Chromosome-specific Markers Research Articles

First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping

BackgroundThe genus Pulmonaria (Boraginaceae) represents a taxonomically complex group of species in which morphological similarity contrasts with striking karyological variation. The presence of different numbers of chromosomes in the diploid state suggests multiple hybridization/polyploidization events followed by chromosome rearrangements (dysploidy). Unfortunately, the phylogenetic relationships and evolution of the genome, have not yet been elucidated. Our study focused on the P. officinalis group, the most widespread species complex, which includes two morphologically similar species that differ in chromosome number, i.e. P. obscura (2n = 14) and P. officinalis (2n = 16). Ornamental cultivars, morphologically similar to P. officinalis (garden escapes), whose origin is unclear, were also studied. Here, we present a pilot study on genome size and repeatome dynamics of these closely related species in order to gain new information on their genome and chromosome structure.ResultsFlow cytometry confirmed a significant difference in genome size between P. obscura and P. officinalis, corresponding to the number of chromosomes. Genome-wide repeatome analysis performed on genome skimming data showed that retrotransposons were the most abundant repeat type, with a higher proportion of Ty3/Gypsy elements, mainly represented by the Tekay lineage. Comparative analysis revealed no species-specific retrotransposons or striking differences in their copy number between the species. A new set of chromosome-specific cytogenetic markers, represented by satellite DNAs, showed that the chromosome structure in P. officinalis was more variable compared to that of P. obscura. Comparative karyotyping supported the hybrid origin of putative hybrids with 2n = 15 collected from a mixed population of both species and outlined the origin of ornamental garden escapes, presumably derived from the P. officinalis complex.ConclusionsLarge-scale genome size analysis and repeatome characterization of the two morphologically similar species of the P. officinalis group improved our knowledge of the genome dynamics and differences in the karyotype structure. A new set of chromosome-specific cytogenetic landmarks was identified and used to reveal the origin of putative hybrids and ornamental cultivars morphologically similar to P. officinalis.

Development and identification of a novel wheat-Thinopyrum ponticum disomic substitution line DS5Ag(5D) with new genes conferring resistance to powdery mildew and leaf rust

BackgroundPowdery mildew (caused by Blumeria graminis f. sp. tritici (Bgt)) and leaf rust (caused by Puccinia triticina (Pt)) are prevalent diseases in wheat (Triticum aestivum L.) production. Thinopyrum ponticum (2n = 10x = 70, EeEeEbEbExExStStStSt) contains genes that confer high levels of resistance to these diseases.ResultsAn elite wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was developed in the Bainong Aikang 58 (AK58) background. The line was assessed using genomic in situ hybridization (GISH), oligo-nucleotide probe multiplex (ONPM) fluorescence in situ hybridization (FISH), and molecular markers. Twenty eight chromosome-specific molecular markers were identified for the alien chromosome, and 22 of them were co-dominant. Additionally, SNP markers from the wheat 660 K SNP chip were utilized to confirm chromosome identification and they provide molecular tools for tagging the chromosome in concern. The substitution line demonstrated high levels of resistance to powdery mildew throughout its growth period and to leaf rust at the adult stage. Based on the resistance evaluation of five F5 populations between the substitution lines and wheat genotypes with different levels of sensitivity to the two diseases. Results showed that the resistance genes located on 5Ag confered stable resistance against both diseases across different backgrounds. Resistance spectrum analysis combined with diagnostic marker detection of known resistance genes of Th. ponticum revealed that 5Ag contained two novel genes, Pm5Ag and Lr5Ag, which conferred resistance to powdery mildew and leaf rust, respectively.ConclusionsIn this study, a novel wheat-Th. ponticum disomic substitution line DS5Ag(5D) was successfully developed. The Th. ponticum chromosome 5Ag contain new resistance genes for powdery mildew and leaf rust. Chromosomic—specific molecular markers were generated and they can be used to track the 5Ag chromosome fragments. Consequently, this study provides new elite germplasm resources and molecular markers to facilitate the breeding of wheat varieties that is resistant to powdery mildew and leaf rust.

Development of chromosome-specific potential intron polymorphism markers in watermelon

Development of chromosome-specific potential intron polymorphism markers in watermelon

Molecular-genetic and cytogenetic analyses of cotton chromosome introgression from Gossypium barbadense L. into the genome of G. hirsutum L. in BC2F1 hybrids.

Substitution lines of the cotton Gossypium hirsutum L. involving chromosomes of the tetraploid species G. barbadense L., G. tomentosum Nutt. ex Seem., and G. mustelinum Miers ex Watt. are a valuable source for breeding, increasing the genetic diversity of G. hirsutum. The substitution of certain G. hirsutum L. chromosomes with G. barbadense chromosomes affect fibre elongation, fibre yield, fibre strength, and micronaire. To increase the efficiency of creating lines, it is necessary to study the nature of the introgression of alien chromosomes into the G. hirsutum L. genome. As a result of molecular genetic analysis of BC2F1 hybrids obtained from crossing monosomic lines of the cotton G. hirsutum from the cytogenetic collection of Uzbekistan with monosomic backcross hybrids BC1F1 G. hirsutum × G. barbadense on the same chromosomes, genetic differences between the hybrids in the profile of chromosome-specific microsatellite SSR markers were found. The predominant introgression of chromosomes 4, 6 and 12 of the At-subgenome and 22 of the Dt-subgenome of G. barbadense was revealed, while chromosomes 2 and 7 of the At-subgenome and 18 of the Dt- subgenome of G. barbadense were characterized by elimination. Among them, chromosomes 7 of the At- subgenome and 18 of the Dt-subgenome of G. barbadense were eliminated in the first backcross generation. In this work, two lines, CS- B06 and CS-B07, from the American cytogenetic collection with a putative substitution involving chromosomes 6 and 7 of the At-subgenome were analysed. The presence of only polymorphic alleles from the species G. hirsutum and the absence of polymorphic alleles from the species G. barbadense were revealed, which showed the absence of substitution involving these chromosomes. BC2F1 hybrids with monosomy for both G. barbadense and G. hirsutum chromosomes were characterized by regular pairing of chromosomes and high meiotic indexes. However, many hybrids were characterized by a decrease in pollen fertility. Two hybrids with monosomy for chromosome 7 of the At-subgenome of G. hirsutum and chromosome 6 of the At-subgenome of G. barbadense had the greatest reduction in pollen viability (70.09 ± 1.57 and 75.00 ± 1.66 %, respectively). Thus, this work shows a specific feature in the introgression of individual chromosomes of the cotton species G. barbadense into the cotton G. hirsutum genome.

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.

All nonhomologous chromosomes and rearrangements in Saccharum officinarum × Saccharum spontaneum allopolyploids identified by oligo-based painting.

Modern sugarcane cultivars (Saccharum spp., 2n = 100~120) are complex polyploids primarily derived from interspecific hybridization between S. officinarum and S. spontaneum. Nobilization is the theory of utilizing wild germplasm in sugarcane breeding, and is the foundation for utilizing S. spontaneum for stress resistance. However, the exact chromosomal transmission remains elusive due to a lack of chromosome-specific markers. Here, we applied chromosome-specific oligonucleotide (oligo)-based probes for identifying chromosomes 1-10 of the F1 hybrids between S. officinarum and S. spontaneum. Then, S. spontaneum-specific repetitive DNA probes were used to distinguish S. spontaneum in these hybrids. This oligo- fluorescence in situ hybridization (FISH) system proved to be an efficient tool for revealing individual chromosomal inheritance during nobilization. We discovered the complete doubling of S. officinarum-derived chromosomes in most F1 hybrids. Notably, we also found defective S. officinarum-derived chromosome doubling in the F1 hybrid Yacheng75-4191, which exhibited 1.5n transmission for all nonhomologous chromosomes. Altogether, these results highlight the presence of variable chromosome transmission in nobilization between S. officinarum and S. spontaneum, including 1.5n + n and 2n + n. These findings provide robust chromosome markers for in-depth studies into the molecular mechanism underlying chromosome doubling during the nobilization, as well as tracing chromosomal inheritance for sugarcane breeding.

The launch of satellite: DNA repeats as a cytogenetic tool in discovering the chromosomal universe of wild Triticeae.

Fluorescence in situ hybridization is a powerful tool that enables plant researchers to perform systematic, evolutionary, and population studies of wheat wild relatives as well as to characterize alien introgression into the wheat genome. This retrospective review reflects on progress made in the development of methods for creating new chromosomal markers since the launch of this cytogenetic satellite instrument to the present day. DNA probes based on satellite repeats have been widely used for chromosome analysis, especially for "classical" wheat probes (pSc119.2 and Afa family) and "universal" repeats (45S rDNA, 5S rDNA, and microsatellites). The rapid development of new-generation sequencing and bioinformatical tools, and the application of oligo- and multioligonucleotides has resulted in an explosion in the discovery of new genome- and chromosome-specific chromosome markers. Owing to modern technologies, new chromosomal markers are appearing at an unprecedented velocity. The present review describes the specifics of localization when employing commonly used vs. newly developed probes for chromosomes in J, E, V, St, Y, and P genomes and their diploid and polyploid carriers Agropyron, Dasypyrum, Thinopyrum, Pseudoroegneria, Elymus, Roegneria, and Kengyilia. Particular attention is paid to the specificity of probes, which determines their applicability for the detection of alien introgression to enhance the genetic diversity of wheat through wide hybridization. The information from the reviewed articles is summarized into the TRepeT database, which may be useful for studying the cytogenetics of Triticeae. The review describes the trends in the development of technology used in establishing chromosomal markers that can be used for prediction and foresight in the field of molecular biology and in methods of cytogenetic analysis.

Cytogenetic characterization and molecular marker development for a wheat-T. boeoticum 4Ab (4B) disomic substitution line with stripe rust resistance.

Triticum boeoticum (2n = 2x = 14, AbAb) is an important relative of wheat. This species tolerates many different types of environmental stresses, including drought, salt, and pathogenic infection, and is lower in dietary fiber and higher in antioxidants, protein (15-18%), lipids, and trace elements than common wheat. However, the gene transfer rate from this species to common wheat is low, and few species-specific molecular markers are available. In this study, the wheat-T. boeoticum substitution line Z1889, derived from a cross between the common wheat cultivar Crocus and Triticum boeoticum line G52, was identified using multicolor fluorescence in situ hybridization (mc-FISH), multicolor genomic in situ hybridization (mc-GISH) and a 55K single nucleotide polymorphism (SNP) array. Z1889 was revealed to be a 4Ab (4B) substitution line with a high degree of resistance to stripe rust pathogen strains prevalent in China. In addition, 22 4Ab chromosome-specific molecular markers and 11 T. boeoticum genome-specific molecular markers were developed from 1145 4Ab chromosome-specific fragments by comparing the sequences generated by specific-length amplified fragment sequencing (SLAF-seq), with an efficiency of up to 55.0%. Furthermore, the specificity of these markers was verified in four species containing the Ab genome. These markers can not only be used for the detection of the 4Ab chromosome but also provide a basis for molecular marker-assisted selection-based breeding in wheat.

HTLV-1 infection of donor-derived T cells might promote acute graft-versus-host disease following liver transplantation

Acute graft versus host disease (aGVHD) is a rare, but severe complication of liver transplantation (LT). It is caused by the activation of donor immune cells in the graft against the host shortly after transplantation, but the contributing pathogenic factors remain unclear. Here we show that human T cell lymphotropic virus type I (HTLV-1) infection of donor T cells is highly associated with aGVHD following LT. The presence of HTLV-1 in peripheral blood and tissue samples from a discovery cohort of 7 aGVHD patients and 17 control patients is assessed with hybridization probes (TargetSeq), mass cytometry (CyTOF), and multiplex immunohistology (IMC). All 7 of our aGVHD patients display detectable HTLV-1 Tax signals by IMC. We identify donor-derived cells based on a Y chromosome-specific genetic marker, EIF1AY. Thus, we confirm the presence of CD4+Tax+EIF1AY+ T cells and Tax+CD68+EIF1AY+ antigen-presenting cells, indicating HTLV-1 infection of donor immune cells. In an independent cohort of 400 patients, we verify that HTLV-1 prevalence correlates with aGVHD incidence, while none of the control viruses shows significant associations. Our findings thus provide new insights into the aetio-pathology of liver-transplantation-associated aGVHD and raise the possibility of preventing aGVHD prior to transplantation.

High-performance LAMP-based method for human sex identification using Y chromosome-specific genetic markers

Finding fast and cheap strategies for DNA typing and human sample identification is of interest in forensic science. We report a new versatile alternative for molecular sex determination using Y-specific targets (TSPY, TTTY, alphoid regions, and Y-Amelogenin). This system uses an isothermal loop-mediated DNA amplification (LAMP) with a set of 6 primers for each target, designed to improve sensibility and specificity, and reducing detection time to only 45 min. Furthermore, detecting the different targets on the Y chromosome either individually or in combination revealed accurate results. Assay sensitivity was determined with a mixture of human female and male DNA at different concentrations to mimic forensic samples. Single primer sets showed high sensitivity at DNA concentrations ranging from 58.6 to 3.7 pg/µL. When a combined primers set was used, sensitivity yielded a detection as low as 0.1 pg/µL of male DNA, making it 10 times more sensitive than qPCR-DNA quantification kits. Finally, high specificity was observed when tested against 6 domestic species.

Development and applications of a collection of single copy gene-based cytogenetic DNA markers in garden asparagus.

Garden asparagus (Asparagus officinalis, 2n = 2x = 20 chromosomes) is an important dioecious vegetable crop and a model species for studying sex chromosome formation and evolution. However, few molecular cytogenetic studies on garden asparagus have been reported because of its small metaphase chromosomes, the scarcity of distinguished cytogenetic markers, and the high content of repetitive sequences. In this study, a set of single copy genes free of repetitive sequences with sizes ranging from 4.3 kb to 8.2 kb were screened and used as probes for fluorescence in situ hybridization (FISH) to identify individual chromosomes of garden asparagus. The chromosome-specific signal distribution patterns of these probes enabled the distinguishment of each pair of chromosomes. The sequence assembly and cytogenetic map were successfully integrated, and the results confirmed that the chromosome 1 representing the sex chromosome in the genome assembly is chromosome 5 in the karyotype analysis. The cytogenetic identification of the male-specific region of the Y chromosome (MSY) was implemented using a mixed probe derived from a number of MSY-specific single copy sequences. In addition, the chromosome orthologous relationship between garden asparagus (A1–A10, karyotypic analysis) and its hermaphrodite close relative, A. setaceus (B1–B10, karyotypic analysis), was analyzed using this collection of chromosome-specific cytological markers. The results showed that B3 is the ortholog of sex chromosome A5 and thus may represent the ancestral autosome of the current sex chromosome in garden asparagus. Chromosomes B5, B4, B1, B8, B7, and B9 are the orthologs of A2, A3, A4, A7, A8, and A10, respectively. The chromosome identification, cytogenetic recognition of MSY, and the orthologous relationship analysis between garden asparagus and A. setaceus are valuable for the further investigation of the sex chromosome emergence and evolutionary mechanism of garden asparagus and genome structure evolution in the Asparagus genus.

Screening and characterization of X chromosome-specific markers in mandarin fish (Siniperca chuatsi)

The mandarin fish (Siniperca chuatsi) is an important aquaculture fish. All-male mandarin fish culture has the advantages of higher profits, more uniform size and lower energy consumption of gonadal development. However, the lack of accurate sex-specific markers has hampered the all-male breeding of this species. Herein, we successfully adopted a next generation sequencing strategy to identify potential X chromosome-specific sequences in mandarin fish. First of all, we performed genome sequencing of three YY super-males, and then align the reads to XX genome to identified candidate X chromosome-specific regions. A total of 49 candidate X chromosome-specific contigs were identified with length ranging from 448 to 1189 bp. After PCR validation, four pairs of primers derived from two X chromosome-specific contigs, Ma-XB1, Ma-XB2, Ma-XC1, Ma-XC2, could successfully produce X chromosome-specific bands of 935 bp, 1105 bp, 850 bp and 1146 bp, respectively, with no band amplified in six YY super-males. In conclusion, based on YY genome sequencing and PCR validation, four X chromosome-specific markers were developed, which would promote the development of all-male breeding of mandarin fish.

Chromosome-specific potential intron polymorphism markers for large-scale genotyping applications in pomegranate.

Despite the availability of whole genome assemblies, the identification and utilization of gene-based marker systems has been limited in pomegranate. In the present study, we performed a genome-wide survey of intron length (IL) markers in the 36,524 annotated genes of the Tunisia genome. We identified and designed a total of 8,812 potential intron polymorphism (PIP) markers specific to 3,445 (13.40%) gene models that span 8 Tunisia chromosomes. The ePCR validation of all these PIP markers on the Tunisia genome revealed single-locus amplification for 1,233 (14%) markers corresponding to 958 (27.80%) genes. The markers yielding single amplicons were then mapped onto Tunisia chromosomes to develop a saturated linkage map. The functional categorization of 958 genes revealed them to be a part of the nucleus and the cytoplasm having protein binding and catalytic activity, and these genes are mainly involved in the metabolic process, including photosynthesis. Further, through ePCR, 1,233 PIP markers were assayed on multiple genomes, which resulted in the identification of 886 polymorphic markers with an average PIC value of 0.62. In silico comparative mapping based on physically mapped PIP markers indicates a higher synteny of Tunisia with the Dabenzi and Taishanhong genomes (>98%) in comparison with the AG2017 genome (95%). We then performed experimental validation of a subset of 100 PIP primers on eight pomegranate genotypes and identified 76 polymorphic markers, with 15 having PIC values ≥0.50. We demonstrated the potential utility of the developed markers by analyzing the genetic diversity of 31 pomegranate genotypes using 24 PIP markers. This study reports for the first time large-scale development of gene-based and chromosome-specific PIP markers, which would serve as a rich marker resource for genetic variation studies, functional gene discovery, and genomics-assisted breeding of pomegranate.

Fine mapping of a recessive leaf rust resistance locus on chromosome 2BS in wheat accession CH1539.

The online version contains supplementary material available at 10.1007/s11032-022-01318-4.

Efficient Anchoring of Erianthus arundinaceus Chromatin Introgressed into Sugarcane by Specific Molecular Markers

Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for the development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones of E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely, Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking of the inherited status of this chromosome in a sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

Molecular and Cytogenetic Identification of Stem Rust Resistant Wheat-Thinopyrum intermedium Introgression Lines.

Thinopyrum intermedium (JJJsJsStSt, 2n = 6x = 42), a wild relative of common wheat, possesses many desirable agronomic genes for wheat improvement. The production of wheat-Thinopyrum intermedium introgression lines is a key step for transferring these beneficial genes into wheat. In this study, we characterized three wheat-Thinopyrum intermedium introgression lines TA3681, TA5566, and TA5567 using non-denaturing fluorescence in situ hybridization, genomic in situ hybridization, PCR-based landmark unique gene, and intron targeting markers. Our results showed that TA3681 is a wheat-Thinopyrum intermedium 1Stdisomic addition line, TA5566 is a wheat-Thinopyrum intermedium non-Robertsonian translocation line carrying two pairs of 3A-7Js translocation chromosomes, and that TA5567 is a wheat-Thinopyrum intermedium non-Robertsonian translocation line carrying a pair of 3A-7Js translocation chromosomes. We developed 13, 36, and 15 Thinopyrum intermedium chromosome-specific markers for detecting the introgressed Thinopyrum chromosomes in TA3681, TA5566, and TA5567, respectively. Stem rust assessment revealed that TA3681 exhibited ahigh level of seedling resistance to Chinese-prevalent Puccinia graminis f. sp. tritici pathotypes, and both TA5566 and TA5567 were highly resistant to Australian P. graminis f. sp. tritici pathotypes, indicating that Thinopyrum intermedium chromosomes 1St and 7Js might carry new stem rust resistance genes. Therefore, the new identified introgression lines may be useful for improving wheat stem rust resistance.

Establishment and identification of six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430.

Six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430 were identified using in situ hybridization and SNP microarray, the homoeologous group and stripe rust resistance of each alien chromosome were determined, and Th. ponticum chromosome-specific markers were developed. Xiaoyan 7430 is a significant partial amphiploid, which is used to set up a bridge for transferring valuable genes from Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey into common wheat. To accelerate the application of these useful genes in enriching the genetic variability of cultivated wheat by chromosome engineering, a complete set of derived addition lines has been created from Xiaoyan 7430. The chromosome composition of each line was characterized by the combination of genomic in situ hybridization and multicolor fluorescence in situ hybridization (mc-FISH), and the homoeology of each alien chromosome was determined by wheat SNP microarray analysis. Addition line WTA55 with alien group-6 chromosome was evaluated resistant to stripe rust isolates at both the seedling and grain-filling stages (Zadoks scale at z.11 and z.73). Diagnostic marker analysis proved that it could carry a novel stripe rust resistance gene derived from Th. ponticum. Furthermore, a FISH probe and 45 molecular markers specific for alien chromosomes were developed based on specific-locus amplified fragment sequencing (SLAF-seq). Of which 27 markers were separately located on single alien chromosome, and some of them could be used to identify the derived translocation lines. This set of addition lines as well as the molecular markers and the FISH probe will promote the introgression of abundant variation from Th. ponticum into wheat in wheat improvement programs.

Introgression of the Triticum timopheevii Genome Into Wheat Detected by Chromosome-Specific Kompetitive Allele Specific PCR Markers.

Triticum timopheevii (2n = 28, AtAtGG) is a tetraploid wild relative species with great potential to increase the genetic diversity of hexaploid wheat Triticum aestivum (2n = 42, AABBDD) for various important agronomic traits. A breeding scheme that propagated advanced backcrossed populations of wheat-T. timopheevii introgression lines through further backcrossing and self-fertilisation resulted in the generation of 99 introgression lines (ILs) that carried 309 homozygous segments from the At and G subgenomes of T. timopheevii. These introgressions contained 89 and 74 unique segments from the At and G subgenomes, respectively. These overlapping segments covered 98.9% of the T. timopheevii genome that has now been introgressed into bread wheat cv. Paragon including the entirety of all T. timopheevii chromosomes via varying sized segments except for chromosomes 3At, 4G, and 6G. Homozygous ILs contained between one and eight of these introgressions with an average of three per introgression line. These homozygous introgressions were detected through the development of a set of 480 chromosome-specific Kompetitive allele specific PCR (KASP) markers that are well-distributed across the wheat genome. Of these, 149 were developed in this study based on single nucleotide polymorphisms (SNPs) discovered through whole genome sequencing of T. timopheevii. A majority of these KASP markers were also found to be T. timopheevii subgenome specific with 182 detecting At subgenome and 275 detecting G subgenome segments. These markers showed that 98% of the At segments had recombined with the A genome of wheat and 74% of the G genome segments had recombined with the B genome of wheat with the rest recombining with the D genome of wheat. These results were validated through multi-colour in situ hybridisation analysis. Together these homozygous wheat-T. timopheevii ILs and chromosome-specific KASP markers provide an invaluable resource to wheat breeders for trait discovery to combat biotic and abiotic stress factors affecting wheat production due to climate change.

Critical review on karyotype diversity in lentil based on classical and molecular cytogenetics.

Lentil is an annual protein rich valuable edible crop with only one cultivated and six wild taxa. Keeping in mind its narrow gene pool, the genus deserves critical assessment of genomic diversity at the chromosomal level. Genetic diversity represents the heritable variation within and between populations of organisms. Over the decades classical and molecular cytogenetics have played an immense role in the field of crop improvement. Lentil, though grown in different countries, country-wise chromosomal information is inadequate. Critical evaluation of more than seven decades chromosomal information has revealed unique karyotype diversity within the landraces of different countries. Application of fluorescent banding and fluorescent in situ hybridization (FISH) has helped to segregate cultivars based on cultivar specific chromosomal markers and landmarks. Selection of cultivated and wild cultivars based on qualitative and diseases related morpho-traits and new information from this critical review especially on molecular cytogenetics may provide more options for crop improvement. More research in the field of molecular cytogenetics from country specific species and cultivars are needed to enrich the repository of gene pool. Alien gene introgression from extended gene pool through the advanced genomics and biotechnological tools could facilitate the path of sustainable improvement of this crop.

Development and application of specific FISH probes for karyotyping Psathyrostachys huashanica chromosomes

BackgroundPsathyrostachys huashanica Keng has long been used as a genetic resource for improving wheat cultivar because of its genes mediating the resistance to various diseases (stripe rust, leaf rust, take-all, and powdery mildew) as well as its desirable agronomic traits. However, a high-resolution fluorescence in situ hybridization (FISH) karyotype of P. huashanica remains unavailable.ResultsTo develop chromosome-specific FISH markers for P. huashanica, repetitive sequences, including pSc119.2, pTa535, pTa713, pAs1, (AAC)5, (CTT)12, pSc200, pTa71A-2, and Oligo-44 were used for a FISH analysis. The results indicated that the combination of pSc200, pTa71A-2 and Oligo-44 probes can clearly identify all Ns genomic chromosomes in the two P. huashanica germplasms. The homoeologous relationships between individual P. huashanica chromosomes and common wheat chromosomes were clarified by FISH painting. Marker validation analyses revealed that the combination of pSc200, pTa71A-2, and Oligo-44 for a FISH analysis can distinguish the P. huashanica Ns-genome chromosomes from wheat chromosomes, as well as all chromosomes (except 4Ns) from the chromosomes of diploid wheat relatives carrying St, E, V, I, P and R genomes. Additionally, the probes were applicable for discriminating between the P. huashanica Ns-genome chromosomes in all homologous groups and the corresponding chromosomes in Psathyrostachys juncea and most Leymus species containing the Ns genome. Furthermore, six wheat–P. huashanica chromosome addition lines (i.e., 2Ns, 3Ns, 4Ns, 7Ns chromosomes and chromosomal segments) were characterized using the newly developed FISH markers. Thus, these probes can rapidly and precisely detect P. huashanica alien chromosomes in the wheat background.ConclusionsThe FISH karyotype established in this study lays a solid foundation for the efficient identification of P. huashanica chromosomes in wheat genetic improvement programs.