Molecular markers and cytogenetics to characterize a wheat-Dasypyrum villosum 3V (3D) substitution line conferring resistance to stripe rust

Dasypyrum villosum has been used as a valuable gene resource for disease resistances, yield increase and quality improvement in wheat. A novel wheat-D. villosum alien introgression line CD-3 was generated through hybridization between the common wheat Chinese Spring (CS) and a CS- D. villosum 3V addition line having considerably high stripe rust resistance, which enable the characterization of a potential new stripe rust resistance gene (s) derived from D. villosum. The results of non-denaturing fluorescent in situ hybridization (ND-FISH) showed that CD-3 contained 42 chromosomes, including a 3V chromosome pair, and the absence of both of the 3D chromosomes. PCR-based Landmark Unique Gene (PLUG) molecular marker analysis supported results from the FISH analysis, revealing CD-3 was a wheat-D. villosum 3V (3D) disomic substitution line. Resistant test of stripe rust on 52 plants of F2 generation (CD-3/CS), CD-3, CS and D.villosum have been conducted at seedling stage. 7 plants of F2 generation possessing two 3V chromosomes exhibited high resistance to stripe rust as CD-3 and D.villosum, 10 plants carrying one 3V chromosome and 35 plants without 3V chromosome were susceptive to stripe rust as CS. The result implied the high stripe rust resistance of CD-3 should be controlled by recessive gene(s) originating from D.villosum. To rapidly detect chromosome 3V in the genetic background of wheat, we developed a novel Sequence Characterized Amplified Region (SCAR) marker specific for 3V chromosome based on the sequence of a grain size-related gene DvGS5 in D. villosum, an orthologue of TaGS5 from wheat. The SCAR marker was designated DvGS5-1443, which could successfully amplify a unique 3V-specific fragment in CD-3 and D. villosum, suggesting that this SCAR marker could facilitate targeting the chromosome 3V in the genetic background of wheat for wheat improvement.


Introduction
Stripe (or yellow) rust, caused by Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst), is considered one of the most dangerous diseases of wheat (Triticum aestivum L.) worldwide, and also in China [1]. It was estimated to commonly reduce crop yield by 10-70%, and even up to 100% [2]. The development of disease-resistant wheat cultivars has been suggested to be the most effective, economical, and environmentally friendly strategy to control stripe rust [3,4]. To date, numerous Yr (yellow rust) genes have been identified, and they have been officially designated as Yr1-Yr78 [5]. However, concerning the coevolution of plants and pathogens, many of the extensively used Yr genes, such as Yr9 and Yr26, did not confer adequate resistance to newly emerging Pst strains [6]. Therefore, there is an urgent need for exploring and identifying novel and effective resistance genes against newly emerged Pst strains. The progenitors and relatives of crops are immensely valuable for modern agriculture, by providing a wide diversity of desirable genetic resources for plant breeding [7]. In particular, a substantial body of evidence supports that wild relatives of wheat constitute a valuable gene pool for disease resistance in wheat [8][9][10]. For example, chromosome arm 1RS of rye harboring powdery resistant genes (Pm8 and Pm17) and rust resistance genes (Sr31, Lr26 and Yr9) [11][12], and 6VS arm of Dasypyrum villosum carrying powdery mildew resistant gene (Pm21) [13] are prevalent in wheat commercial cultivars.
Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum L. Candargy, 2n = 2x = 14, VV) is of interest as a genetic germplasm source, possessing many agronomically important traits for wheat improvement, such as tolerance to biotic and abiotic stresses, and high nutritional and bread-making quality [14][15]. Since the development of the first set (#1) of Triticum-Dasypyrum alien lines [16], the chromosomes of different D. villosum accessions have been introduced into wheat (set #2 to set #4) [17]. The desirable genes present on V genome chromosomes have been identified and characterized in the genetic background of wheat. For example, the well-known powdery mildew resistant gene Pm21, located on 6VS, has been cloned [18][19] and further used in wheat breeding [20]. However, limited progress has been made in the exploration of stripe rust resistant gene (s) in D. villosum [21].
An effective strategy for utilizing plant genetic resources by employing conventional breeding, molecular genetics, and transformation is gaining ground nowadays [22]. In this study, using functional molecular markers and cytogenetic methods, we characterized a new wheat-D. villosum 3V (3D) substitution line CD-3 showing high resistance to stripe rust.

Plant materials
D. villosum accession PI 257477 (genome VV, 2n = 2x = 14) was obtained from the National Genetic Resources Program, United States Department of Agriculture. Chinese Spring (CS)-D. villosum addition lines (# 3) Additions 1V, 2V, and 4V-7V were provided by the School of Life Science and Technology, University of Electronic Science and Technology of China (the D. villosum accession used to develop this set (#3) of additional line was TA10220). Line CD-3 and the sixty plants of CD-3 used for stripe rust test were the F 6 progeny derived from hybridization of CS and CS-D. villosum 3V addition line (# 3). F 2 population used for genetic analysis of resistance to stripe rust was derived from crosses between CS and CD-3. Other accessions, including common wheat cultivars CS, Chuanmai49, Chuanmai50, Chuanmai60, and rye cultivar JZHM were maintained by our laboratory.

Non-denaturing fluorescent in situ hybridization (ND-FISH) procedures
Root tips from 60 individual seedlings of CD-3 were collected, treated with nitrous oxide for 2h and fixed with 90% acetic acid for 8-10 min. Then, the root tips were washed quickly with  and 400 nmol of each primer. The amplification protocol as follows: initial denaturation at 94˚C for 3 min; followed by 35 cycles of denaturation at 94˚C for 1 min, annealing at 55˚C (dependent on different primer sets) for 1 min, extension at 72˚C for 2 min, and final extension at 72˚C for 10 min. The PCR products were separated on 2% (w/v) agarose gels, and visualized by EtBr staining.

Stripe rust resistance tests
The sixty plants of CD-3 identified by ND-FISH were grown in the field and used for stripe rust resistance tests. During the cropping seasons in 2014, 2015, 2016 and 2017, field tests were conducted in Pixian city, Sichuan, China. The mixed Pst strains, mainly consisting of CYR32, CYR33, and CYR34, were used to infect adult plants of CD-3, CS, and D. villosum. The infection type (IT) were recorded 18-20 days after inoculation. Genetic analysis of resistance to stripe rust was conducted on 52 plants of F 2 (CD-3/CS). After identified by ND-FISH, seeds were grown in small pots(5×5×5cm), and one pot was for one seed. Seedlings at two-leaf stage were inoculated with mixed urediniospores and were kept in dew chamber at 9-12˚C for 24 h without light. And then, the seedlings were transferred to rust-free greenhouse with daily cycle of 12 h of light and 12 h of dark at 11˚C-17˚C. The mixed Pst strains mentioned above were used to infect seedling plant. The infection types were recorded 15 days after inoculation.
Infection types were recorded on 1-9 scale as described by Line and Qayoum [30], where IT 0-3 were resistant, IT 4-6 were intermediate, and IT 6-9 were susceptible. The Pst strains used were provided by the Plant Protection Institute, Sichuan Academy of Agricultural Sciences, China, and the Plant Protection Institute, Gansu Academy of Agricultural Sciences, China.

Development of 3V-specific molecular markers
To monitoring the 3V chromosomes from D.villosum, we chose TaGS5 genes mapped on 3AS and 3DS of wheat for the developing the 3V-specific marker. Five primer pairs for TaGS5 genes (TaGS5-P5, TaGS5-P6, TaGS5-P7, TaGS5-P8, and TaGS5-P9), were used in this study, as described by Wang et al. [31]. PCR was conducted using a T100 TM Thermal cycler (Bio-RAD Laboratories). The reaction system and procedure were in accordance with the description of Wang et al. [31]. The amplified products were separated on 2% (w/v) agarose gels. D. villosum-specific bands were excised and purified using gel extraction kit (TianGen Biotech) following the manufacturer's instructions. After introducing the purified product into the vector pMD19-T (TaKaRa Biotechnology, Dalian, China) following the manufacturer's instructions, the modified vector was transformed into competent cells of Escherichia coli strain DH-5α. The obtained clones were screened by PCR using M 13 universal primers, and three positive clones were randomly chosen for double end sequencing at Shanghai Sangon Biotech Co., Ltd, Shanghai, China. Sequences obtained were assembled by DNAman (Lynnon Biosoft, San Ramon, CA, USA).Based on sequence alignment between DaGS5 and TaGS5, putative SCAR primers were designed based on the low-homology region using Primer Premier 5.0 (PRE-MIER Biosoft, Palo Alto, CA, USA), followed by synthesis at Shanghai Sangon Biotech Co., Ltd. PCR was conducted by a T100 TM Thermal cycler (Bio-RAD Laboratories), using a 25 μL reaction system, containing 2.5 μL of 10× buffer (50 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.3), 40-100 ng of genomic DNA, 200 nmol of each primer, and 1 U of Taq DNA polymerase (TianGen Biotech). The PCR protocol was as follows: initial denaturation at 94˚C for 5 min, 35 cycles of denaturation at 94˚C for 1 min, annealing at 56˚C for 0.5 min, extension at 72˚C for 0.5 min, and final extension at 72˚C for 10 min. The amplicons were separated on a 1% (w/v) agarose gel, and visualized by EtBr staining.

Chromosomal characterization
Sequential ND-FISH was conducted on the mitotic spread chromosomes of CD-3 using probes of Oligo-pSc119.2, Oligo-pTa535, Oligo-pHv62-1 and Oligo-(GAA) 7 (Fig 1A-1C). As shown in Fig 1D, we observed that all wheat and D. villosum chromosomes could be accurately distinguished using the probes mentioned above. The Oligo-pSc119.2 and Oligo-pTa535 probe combination could easily distinguish all 42 wheat chromosomes ( Fig 1A). As shown in Fig 1B, Oligo-pHv62-1 highlighted a pair of 3V chromosomes of D. villosum with strong hybridization signals on the terminal regions of both chromosomal arms, and a faint signal in the centromeric region. Oligo-(GAA) 7 was mainly located in centromeric or sub-terminal regions of the B genome of wheat ( Fig 1C). The chromosome number of CD-3 was 42, containing 40 wheat chromosomes and a pair of 3V chromosomes. By comparing the standard wheat karyotype obtained by the combined use of Oligo-pSc119.2 and Oligo-pTa535 as probes [26], we deduced that wheat chromosome 3D was absent in the line CD-3.

PLUG marker analysis
D. villosum was firstly analyzed by employing 30 PLUG markers specific for wheat homoeologous group 3 chromosomes. Five pairs (three were located on the short arm, and two were located on the long arm) generated stable, clear bands in CS, D. villosum, CD-3, and some CS-D. villosum addition lines (Table 1). Among the five PLUG primers pairs, three primer pairs (TNAC1248, TNAC1294 and TNAC1267) generated D. villosum-specific bands in both D. villosum and CD-3, excluding one fragment from CS, while the remaining two primers (TNAC1301 and TNAC1277) amplified all fragments from D. villosum and CS (Fig 2). By comparing the CS band pattern with the standard bands obtained in nullisomic-tetrasomic lines of CS using the same primer pairs (TNAC1294 and TNAC 1267) described by Ishikawa et al. (2009), we observed that the fragment absented in CD-3 belonged to chromosome 3D.
These results showed that CD-3 contained the D. villosum 3V chromosome, and had lost a pair of 3D chromosomes.

Evaluation of stripe rust resistance
After field resistance evaluations were conducted in four successive seasons, we found that all 60 individuals of CD-3 and the D. villosum plants were highly resistant to the epidemic Pst strains, including CYR32, CYR33 and CYR34, whereas the recipient parent CS was susceptible (Fig 3A-3C). The resistance tests on 52 plants of F 2 were conducted at seedling stage. We observed that 7 plants possessing two 3V chromosomes were highly resistant to stripe rust, 10 plants carrying one 3V chromosome and 35 plants without 3V chromosome were susceptible to stripe rust (Fig 3D-3J). These results showed that stripe rust resistance of CD-3 probably originated from recessive gene(s) on the 3V chromosome of D. villosum.

Isolation of GS5 gene from D. villosum and development of 3V-specific marker
The yield-related gene TaGS5, present in 3AS and 3DS of common wheat, was used in this study for developing a 3V-specific SCAR marker. PCR analysis was conducted for CS, D. villosum and CD-3 using five primer pairs for TaGS5 genes (TaGS5-P5, TaGS5-P6, TaGS5-P7, TaGS5-P8, and TaGS5-P9) as described by Wang et al.

Discussion
Due to its resistance to several serious wheat diseases, including powdery mildew, rust, eyespot, take-all, and so on [17], D. villosum has been extensively used as a valuable genetic resource for wheat improvement. In the past few decades, D. villosum chromatin from several  [9]. To date, a total of 78 stripe rust resistance genes in wheat have been officially designated (Yr1-Yr78). Among them, Yr30 and Yr57 have been mapped on chromosome 3B [40][41], Yr45 and Yr71 has been placed on 3D [42][43], and Yr76 has been mapped on 3A [44]. The resistance genes mentioned above, located on the Triticeae homoeologous group 3 chromosomes, all originated from hexaploid landraces. No previous studies on stripe rust resistance gene(s) present on chromosome 3V originating from the relative of wheat, D. villosum, have been reported.    chromosome 3V of CD-3 probably carried high degree of stripe rust resistance which should be controlled by recessive gene(s), and the resistance gene(s) could function in the genetic background of wheat. Therefore, CD-3 should be considered as a valuable resource for further exploration and utilization in wheat breeding. PCR-based species-specific markers have proven effective tools to monitor alien chromatin harboring valuable genes in the genetic background of wheat [46]. To date, continuing efforts have been made to develop V genome-specific SCAR markers, as well as a few V chromosome-specific SCAR markers [47][48][49][50]. However, no studies on the development of 3V-specific SCAR markers have been reported. In this study, we identified a 155-bp insertion into DvGS5, an orthologue of TaGS5, locating on 3AS and 3DS of common wheat. This helped us in developing a marker to target 3V chromosome in the further transferring of 3V chromosome carrying stripe rust resistance to various wheat genetic background. Based on the polymorphism between DvGS5 and TaGS5 we developed a SCAR marker, designated DvGS5-1443, which could generate a 443-bp band specific to 3V chromosome. We demonstrated that it could indicate the presence or absence of the 3V chromosome in the background of wheat reliably, easily and efficiently. Therefore, it could be used as an efficient tool for monitoring the D. villosum 3V chromosome carrying stripe rust resistant gene (s) for use in wheat breeding programs.