Gametocidal genes: from a discovery to the application in wheat breeding.

Some species of the genus Aegilops, a wild relative of wheat, carry chromosomes that after introducing to wheat exhibit preferential transmission to progeny. Their selective retention is a result of the abortion of gametes lacking them due to induced chromosomal aberrations. These chromosomes are termed Gametocidal (Gc) and, based on their effects, they are categorized into three types: mild, intense or severe, and very strong. Gc elements within the same homoeologous chromosome groups of Aegilops (II, III, or IV) demonstrate similar Gc action. This review explores the intriguing dynamics of Gc chromosomes and encompasses comprehensive insights into their source species, behavioral aspects, mode of action, interactions, suppressions, and practical applications of the Gc system in wheat breeding. By delving into these areas, this work aims to contribute to the development of novel plant genetic resources for wheat breeding. The insights provided herein shed light on the utilization of Gc chromosomes to produce chromosomal rearrangements in wheat and its wild relatives, thereby facilitating the generation of chromosome deletions, translocations, and telosomic lines. The Gc approach has significantly advanced various aspects of wheat genetics, including the introgression of novel genes and alleles, molecular markers and gene mapping, and the exploration of homoeologous relationships within Triticeae species. The mystery lies in why gametes possessing Gc genes maintain their normality while those lacking Gc genes suffer abnormalities, highlighting an unresolved research gap necessitating deeper investigation.

A chromosome arm from Thinopyrum intermedium × Thinopyrum ponticum hybrid confers increased tillering and yield potential in wheat.

Tiller number is a key component of wheat plant architecture having a direct impact on grain yield. Because of their viability, biotic resistance, and abiotic stress tolerance, wild relative species are a valuable gene source for increasing wheat genetic diversity, including yield potential. Agropyron glael, a perennial hybrid of Thinopyrum intermedium and Th. ponticum, was created in the 1930s. Recent genome analyses identified five evolutionarily distinct subgenomes (J, Jst, Jvs, Jr, and St), making A. glael an important gene source for transferring useful agronomical traits into wheat. During a bread wheat × A. glael crossing program, a genetically stable translocation line, WT153397, was developed. Sequential in situ hybridizations (McGISH) with J-, St-, and D-genomic DNA probes and pSc119.2, Afa family, pTa71, and (GAA)7 DNA repeats, as well as molecular markers specific for the wheat 6D chromosome, revealed the presence of a 6DS.6Jvs Robertsonian translocation in the genetic line. Field trials in low-input and high-input breeding nurseries over four growing seasons demonstrated the Agropyron chromosome arm's high compensating ability for the missing 6DL, as spike morphology and fertility of WT153397 did not differ significantly from those of wheat parents, Mv9kr1 and 'Mv Karizma.' Moreover, the introgressed 6Jvs chromosome arm significantly increased the number of productive tillers, resulting in a significantly higher grain yield potential compared to the parental wheat cultivars. The translocated chromosome could be highly purified by flow cytometric sorting due to the intense fluorescent labeling of (GAA)7 clusters on the Thinopyrum chromosome arm, providing an opportunity to use chromosome genomics to identify Agropyron gene variant(s) responsible for the tillering capacity. The translocation line WT153397 is an important genetic stock for functional genetic studies of tiller formation and useful breeding material for increasing wheat yield potential. The study also discusses the use of the translocation line in wheat breeding. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-024-01439-y.

Chromosome genomics facilitates the marker development and selection of wheat-Aegilops biuncialis addition, substitution and translocation lines.

The annual goatgrass, Aegilops biuncialis is a rich source of genes with considerable agronomic value. This genetic potential can be exploited for wheat improvement through interspecific hybridization to increase stress resistance, grain quality and adaptability. However, the low throughput of cytogenetic selection hampers the development of alien introgressions. Using the sequence of flow-sorted chromosomes of diploid progenitors, the present study enabled the development of chromosome-specific markers. In total, 482 PCR markers were validated on wheat (Mv9kr1) and Ae. biuncialis (MvGB642) crossing partners, and 126 on wheat-Aegilops additions. Thirty-two markers specific for U- or M-chromosomes were used in combination with GISH and FISH for the screening of 44 Mv9kr1 × Ae. biuncialis BC3F3 genotypes. The predominance of chromosomes 4M and 5M, as well as the presence of chromosomal aberrations, may indicate that these chromosomes have a gametocidal effect. A new wheat-Ae. biuncialis disomic 4U addition, 4M(4D) and 5M(5D) substitutions, as well as several introgression lines were selected. Spike morphology and fertility indicated that the Aegilops 4M or 5M compensated well for the loss of 4D and 5D, respectively. The new cytogenetic stocks represent valuable genetic resources for the introgression of key genes alleles into wheat.

Flow karyotyping of wheat-Aegilops additions facilitate dissecting the genomes of Ae. biuncialis and Ae. geniculata into individual chromosomes.

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

Transfer of the ph1b Deletion Chromosome 5B From Chinese Spring Wheat Into a Winter Wheat Line and Induction of Chromosome Rearrangements in Wheat-Aegilops biuncialis Hybrids.

Effective utilization of genetic diversity in wild relatives to improve wheat requires recombination between wheat and alien chromosomes. However, this is suppressed by the Pairing homoeologous gene, Ph1, on the long arm of wheat chromosome 5B. A deletion mutant of the Ph1 locus (ph1b) has been used widely to induce homoeologous recombination in wheat × alien hybrids. However, the original ph1b mutation, developed in Chinese Spring (CS) background has poor agronomic performance. Hence, alien introgression lines are first backcrossed with adapted wheat genotypes and after this step, alien chromosome segments are introduced into breeding lines. In this work, the ph1b mutation was transferred from two CSph1b mutants into winter wheat line Mv9kr1. Homozygous genotypes Mv9kr1 ph1b/ph1b exhibited improved plant and spike morphology compared to Chinese Spring. Flow cytometric chromosome analysis confirmed reduced DNA content of the mutant 5B chromosome in both wheat genotype relative to the wild type chromosome. The ph1b mutation in the Mv9kr1 genotype allowed wheat-alien chromosome pairing in meiosis of Mv9kr1ph1b_K × Aegilops biuncialis F1 hybrids, predominantly with the Mb-genome chromosomes of Aegilops relative to those of the Ub genome. High frequency of wheat-Aegilops chromosome interactions resulted in rearranged chromosomes identified in the new Mv9kr1ph1b × Ae. Biuncialis amphiploids, making these lines valuable sources for alien introgressions. The new Mv9kr1ph1b mutant genotype is a unique resource to support alien introgression breeding of hexaploid wheat.

Identification of New QTLs for Dietary Fiber Content in Aegilops biuncialis.

Grain dietary fiber content is an important health-promoting trait of bread wheat. A dominant dietary fiber component of wheat is the cell wall polysaccharide arabinoxylan and the goatgrass Aegilops biuncialis has high ß-glucan content, which makes it an attractive gene source to develop wheat lines with modified fiber composition. In order to support introgression breeding, this work examined genetic variability in grain ß-glucan, pentosan, and protein content in a collection of Ae. biuncialis. A large variation in grain protein and edible fiber content was revealed, reflecting the origin of Ae. biuncialis accessions from different eco-geographical habitats. Association analysis using DArTseq-derived SNPs identified 34 QTLs associated with ß-glucan, pentosan, water-extractable pentosan, and protein content. Mapping the markers to draft chromosome assemblies of diploid progenitors of Ae. biuncialis underlined the role of genes on chromosomes 1Mb, 4Mb, and 5Mb in the formation of grain ß-glucan content, while other QTLs on chromosome groups 3, 6, and 1 identified genes responsible for total- and water-extractable pentosan content. Functional annotation of the associated marker sequences identified fourteen genes, nine of which were identified in other monocots. The QTLs and genes identified in the present work are attractive targets for chromosome-mediated gene transfer to improve the health-promoting properties of wheat-derived foods.

Development of DNA Markers From Physically Mapped Loci in Aegilops comosa and Aegilops umbellulata Using Single-Gene FISH and Chromosome Sequences.

Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat (Triticum aestivum), the species of genus Aegilops are a particularly attractive source of new genes and alleles. However, during the evolution of the Aegilops and Triticum genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of Aegilops. The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect Aegilops chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence in situ hybridization (FISH) maps for M- and U-genome progenitors, Aegilops comosa and Aegilops umbellulata, respectively. Forty-three ortholog genes were located on 47 loci in Ae. comosa and on 52 loci in Ae. umbellulata using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While Ae. umbellulata chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of Aegilops markgrafii. To develop molecular markers with exact physical positions on chromosomes of Aegilops, the single-gene FISH data were validated in silico using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in Ae. comosa and 40 of the 52 map positions in Ae. umbellulata. Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat-Aegilops disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on Aegilops chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of Aegilops genes into wheat and their cloning.

Unlocking the Genetic Diversity and Population Structure of a Wild Gene Source of Wheat, Aegilops biuncialis Vis., and Its Relationship With the Heading Time.

Understanding the genetic diversity of Aegilops biuncialis, a valuable source of agronomical useful genes, may significantly facilitate the introgression breeding of wheat. The genetic diversity and population structure of 86 Ae. biuncialis genotypes were investigated by 32700 DArT markers with the simultaneous application of three statistical methods- neighbor-joining clustering, Principal Coordinate Analysis, and the Bayesian approach to classification. The collection of Ae. biuncialis accessions was divided into five groups that correlated well with their eco-geographic habitat: A (North Africa), B (mainly from Balkans), C (Kosovo and Near East), D (Turkey, Crimea, and Peloponnese), and E (Azerbaijan and the Levant region). The diversity between the Ae. biuncialis accessions for a phenological trait (heading time), which is of decisive importance in the adaptation of plants to different eco-geographical environments, was studied over 3 years. A comparison of the intraspecific variation in the heading time trait by means of analysis of variance and principal component analysis revealed four phenotypic categories showing association with the genetic structure and geographic distribution, except for minor differences. The detailed exploration of genetic and phenologic divergence provides an insight into the adaptation capacity of Ae. biuncialis, identifying promising genotypes that could be utilized for wheat improvement.

Uncovering homeologous relationships between tetraploid Agropyron cristatum and bread wheat genomes using COS markers.

KEY MESSAGE: Using COS markers, the study reveals homeologous relationships between tetraploid Agropyron cristatum and bread wheat to support alien introgression breeding of wheat. Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat that possesses many genes that are potentially useful in wheat improvement. The species comprises a complex of diploid, tetraploid and hexaploid forms. In this study, wheat-A. cristatum chromosome, telosome and translocation lines were used to characterize syntenic relationships between tetraploid A. cristatum and bread wheat. Prior to mapping COS markers, the cytogenetic stock lines were characterized for fertility and by FISH and GISH for karyotype stability. Out of 328 COS markers selected for the study, 279 consistently amplified products in tetraploid A. cristatum, and, out of these, 139 were polymorphic between tetraploid crested wheatgrass and wheat. Sixty-nine markers were found to be suitable for the detection of tetraploid A. cristatum chromosomes 1P-6P in wheat, ranging from 6 to 17 markers per chromosome. BLASTn of the source ESTs resulted in significant hits for 67 markers on the wheat pseudomolecules. Generally, COS markers of the same homeologous group were detected on similar arms in both Agropyron and wheat. However, some intragenomic duplications and chromosome rearrangements were detected in tetraploid A. cristatum. These results provide new insights into the structure and evolution of the tetraploid A. cristatum genome and will facilitate the exploitation of the wild species for introgression breeding of bread wheat.

Out-of-position telomeres in meiotic leptotene appear responsible for chiasmate pairing in an inversion heterozygote in wheat (Triticum aestivum L.).

Chromosome pairing in meiosis usually starts in the vicinity of the telomere attachment to the nuclear membrane and congregation of telomeres in the leptotene bouquet is believed responsible for bringing homologue pairs together. In a heterozygote for an inversion of a rye (Secale cereale L.) chromosome arm in wheat, a distal segment of the normal homologue is capable of chiasmate pairing with its counterpart in the inverted arm, located near the centromere. Using 3D imaging confocal microscopy, we observed that some telomeres failed to be incorporated into the bouquet and occupied various positions throughout the entire volume of the nucleus, including the centromere pole. Rye telomeres appeared ca. 21 times more likely to fail to be included in the telomere bouquet than wheat telomeres. The frequency of the out-of-bouquet rye telomere position in leptotene was virtually identical to the frequency of telomeres deviating from Rabl's orientation in the nuclei of somatic cells, and was similar to the frequency of synapsis of the normal and inverted chromosome arms, but lower than the MI pairing frequency of segments of these two arms normally positioned across the volume of the nucleus. Out-of-position placement of the rye telomeres may be responsible for reduced MI pairing of rye chromosomes in hybrids with wheat and their disproportionate contribution to aneuploidy, but appears responsible for initiating chiasmate pairing of distantly positioned segments of homology in an inversion heterozygote.

Identification of COS markers specific for Thinopyrum elongatum chromosomes preliminary revealed high level of macrosyntenic relationship between the wheat and Th. elongatum genomes.

Thinopyrum elongatum (Host) D.R. Dewey has served as an important gene source for wheat breeding improvement for many years. The exact characterization of its chromosomes is important for the detailed analysis of prebreeding materials produced with this species. The major aim of this study was to identify and characterize new molecular markers to be used for the rapid analysis of E genome chromatin in wheat background. Sixty of the 169 conserved orthologous set (COS) markers tested on diverse wheat-Th. elongatum disomic/ditelosomic addition lines were assigned to various Th. elongatum chromosomes and will be used for marker-assisted selection. The macrosyntenic relationship between the wheat and Th. elongatum genomes was investigated using EST sequences. Several rearrangements were revealed in homoeologous chromosome groups 2, 5, 6 and 7, while chromosomes 1 and 4 were conserved. Molecular cytogenetic and marker analysis showed the presence of rearranged chromosome involved in 6ES and 2EL arms in the 6E disomic addition line. The selected chromosome arm-specific COS markers will make it possible to identify gene introgressions in breeding programmes and will also be useful in the development of new chromosome-specific markers, evolutionary analysis and gene mapping.

Molecular cytogenetic (FISH) and genome analysis of diploid wheatgrasses and their phylogenetic relationship.

This paper reports detailed FISH-based karyotypes for three diploid wheatgrass species Agropyron cristatum (L.) Beauv., Thinopyrum bessarabicum (Savul.&Rayss) A. Löve, Pseudoroegneria spicata (Pursh) A. Löve, the supposed ancestors of hexaploid Thinopyrum intermedium (Host) Barkworth & D.R.Dewey, compiled using DNA repeats and comparative genome analysis based on COS markers. Fluorescence in situ hybridization (FISH) with repetitive DNA probes proved suitable for the identification of individual chromosomes in the diploid JJ, StSt and PP genomes. Of the seven microsatellite markers tested only the (GAA)n trinucleotide sequence was appropriate for use as a single chromosome marker for the P. spicata AS chromosome. Based on COS marker analysis, the phylogenetic relationship between diploid wheatgrasses and the hexaploid bread wheat genomes was established. These findings confirmed that the J and E genomes are in neighbouring clusters.