Development of Sequence-Tagged Site Marker Set for Identification of J, JS, and St Sub-genomes of Thinopyrum intermedium in Wheat Background

Abstract

Thinopyrum intermedium (2n = 6x = 42, JJJ^SJ^SStSt) is one of the important resources for the wheat improvement. So far, a few Th. intermedium (Thi)-specific molecular markers have been reported, but the number is far from enough to meet the need of identifying alien fragments in wheat-Th. intermedium hybrids. In this study, 5,877,409 contigs were assembled using the Th. intermedium genotyping-by-sequencing (GBS) data. We obtained 5,452 non-redundant contigs containing mapped Thi-GBS markers with less than 20% similarity to the wheat genome and developed 2,019 sequence-tagged site (STS) molecular markers. Among the markers designed, 745 Thi-specific markers with amplification products in Th. intermedium but not in eight wheat landraces were further selected. The distribution of these markers in different homologous groups of Th. intermedium varied from 47 (7/12/28 on 6J/6St/6J^S) to 183 (54/62/67 on 7J/7St/7J^S). Furthermore, the effectiveness of these Thi-specific markers was verified using wheat-Th. intermedium partial amphidiploids, addition lines, substitution lines, and translocation lines. Markers developed in this study provide a convenient, rapid, reliable, and economical method for identifying Th. intermedium chromosomes in wheat. In addition, this set of Thi-specific markers can also be used to estimate genetic and physical locations of Th. intermedium chromatin in the introgression lines, thus providing valuable information for follow-up studies such as alien gene mining.

Keywords: STS markers; Thinopyrum intermedium; chromosome identification; physical location; specificity.

Figure 1

Amount and distribution of the GBS-contigs and markers developed in Th. intermedium. The number of Thi-specific markers on each chromosome is listed.

Figure 2

A linkage map of 745 Thi-specific STS markers derived from the Thi-GBS linkage map reported in Kantarski et al. (2017). Black dots next to the markers indicate that these markers have amplification products in Th. elongatum (The), Th. bessarabicum (Thb), Ps. spicata (Ps), and D. villosum (Dv).

Figure 3

Amplification of Thi-specific markers in wheat-Th. intermedium introgression lines. (A) partial amphiploid TAF46 (ABD+1J+2St+3J+4St+5J+6St+7J); (B) partial amphiploid TE-3 (ABD+1St+2J^s+3J+4J+4J^s+5J^s+6St+7St); (C) substitution line AS1677 [ABD+1St(1D)]; (D) addition line X24C14 (ABD+2J^s); (E) addition line A1082 (ABD+3J); (F) substitution line A1125 [ABD+4J^s(4B)]; (G) translocation line A39 (ABD+T4BS.5J^sL); (H) substitution line XM-4 [ABD+6J^s(6B)]; (I) translocation line Z4 (ABD+T7J^sS-3AS.3AL+T7J^sS.7J^sL-3AL). The sub-genomes with the largest proportion of positive markers were labeled, and the numbers on the column were significantly different from the two other observations within the same homologous group (HG) at p ≤ 0.0125 level (Bonferroni corrected).

Figure 4

Sequential FISH and GISH patterns of six wheat-Th. intermedium introgressions with single alien sub-genome from Thi-HG1-6, respectively. (A,B) substitution line AS1677 [ABD+1St(1D)]; (C,D) addition line X24C14 (ABD+2J^s); (E,F) addition line A1082 (ABD+3J); (G,H) substitution line A1125 [ABD+4J^s(4B)]; (I,J) translocation line A39 (ABD+T4BS.5J^sL); and (K,L) substitution line XM-4 [ABD+6J^s(6B)]. The probes for FISH were Oligo-pSc119.2 (green) + Oligo-pTa535 (red) (A,C,E,G,I,K). The probe (yellow-green) for GISH analysis was Th. intermedium total genomic DNA (B,D,F,H,J,L). Bars, 10 μm.

Figure 5

Identification of wheat-Th. intermedium translocation line T1332. The probes were Oligo-k288 (red) + Oligo-B11 (green) (A) and Oligo-pSc119.2 (green) + Oligo-pTa535 (red) (B). Bars, 10 μm. The chromosomes in the karyotype (C) were 4B, 4J, and T4BS/4JL, respectively, and the arrows pointed to the translocation breakpoints. (D) Amplification of 4J-, 4St-, and 4J^s-specific markers in Th. intermedium and six wheat-Thi introgression lines. (E) The position of 21 Thi-specific markers on 4J physical and linkage maps. Red markers are 4J-specific, four of which with amplification products in translocation line T1332 are marked with plus sign.

Figure 6

Amplification of Thi-specific markers in the J^e/J^b/St/V genomes. (A) Phylogenetic analysis and proportion of the positive Thi-specific markers in Th. elongatum (The), Th. bessarabicum (Thb), Ps. spicata (Ps), and D. villosum (Dv). (B) Number of the negative and positive Thi-specific markers in Th. elongatum, Th. bessarabicum, Ps. spicata, and D. villosum.