New molecular markers and cytogenetic probes enable chromosome identification of wheat-Thinopyrum intermedium introgression lines for improving protein and gluten contents.

New molecular markers were developed for targeting Thinopyrum intermedium 1St#2 chromosome, and novel FISH probe representing the terminal repeats was produced for identification of Thinopyrum chromosomes. Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. A wheat-Th. intermedium ssp. trichophorum chromosome 1St#2 substitution and translocation has displayed superior grain protein and wet gluten content. With the aim to develop a number of chromosome 1St#2 specific molecular and cytogenetic markers, a high throughput, low-cost specific-locus amplified fragment sequencing (SLAF-seq) technology was used to compare the sequences between a wheat-Thinopyrum 1St#2 (1D) substitution and the related species Pseudoroegneria spicata (St genome, 2n = 14). A total of 5142 polymorphic fragments were analyzed and 359 different SLAF markers for 1St#2 were predicted. Thirty-seven specific molecular markers were validated by PCR from 50 randomly selected SLAFs. Meanwhile, the distribution of transposable elements (TEs) at the family level between wheat and St genomes was compared using the SLAFs. A new oligo-nucleotide probe named Oligo-pSt122 from high SLAF reads was produced for fluorescence in situ hybridization (FISH), and was observed to hybridize to the terminal region of 1St#L and also onto the terminal heterochromatic region of Th. intermedium genomes. The genome-wide markers and repetitive based probe Oligo-pSt122 will be valuable for identifying Thinopyrum chromosome segments in wheat backgrounds.