than ph1b-mediated recombination or endosperm morphology were responsible for the variation in kernel hardness.

Characterization of a new hexaploid triticale 6D(6A) substitution line with increased grain weight and decreased spikelet number

Hexaploid triticale (×Triticosecale, AABBRR) is an important forage crop and a promising energy plant. Transferring D-genome chromosomes or segments from common wheat (Triticum aestivum) into hexaploid triticale is attractive in improving its economically important traits. Here, a hexaploid triticale 6D(6A) substitution line Lin 456 derived from the cross between the octoploid triticale line H400 and the hexaploid wheat Lin 56 was identified and analyzed by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and molecular markers. The GISH analysis showed that Lin 456 is a hexaploid triticale with 14 rye (Secale cereale) chromosomes and 28 wheat chromosomes, whereas non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular marker analysis revealed that it is a 6D(6A) substitution line. In contrast to previous studies, the signal of Oligo-pSc119.2 was observed at the distal end of 6DL in Lin 456. The wheat chromosome 6D was associated with increased grain weight and decreased spikelet number using the genotypic data combined with the phenotypes of the F2 population in the three environments. The thousand-grain weight and grain width in the substitution individuals were significantly higher than those in the non-substitution individuals in the F2 population across the three environments. We propose that the hexaploid triticale 6D(6A) substitution line Lin 456 can be a valuable and promising donor stock for genetic improvement during triticale breeding.

Development and identification of a dwarf wheat-Leymus mollis double substitution line with resistance to yellow rust and Fusarium head blight

Leymus mollis (Trin.) Pilger (2n = 4x = 28, NsNsXmXm,), a wild relative of common wheat, possesses many potentially valuable traits for genetic improvement of wheat, including strong, short stems, long spikes with numerous spikelets, tolerance to drought and cold stresses, and resistance to many fungal and bacterial diseases. In the present study, a wheat–L. mollis double substitution line DM96 was selected from a F6 progeny of a cross between M842-16 (an octoploid Tritileymus line) and D4286 (a Triticum durum line) using genomic in situ hybridization (GISH), simple sequence repeat (SSR) markers, and expressed sequence tagged sequence site (EST-STS) markers. Chromosome analysis at mitosis and meiosis showed that DM96 had a chromosome constitution of 2n = 42 = 21II. GISH analysis indicated that DM96 carried 38 chromosomes from wheat and two homologous pairs of Ns chromosomes from L. mollis. Fluorescent in situ hybridization (FISH) showed that chromosomes 2Ns and 3Ns from L. mollis had replaced wheat chromosomes 2D and 3D in DM96, which was confirmed by SSR and STS markers. The newly developed substitution line DM96 has shorter height, longer spikes and more kernels than its parents and showed high resistance to stripe rust and Fusarium head blight (FHB). Thus, this line is a new bridge material for the production of useful translocation lines for wheat genetic research and genetic improvement of wheat yield and disease resistance in breeding programs.

Origin and parental genome characterization of the allotetraploid Stylosanthes scabra Vogel (Papilionoideae, Leguminosae), an important legume pasture crop.

The genus Stylosanthes includes nitrogen-fixing and drought-tolerant species of considerable economic importance for perennial pasture, green manure and land recovery. Stylosanthes scabra is adapted to variable soil conditions, being cultivated to improve pastures and soils worldwide. Previous studies have proposed S. scabra as an allotetraploid species (2n = 40) with a putative diploid A genome progenitor S. hamata or S. seabrana (2n = 20) and the B genome progenitor S. viscosa (2n = 20). We aimed to provide conclusive evidence for the origin of S. scabra. We performed fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) experiments and Illumina paired-end sequencing of S. scabra, S. hamata and S. viscosa genomic DNA, to assemble and compare complete ribosomal DNA (rDNA) units and chloroplast genomes. Plastome- and genome-wide single nucleotide variation detection was also performed. GISH and phylogenetic analyses of plastid DNA and rDNA sequences support that S. scabra is an allotetraploid formed from 0.63 to 0.52 million years ago (Mya), from progenitors with a similar genome structure to the maternal donor S. hamata and the paternal donor S. viscosa. FISH revealed a non-additive number of 35S rDNA sites in S. scabra compared with its progenitors, indicating the loss of one locus from A genome origin. In S. scabra, most 5S rDNA units were similar to S. viscosa, while one 5S rDNA site of reduced size most probably came from an A genome species as revealed by GISH and in silico analysis. Our approach combined whole-plastome and rDNA assembly with additional cytogenetic analysis to shed light successfully on the allotetraploid origin of S. scabra. We propose a Middle Pleistocene origin for S. scabra involving species with maternal A and paternal B genomes. Our data also suggest that variation found in rDNA units in S. scabra and its progenitors reveals differences that can be explained by homogenization, deletion and amplification processes that have occurred since its origin.