Characterization of Chromosomal Rearrangement in New Wheat—Thinopyrum intermedium Addition Lines Carrying Thinopyrum—Specific Grain Hardness Genes

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Abstract

The wild species, Thinopyrum intermedium. (Genome StStJSJSJJ), serves as a valuable germplasm resource providing novel genes for wheat improvement. In the current study, non-denaturing fluorescence in situ hybridization (ND-FISH) with multiple probes and comparative molecular markers were applied to characterize two wheat-Th. intermedium chromosome additions. Sequential ND-FISH with new labeled Th. intermedium specific oligo-probes were used to precisely determine the chromosomal constitution of Th. intermedium, wheat—Th. intermedium partial amphiploids and addition lines Hy36 and Hy37. The ND-FISH results showed that the added JS-St translocated chromosomes in Hy36 had minor Oligo-5S ribosomal DNA (rDNA) signals at the short arm, while a pair of J-St chromosomes in Hy37 had major Oligo-pTa71 and minor Oligo-5S rDNA signals. The 90K SNP array and PCR-based molecular markers that mapped on wheat linkage group 5 and 3 facilitated the identification of Thinopyrum chromosome introgressions in the addition lines, and confirmed that added chromosomes in Hy36 and Hy37 were 5JSS.3StS and 5JS.3StS, respectively. Complete coding sequences at the paralogous puroindoline-a (Pina) loci from Th. intermedium were cloned and localized on the short arm of chromosome 5JS of Hy36. Line Hy36 showed a reduction in the hardness index, which suggested that Th. intermedium-specific Pina gene sequences may be associated with the softness trait in wheat background. The molecular cytogenetic identification of novel wheat—Th. intermedium derivatives indicated that the frequent chromosome rearrangement occurred in the progenies of wheat-Thinopyrum hybridization. The new wheat-Thinopyrum derived lines may increase the genetic diversity for wheat breeding.

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Characterization of polyploid wheat genomic diversity using a high-density 90 000 single nucleotide polymorphism array

Shichen Wang, Debbie Wong, Kerrie Forrest … (2014)

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker–trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence–absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

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Cloning and characterization of ribosomal RNA genes from wheat and barley.

W Gerlach, J R Bedbrook (1979)

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.

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Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis.

Zongxiang Tang, Zujun Yang, Shulan Fu (2014)

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.