Cytological and molecular characterization of wheat lines carrying leaf rust and stem rust resistance genes Lr24 and Sr24

Previous studies showed that Australian wheat cultivars Janz and Sunco carry leaf rust and stem rust resistance genes Lr24 and Sr24 derived from Thinopyrum ponticum chromosome arm 3AgL. However, the size of the alien segments carrying Lr24 and Sr24 in the lines were not determined. In this study, we used non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and PCR-based landmark unique gene (PLUG) markers to visualize the alien segments in Janz and Sunco, and further compared them with the segments in US cultivars Agent and Amigo. The fraction length (FL) of the alien translocation in Agent was 0.70–1.00, whereas those in Janz, Sunco, and Amigo were smaller, at FL 0.85–1.00. It was deduced that the alien gene RAg encoding for red grain color and rust resistance genes Lr24 and Sr24 on chromosome arm 3AgL were in bins of FL 0.70–0.85 and 0.85–1.00, respectively. We retrieved and extracted nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor genes corresponding to the region of Lr24 and Sr24 on chromosomes 3E, and 3J, 3Js and 3St from the reference genome sequences of Th. elongatum and Th. intermedium, respectively. A set of molecular markers developed for Lr24 and Sr24 from those extracted NBS-LRR genes will provide valuable information for fine mapping and cloning of these genes.

Figure 1.FISH-GISH images of metaphase chromosomes of four wheat lines carrying Lr24 and Sr24, Agent (a,b), Janz (c,d), Sunco (e,f) and Amigo (g,h).Probes Oligo-pSc119.2-1(green) and Oligo-pTa535-1 (red) were used in FISH (a,c,e,g) and Pseudoroegneria stipifolia (St) genomic DNA was used as the probe (yellow-green) for GISH (b,d,f,h).(a-f) Arrows point to the breakpoint in T3DS.3DL-3AgL.(g,h) Arrows point to the breakpoint in T1BL.1BS-3AgL, and arrowheads point to the breakpoint for T1RS.1AL.Bars, 10 μm.Table 1.Analysis of 3AgL chromosomal segment sizes and breakpoint positions in four wheat lines carrying Lr24/Sr24.The position of the centromere was considered 0, and the terminal end of long arm of T3DS.3DL-3AgL in Agent was considered 1.00.L and S represent long and short arm, respectively.The column "FL" shows the fraction length.± Standard error.

Discussion
Although neither Lr24 nor Sr24 is a source of durable rust resistance, they are still useful for resistance breeding in many parts of the world, especially in combination with other genes.Several publications reported the development of molecular markers linked to Lr24 and/or Sr24 [11][12][13][14] .These resistance genes originated from Th. ponticum mainly in the form of chromosome T3DS.3DL-3AgLtranslocations 10,15 .Janz and Sunco are white-seeded Australian wheat cultivars with CS 3D-3Ag#3 and CS 3D-3Ag#14 as donors of Lr24 and Sr24.Although Sears 3 predicted that CS 3D-3Ag#3 and CS 3D-3Ag#14 might carry smaller alien segments than that in Agent based on chromosome pairing studies, their exact chromosomal structures were not determined.Zwart et al. 16 constructed a genetic linkage map of chromosome 3D using 111 doubled haploid lines derived from a cross between synthetic hexaploid wheat accession CPI133872 and Janz.Their finding that Lr24 was flanked by P37M54e and Xgwm169b at 0.4 cM and 1.0 cM, respectively, inspired our curiosity to characterize the 3AgL chromosomal segment size in Janz and Sunco.In the present study, ND-FISH and GISH revealed that different translocation events in Janz (Fig. 1c,d) and Sunco (Fig. 1e,f) were of similar size and significantly smaller than the translocation in Agent (Fig. 1a,b), as predicted by Sears 3 .Based on the cytological results, R Ag and Lr24 and Sr24 were in 3AgL bins FL 0.70-0.85and 0.85-1.00,respectively (Fig. 2).Combined with the PLUG marker results, the 3AgL bin FL 0.85-1.00containing Lr24 and Sr24 was estimated to be at least 60 Mb (Table S1).Due to the presence of the Ph1 (pairing homoeologous 1) gene in wheat, it is highly unlikely to have recombination between the Th.ponticum segment in the translocation chromosome T3DS.3DL-3AgLand homoeologous wheat chromosome regions in Janz and Sunco, as also mentioned by others 2,11,12,14 .based on orthologous gene conservation between rice and wheat have proven to be highly efficient and reliable molecular markers for confirming homoeologous relationships between wheat and alien chromosomes [18][19][20] .In this study, we used 24 PLUG primer pairs identified at the distal ends of the long arms of homoeologous group 3 chromosomes to genotype Agent, Janz, Sunco and Amigo, and found that five markers amplified Thinopyrum-specific bands.Two additional PLUG markers amplified fragments specific to Agent (Fig. 3, Table 2), indicating that: (1) the translocated Thinopyrum chromosomal segments in Agent, Janz, Sunco and Amigo belonged to homeologous group 3 (3AgL); and (2) the 3AgL chromosomal segments in Janz, Sunco and Amigo were similar in size, but smaller than that in Agent.
With the rapid development of sequencing technologies and availability of whole genome sequences of T. aestivum, Hordeum vulgare L. and Secale cereale, comparative genomic analysis is becoming increasingly achievable in developing molecular markers for use in non-sequenced species or in fine mapping of target genes [21][22][23][24] .In this study, based on the syntenic relationships among Th.elongatum chromosome 3EL, Th. intermedium chromosomes 3JL, 3J s L and 3StL, and the current Th.ponticum chromosome 3AgL, we developed four pairs of chromosome 3AgL-specific NBS-LRR-related primers (Fig. 4, Table S1) from the coding sequences (CDS) of three NBS-LRR genes in the Lr24/Sr24 region.Moreover, the eight Th.ponticum chromosome 3AgL-specifc molecular (five PLUG and three NBS-LRR) markers developed in this study will be useful in future fine mapping and cloning Lr24 and/or Sr24.No study thus far has separated the two genes indicating relatively close linkage, but we have no reason to believe they are the same gene.
Grain color is an important trait affecting flour yield and quality in wheat.Previous studies indicated that red-seeded wheats are more tolerant to preharvest sprouting than white-seeded wheats 25 .Hence Agent or another Uncropped gel images are provided in Supplementary Information (Supplementary Fig. 1).Table 3. Primer sequences of newly developed chromosome arm 3AgL-specific NBS-LRR-related makers.ATT CGT GGA GCC TCT CCA GT of Sears' translocation lines may be preferred as donors of Sr24 and Lr24 in locations where red seeded wheat is preferred.In countries such as Australia and India where white seededness is considered a key aspect of quality or marketing advantage the use of these resistance genes was made possible only by their separation from R Ag .In hindsight, mutation of R Ag in an appropriate derivative lacking R2 and R3 from Agent or in other Sears' derivatives other than CS 3D-3Ag#3 and CS 3D-3Ag#14 could have achieved the same result.

Plant materials
The materials used in this study included four common wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42) cultivars Aroona, Chinese Spring, Avocet S and Schomburgk, and four cultivars carrying Lr24 and Sr24, namely Agent, Janz, Sunco and Amigo.Agent is a hard red winter wheat cultivar carrying a spontaneous wheat-Th.ponticum translocation T3DS.3DL-3AgL 1 .Janz and Sunco are white-seeded Australian wheat cultivars with CS 3D-3Ag#3 and CS 3D-3Ag#14 translocations, respectively, as donors of Sr24 and Lr24.The red-seeded wheat cultivar Amigo with a T1AL.1RSRobertsonian translocation also carried a 3Ag chromosome segment, but as a T1BL.1BS-3AgLtranslocation 9 .The above materials are maintained at the Plant Breeding Institute, The University of Sydney.

Chromosome preparation
Slides for examination of mitotic metaphase chromosomes of Agent, Janz, Sunco and Amigo were prepared according to the procedure in Lang et al. 26 with minor modifications.

Genomic in situ hybridization (GISH)
Sequential GISH was performed after stripping off the oligo probes in 50% FA/1 × SSC and 50% FA/0.5 × SSC for 10 min each at 42 °C.GISH followed the procedure of Zhang et al. 28 .Total genomic DNA of Ps. stipifolia (StSt, 2n = 2x = 14, PI 314058, The National Small Grains Collection, USDA-ARS; kindly provided by Dr. L. Qi, USDA-ARS, ND) was used as probe, which was labelled with biotin-16-dUTP (Roche Diagnostics Australia, Castle Hill, NSW, Australia) using nick translation.Unlabeled total genomic DNA of CS wheat was used as blocker with a probe:blocker ratio of 1:120.Signals were detected with fluorescein-avidin DN (Vector Laboratories, Burlingame, CA).Chromosomes were counterstained with DAPI and pseudo-colored red.

Alien segment size and breakpoints in translocation lines
To estimate the size and breakpoints of the Th.ponticum 3AgL chromosomal segments in lines carrying Lr24 and Sr24, 10 cells at mitotic metaphase from each translocation line were photographed.Arm lengths were measured using ImageJ v2.0.0 software 29 , and the ratio between the length of 3AgL chromosomal segments and the long arm of translocation chromosome T3DS.3DL-3AgL in Agent was calculated as described by Endo and Gill 30 .
The position of the centromere was considered zero, and the terminal end of long arm of T3DS.3DL-3AgL in Agent was considered one.Standard error calculations for fraction length (FL) measurements were performed with the software Excel 2021 (IBM Corporation, New York).

PLUG marker analysis
Genomic DNA was extracted from young leaves of Aroona, CS, AvS, Schomburgk, Agent, Janz, Sunco, and Amigo.Twenty-four PCR-based landmark unique gene (PLUG) markers from the long arm of wheat homoeologous group 3 17 were selected and synthesized (Sigma-Aldrich, Inc., St. Louis, MO, USA).PCR were performed according to the procedure in Li et al. 31 .PCR products were digested with TaqI (at 65 °C) or HaeIII (at 37 °C) (Genesearch Pty Ltd, Arundel, QLD, Australia) for improving levels of polymorphism, and separated in 1.5% agarose gels.PLUG markers present in Agent, Janz, Sunco and Amigo but absent in Aroona, CS, AvS and Schomburgk were identified as chromosome 3AgL-specific.The physical locations of PLUG markers were searched in the reference genome sequence of CS (IWGSC RefSeq v.2.1; https:// urgi.versa illes.infra.fr/ blast/).

Ethical approval
We comply with relevant guidelines and legislation regarding the sample collection and use in the present study.
All materials in the present study are not endangered.

Figure 2 .
Figure 2. Chromosomal locations of the red grain color gene R Ag and Lr24/Sr24 genes in chromosome arm 3AgL.Dashed lines separate chromosome 3AgL-specific molecular markers specific for Lr24/Sr24 and R Ag , respectively.Green, blue, and red colors indicate Th. ponticum chromosome 3AgL segments, and wheat chromosomes 3D and 1B, respectively.+/+ (R) indicates that the line carries Lr24 and Sr24.Red and white, grain colors.

Table 2 .
Sequences of seven pairs of Thinopyrum ponticum chromosome arm 3AgL-specific PLUG primers.