Disequilibrium linkage (gametic disequilibrium) of structural genes in the bovine population

In the population of Kholmogor cattle (n=824), gametic disequilibrium was studied for alleles of the polymorphic loci β-Lg (LGB), β-Cn (CSN2) and æ-Cn (CSN3). In purebred Kholmogor cattle, the β-casein polymorphism is determined by three alleles with the frequencies of CSN2А 1 – 0.368±0.0076, CSN2А 2 – 0.497±0.0078, CSN2B – 0.136±0.0056 and the frequencies of acasein of CSN3А – 0.689 ±0.0061, CSN3B – 0.311±0.0061, respectively. The beta-lactoglobulin polymorphism is controlled by two alleles LGBA and LGBB with a frequency of 0.237±0.0048 and 0.763±0.0048, respectively. In the population of breeding plants, the gametic disequilibrium was revealed according to the alleles of loci CSN2 and LGB; CSN2 and CSN3. The factor that caused the gametic disequilibrium was probably the initial difference between native Russian cattle and black-and-white cattle of Western Europe on the basis of which the Kholmogor breed gene pool was developed. The population showed an excess of haplotypes β-CnВæ-CnВ, β-CnВβ-LgВ (the "attraction" phase), apparently inherited mainly from native Russian cattle, and β-CnА 1æ-CnА, β-CnА 2β-LgА characteristic mainly for black-and-white cattle of Western Europe and a lack of β-CnВæ-CnА, β-CnВβ-LgА (the "repulsion" phase), probably due to the low frequency of these haplotypes in both initial breeds. The role of various factors in the occurrence and maintenance of gamete disequilibrium is discussed. It is suggested that for alleles of non-linked loci, longtime retention of gametic disequilibrium in population is most likely supported by selection

A Robust and Rapid Candidate Gene Mapping Pipeline Based on M2 Populations

The whole-genome sequencing-based bulked segregant analysis (WGS-BSA) has facilitated the mapping candidate causal variations for cloning target plant genes. Here, we report an improved WGS-BSA method termed as M2-seq to expedite the mapping candidate mutant loci by studying just M2 generation. It is an efficient mutant gene mapping tool, rapid, and comparable to the previously reported approaches, such as Mutmap and Mutmap+ that require studying M3 or advanced selfed generations. In M2-seq, background variations among the M2 populations can be removed efficiently without knowledge of the variations of the wild-type progenitor plant. Furthermore, the use of absolute delta single-nucleotide polymorphism (SNP) index values can effectively remove the background variation caused by repulsion phase linkages of adjacent mutant alleles; and thereby facilitating the identification of the causal mutation in target genes. Here, we demonstrated the application of M2-seq in successfully mapping the genomic regions harboring causal mutations for mutant phenotypes among 10 independent M2 populations of soybean. The mapping candidate mutant genes just in M2 generation with the aid of the M2-seq method should be particularly useful in expediting gene cloning especially among the plant species with long generation time.

Disease Resistance Evaluation of Elite CIMMYT Wheat Lines Containing the Coupled Fhb1 and Sr2 Genes

Fusarium head blight (FHB) and stem rust are among the most devastating diseases of wheat worldwide. Fhb1 is the most widely utilized and the only isolated gene for FHB resistance, while Sr2 is a durable stem rust resistance gene used in rust-prone areas. The two loci are closely linked on the short arm of chromosome 3B and the two genes are in repulsion phase among cultivars. With climate change and the shift in Fusarium populations, it is imperative to develop wheat cultivars resistant to both diseases. The present study was dedicated to developing wheat germplasm combining Fhb1 and Sr2 resistance alleles in the International Maize and Wheat Improvement Center (CIMMYT)’s elite cultivars’ backgrounds. Four recombinant inbred lines (RILs) in Hartog background that have the resistant Fhb1 and Sr2 alleles in coupled phase linkage were crossed with seven CIMMYT bread wheat lines, resulting in 208 lines. Molecular markers for both genes were employed in addition to the use of pseudo-black chaff (PBC) as a phenotypic marker for the selection of Sr2. At various stages of the selection process, progeny lines were assessed for FHB index, Fusarium damaged kernels (FDK), stem rust, and PBC expression as well as other diseases of interest (stripe rust and leaf spotting diseases). The 25 best lines were selected for CIMMYT’s wheat breeding program. In addition to expressing resistance to FHB, most of these 25 lines have an acceptable level of resistance to other tested diseases. These lines will be useful for wheat breeding programs worldwide and potentially speed up the resistance breeding efforts against FHB and stem rust.

Semidwarf Gene d60 Affected by Ubiquitous Gamete Lethal Gene gal Produced Rare Double Dwarf with d30 via Recombination Breaking Repulsion-Phase Linkage on Rice Chromosome 2

The genotype of gal and d60 were investigated in 33 rice varieties chosen from representative semidwarf and dwarf rice varieties. These were crossed with three tester lines, the d60Gal line (genotype d60d60GalGal), the D60gal line (Koshihikari, D60D60galgal), and the D60Gal line (D60D60GalGal). Each F1 plant was measured for culm length, and seed fertility. As a result, all F1 lines with the d60Gal line showed tallness and partial sterility, reduced by 25% in average from those with the D60gal line (Koshihikari) and the D60Gal line. These data indicated that the genotype of the 33 varieties is D60D60galgal and that the d60 locus is not allelic to those of sd1, d1, d2, d6, d18k, d29, d30, d35, d49, d50, and qCL1 involved in the 33 varieties. In addition, the gal gene is not complementarily activated with the semidwarf and dwarf genes described above, other than d60. The Gal gene will be ubiquitously distributed in rice. It is emphasized that Gal is a rare and valuable mutant gene essential to the transmission of d60. The double dwarf genotype of homozygous d30d60 was rarely gained in the F3 of the d30 line × d60 line by breaking their repulsion d60-D30 linkage on chromosome 2.

EphB2 and ERK signaling are required for heterotypic contact inhibition of locomotion to drive cell sorting

SummaryInteractions between different cell-types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesized to control population-wide behaviors during embryogenesis [1, 2]. However, our understanding of the signals that lead to cell-type specific repulsion, and the precise capacity of heterotypic CIL responses to drive emergent behaviors is lacking. Using a new in vitro model of heterotypic CIL between epithelial and mesenchymal cells, we show that fibrosarcoma cells (HT1080), but not fibroblasts (NIH3T3), are actively repelled by epithelial cells in culture. We show that knocking down EphB2 in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. Furthermore, this heterotypic interaction requires ERK activation, downstream of EphB2 signaling. We also examine the population-wide effects when these various cell combinations, and their specific heterotypic CIL responses, are allowed to interact in culture. Mixtures of fibrosarcoma and epithelial cells – unlike fibroblasts and epithelial cells – lead to complete sorting and segregation of the two populations, and inhibiting their distinct CIL response by knocking down EphB2 or ERK in fibrosarcoma cells disrupts this emergent sorting behavior. Our understanding of the mechanisms underlying developmental behaviors such as cell sorting is lacking as predominant sorting hypotheses, such as differential adhesion, have recently been found inadequate in predicting the sorting of mesenchymal cells [3, 4]. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations during embryogenesis.

Multiple Resistance to Meloidogyne spp. and to Bipartite and Monopartite Begomovirus spp. in Wild Solanum (Lycopersicon) Accessions

The Ty-1 locus confers tolerance to monopartite and bipartite Begomovirus spp. (genus Begomovirus, family Geminiviridae) and this phenotype is improved in homozygous tomato lines. However, the gene Mi (Meloidogyne spp. resistance) is in repulsion phase linkage with Ty-1, which hampers the large-scale development of multiresistant inbred lines. Seventy-one Solanum (section Lycopersicon) accessions were whitefly inoculated with the bipartite Begomovirus sp. Tomato rugose mosaic virus (ToRMV) and simultaneously infested with a mixture of Meloidogyne incognita and M. javanica under greenhouse conditions in Brazil. Accessions were then transplanted into a nematode-infested field with natural ToRMV infection. A severity index was used to evaluate ToRMV reaction. Nematode evaluation was done by counting the number of galls per root system. Seventeen accessions with Meloidogyne spp. and ToRMV resistance were selected and evaluated in Spain against three monopartite Begomovirus spp. associated with the tomato yellow leaf curl virus disease, using infectious clones. Systemic infection was monitored by DNA hybridization. Five S. peruvianum accessions (PI-306811, PI-365951, LA-1609, LA-2553, and CNPH-1194) displayed nematode and broad-spectrum resistance to all Begomovirus spp. tested in both continents. From the breeding standpoint, accessions combining resistance to Meloidogyne spp. and to bipartite and monopartite Begomovirus spp. would be useful for the development of elite lines expressing all traits in homozygous condition.

Development of SCAR markers linked to zt-2, one of the genes controlling absence of tannins in faba bean

Faba beans (Vicia faba L.) have a great potential as a protein-rich fodder crop, but anti-nutritional factors such as condensed tannins reduce the biological value of their protein. Tannins can be removed from seeds by any of the two complementary genes, zt-1 and zt-2, which also determine white-flowered plants. The less common gene, zt-2, is also associated with increased protein levels and energy values and reduced fibre content of the seeds. To identify a cost-effective marker linked to zt-2, we analysed a segregating F2 population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-2 line. By using Bulked Segregant Analysis (BSA), five RAPD markers linked in coupling and repulsion phase to zt-2 were identified and their conversion into Sequence Characterised Amplified Regions (SCARs) was attempted. Amplification of the SCARS was more consistent, although the initial polymorphism was lost. Restriction digestion of SCAR SCAD16589 with AluI (SCAD16-A), Bsp120I (SCAD16-B) and HinfI (SCAD16-H) revealed clear differences due to the amplification of different loci. The consensus sequence of these CAPs (Cleavage Amplification Polymorphisms) markers allowed discrimination of three bands from which two new forward SCAR primers were developed based on specific sequences from zero tannin and high tannin content genotypes. To improve the efficiency of the marker screening, a multiplex PCR was developed that allowed the simultaneous amplification of the SCAR with the same advantages as a codominant marker. Marker validation was carried out with a new F2 population segregating for flower colour and tannin content, underscoring the potential of these markers in breeding selection to introgress the zt-2 gene for the development of new tannin free faba bean cultivars.