Using the 6RLKu Minichromosome of Rye (Secale cereale L.) to Create Wheat-Rye 6D/6RLKu Small Segment Translocation Lines with Powdery Mildew Resistance

Long arms of rye (Secale cereale L.) chromosome 6 (6RL) carry powdery mildew resistance genes. However, these sources of resistance have not yet been successfully used in commercial wheat cultivars. The development of small segment translocation chromosomes carrying resistance may result in lines carrying the 6R chromosome becoming more commercially acceptable. However, no wheat-rye 6RL small segment translocation line with powdery mildew resistance has been reported. In this study, a wheat-rye 6RLKu minichromosome addition line with powdery mildew resistance was identified, and this minichromosome was derived from the segment between L2.5 and L2.8 of the 6RLKu chromosome arm. Following irradiation, the 6RLKu minichromosome divided into two smaller segments, named 6RLKumi200 and 6RLKumi119, and these fragments participated in the formation of wheat-rye small segment translocation chromosomes 6DS/6RLKumi200 and 6DL/6RLKumi119, respectively. The powdery mildew resistance gene was found to be located on the 6RLKumi119 segment. Sixteen 6RLKumi119-specific markers were developed, and their products were cloned and sequenced. Nucleotide BLAST searches indicated that 14 of the 16 sequences had 91–100% similarity with nine scaffolds derived from 6R chromosome of S. cereale L. Lo7. The small segment translocation chromosome 6DL/6RLKumi119 makes the practical utilization in agriculture of powdery mildew resistance gene on 6RLKu more likely. The nine scaffolds are useful for further studying the structure and function of this small segment.


Introduction
It has already been reported that the long arms of rye (Secale cereale L.) chromosome 6 (6RL) carry powdery mildew resistance gene Pm20 [1], and this gene was introduced into wheat background in the form of a 6BS/6RL translocation chromosome [1]. The gene Pm20 still has a broad spectrum of resistance to Blumeria graminis f. sp. tritici (Bgt) isolates [2,3]. The 6RL chromosome arm that carries Pm20 was derived from S. cereale L. cv. Prolific [1]. Recently, some reports indicated that 6RL arms derived from S. cereale cv. Jingzhouheimai, S. cereale cv. German White, and S. cereale cv. Kustro also carried powdery mildew resistance genes [3][4][5][6]. It has already been established that the powdery mildew resistance gene on 6RL of German White was different from the gene Pm20 [3], and this indicates that different 6RL arms may also display genetic diversity for powdery mildew resistance genes. However, the powdery mildew resistance genes on 6RL arms have not been successfully used in commercial wheat cultivars because of agronomic disadvantages, possibly caused by non-compensation and linkage drag of the 6RL arm. The development of small segment translocations between wheat chromosomes and 6RL, which causes minimal loss of indispensable wheat genes, may resolve this problem [3,4,7,8]. Only one translocation chromosome carrying a small segment of a 6RL arm, Ti4AS.4AL-6RL-4AL, has been reported. In this case, the 6RL segment was derived from the telomeric region and carried the Hessian fly-resistant gene H25 [9,10]. So far, no wheat-rye 6RL small segment translocation lines with powdery mildew resistance have been reported. In this study, a wheat-rye 6RL Ku minichromosome addition line was developed, and 6D/6RL Ku small segment translocation lines were identified from the irradiated seeds of this minichromosome addition line.

Transmission of 6RL Ku Minichromosome
Thirty-four seeds were randomly selected from the self-fertilized progeny of line MiA6RL Ku for ND-FISH analysis. Among the 34 plants, 24 had no 6RL Ku minichromosome; nine contained one 6RL Ku minichromosome; and one plant, 13FT104-7, contained a pair of this minichromosome ( Figure 1C). From the self-fertilized progeny of line 13FT104-7, 100 seeds were randomly selected for ND-FISH analysis; 25 plants contained two 6RL Ku minichromosomes, 62 plants contained one 6RL Ku minichromosomes, and the remaining 13 plants had none of this minichromosome. The progeny of 13FT104-7 were named 15T154, and some of these plants were used for developing additional 6RL Ku minichromosome-specific markers and producing wheat-rye small segment translocations.

Development of 6D/6RL Ku Small Segment Translocation Lines
Some seeds that were derived from line 14T154-35 with two 6RL Ku minichromosomes were exposed to 60 Co-γ rays. A total of 1428 M1 seeds were analyzed using ND-FISH, and ten wheat-rye 6D/6RL Ku small segment translocation lines were detected and named 16T379 or 16T380 (Table 1, Figure 2). In these 6D/6RL Ku small segment translocation lines, the 6RL Ku minichromosome divided into two smaller segments. One small segment, with the Oligo-pSc200 and Oligo-pSc250 signals and a strong Oligo-pSc119.2-1 signal, was named 6RL Kumi119 . The other small segment, with the Oligo-pSc200 and Oligo-pSc250 signals and a weak Oligo-pSc119.2-1 signal, was named 6RL Kumi200 . The segment 6RL Kumi200 had been translocated onto the 6DS arm, while the segment 6RL Kumi119 was translocated onto the 6DL arm ( Figure 2). The small segment translocation chromosome fused with 6DS was named 6DS/6RL Kumi200 , and the other translocation attached with 6DL was named 6DL/6RL Kumi119 ( Figure 2).

Marker
Forward (

Sequence Characteristics of the Products Amplified by the 16 Markers
The sequences amplified by the 16 6RL Kumi119 -specific markers were isolated. These sequences have been deposited in the GenBank Database (GenBank accession numbers: MK051036-MK051051). Sequence alignment using the BLAST tool in NCBI and gene annotation indicated that all the 16 sequences did not belong to any gene sequence; therefore, they are probably not involved in active genes. Analysis using RepeatMasker software (http://repeatmasker.org/) also indicated that these sequences were not involved in repetitive DNA sequences. Nucleotide BLAST searches indicated that 14 of the 16 sequences had 91-100% similarity with the scaffolds from the 6R chromosome of S. cereale L. Lo7, and the other two sequences had 99-100% similarity with the scaffolds derived from 0R of S. cereale L. Lo7 scaffolds ( Table 2). It can be noted that all the five sequences (MK051040, MK051042, MK051044, MK051045, and MK051047) were located in the scaffold Lo7_v2_scaffold_445202 6R, and both MK051050 and MK051051 were located in the scaffold Lo7_v2_scaffold_445253 6R (Table 2). According to the gene annotation of these scaffolds published by Bauer et al. [12], they did not contain genes; however, these scaffolds were still useful for the future study on the structure and function of the 6RL Kumi119 segment.

Powdery Mildew Resistance
Powdery mildew resistance testing indicated that parental wheat MY11, lines without 6RL Ku minichromosome, and lines with only one 6DS/6RL Kumi200 chromosome were highly susceptible to powdery mildew ( Figure 6). Lines with both the 6DS/6RL Kumi200 and 6DL/6RL Kumi119 chromosomes and lines with only one 6DL/6RL Kumi119 chromosome displayed high resistance to powdery mildew ( Figure 6). These results indicated that the powdery mildew resistance gene on 6RL Ku was located on the small segment 6RL Kumi119 .

Extending Genetic Basis of Powdery Mildew Resistance Genes
Several reports have highlighted the current narrowness of genetic diversity of powdery mildew resistance genes in wheat breeding programs in China [13][14][15]. For example, the wheat cultivars (lines) from the Yangtze River region mainly contained genes Pm4a and Pm21 [13]. The gene Pm21 is widely used in wheat cultivars from Sichuan and Guizhou provinces [14], while the wheat cultivars (lines) from Henan province mainly carry genes Pm2, Pm4, Pm21and unknown gene(s) located on a new 1RS.1BL translocation chromosome [15]. It should be noted that gene Pm21 has played an important role in wheat powdery mildew resistance breeding programs in China, but there is the risk of pathogen directional selection caused by the high frequency of Pm21 gene in wheat breeding programs [15]. Therefore, extending the genetic basis of powdery mildew resistance genes in wheat breeding programs in China must be an urgent priority. 6R chromosomes of rye (S. cereale L.) carry powdery mildew resistance genes, and still display effective resistance to powdery mildew pathotypes in China [1][2][3][4][5][6]16]. In addition, the powdery mildew resistance genes on 6R chromosomes have genetic diversity [3,16], which may provide recipient wheat lines with resistance to future virulent powdery mildew races that might defeat existing genes. However, these 6R-derived powdery mildew resistance genes have not been successfully used in commercial wheat cultivars because of the loss of indispensable wheat genes and alien chromosome-associated linkage drag. Therefore, the development of new wheat-rye 6RL small segment translocation chromosomes with powdery mildew resistance gene(s) is of pressing concern [3,4,8]. It has been reported that few recombinants (sometime none) can be recovered between 6R and wheat chromosomes, even in a ph1b mutant background. Furthermore, it is difficult to produce suitable wheat-rye 6RL small segment translocations [17]. In this study, a wheat-rye small segment translocation chromosome 6DL/6RL Kumi119 with powdery mildew resistance was developed using a 6RL Ku minichromosome. According to the fraction length standard of the 6RL arm constructed by Mukai et al. [11], the size of the 6RL Kumi119 segment might be slightly larger than the small 6RL segment in the Ti4AS.4AL-6RL-4AL translocation chromosome, in which the 6RL segment occupied 10% of the total length of 6RL [11]. Therefore, the occurrence of possible deleterious genes on the 6RL Ku arm may have been greatly reduced. Additionally, the 6DS/6RL Kumi200 and 6DL/6RL Kumi119 chromosomes showed high transmission to progeny. These characteristics make the successful utilization of the powdery mildew resistance gene on 6RL Ku more likely, although the agronomic traits of the lines with 6DS/6RL Kumi200 and 6DL/6RL Kumi119 chromosomes have not been investigated.

6RL Ku Minichromosome Specific Markers
For the effective utilization of elite genes in wild germplasm, it is necessary to increase our understanding of the molecular basis of target alien chromosome regions [18,19]. In this study, the small segment translocation chromosome 6DL/6RL Kumi119 provides an opportunity to understand the molecular basis of the small segment 6RL Kumi119 carrying powdery mildew resistance. Additionally, 16 6RL Ku minichromosome-specific markers were developed, and these markers were subsequently located onto the segment 6RL Kumi119 with powdery mildew resistance. These markers represent convenient tools to use the 6DL/6RL Kumi119 translocation chromosome in wheat breeding programs. Using the sequences amplified by the 16 markers, 11 scaffolds derived from winter rye inbred line S. cereale L. Lo7 [12] were located on the segment 6RL Kumi119 . These scaffolds are useful for the further detailed studying of this segment. However, published data indicated that no active genes could be found among the 11 scaffolds [12]. There are two possible explanations for this phenomenon. First, the 6RL Ku minichromosome-specific markers may be insufficient in number. Second, large gaps may exist in these scaffolds. Therefore, a greater number of 6RL Ku minichromosome-specific markers are required and more complete rye genomic sequences are needed.

Plant Materials
A monotelosomic addition line MTA6RL Ku was developed according to the methods described by Qiu et al. [20]. From the self-pollinated progeny of MTA6RL Ku , a 6RL Ku minichromosome addition line MiA6RL Ku was identified. Some seeds selected from the progeny of MiA6RL Ku were irradiated with 60 Co-γ rays at the Biotechnology and Nuclear Technology Research Institute, Sichuan Academy of Agricultural Sciences, China. Common wheat (Triticum aestivum L.) Mianyang 11 (MY11) and Chinese Spring (CS) were used as controls.

Cytological Analysis
The root-tip metaphase cells were analyzed using non-denaturing fluorescence in situ hybridization (ND-FISH) technology. Oligonucleotide (oligo) probes Oligo-1162, Oligo-pSc200, Oligo-pSc250, Oligo-pSc119.2-1, and Oligo-pTa535-1 [21,22] were used. These oligo probes were 5 end-labelled with 6-carboxyfluorescein (6-FAM) or 6-carboxytetramethylrhodamine (Tamra). Root-tip metaphase chromosomes were prepared following the methods described by Han et al. [23]. ND-FISH analysis was performed following the methods described by Fu et al. [22] with the following minor modification. When dropped onto cell spreads, the probe mixture containing Oligo-1162 around the slides was kept above 28 • C, and the slides were immediately placed into a moist box that was incubated at 42 • C in advance for 1-2 h. Then, the slides were washed 15-20 s in 2 × SSC at 42 • C.
The pair-end reads with high wheat homology were discarded. At last, after comparing specific pair-end reads of Kustro and MiA6RL Ku , the 6RL Ku minichromosome-specific pair-end reads were obtained. Primers were designed according to the 6RL Ku minichromosome-specific pair-end reads using the software Primer 3 (version 4.0). The optimal melting temperature and size values of primers were set following the methods described by Duan et al. [24].

PCR Analysis and Sequence Cloning
The PCR amplifications were carried out according to the procedure described by Li et al. [6]. Agarose gel (2%) electrophoresis was used to detect the amplicons in 1 × TAE buffer. The products amplified by the 6RL Kumi119 -specific primers were recovered using Universal DNA Purification Kit (Tiangen Biotech Co., Ltd, Beijing, China) and cloned using pClone007 Vector Kit (TsingKe Biotech Co., Ltd, Beijing, China). Inserts were sequenced by TsingKe Biotech (Beijing) Co., Ltd. These sequences were deposited in the GenBank Database. Finally, these sequences were used for Nucleotide BLAST searches against the S. cereale L. Lo7 scaffolds database using the BLAST tool in GrainGenes (https://wheat.pw.usda. gov/cgi-bin/seqserve/blast_rye.cgi) and gene annotation was carried out according to the data sets related to the publication by Bauer et al. [12]. Additionally, RepeatMasker software (http://repeatmasker.org/) was used to identify whether these sequences amplified by the 6RL Kumi119 -specific primers are repetitive DNA.

Powdery Mildew Resistance Test
The resistance of line MiA6RL Ku , the progeny of MiA6RL Ku , 6D/6RL Ku small segment translocation lines, and parental wheat MY11 to powdery mildew were evaluated. Plants were grown in Qionglai, Sichuan, China. The materials were naturally inoculated by locally occurring field derived powdery mildew, and infection types (IT) were scored according to the standard described by Fu et al. [5].

Conclusions
In conclusion, a new wheat-rye small segment translocation chromosome 6DL/6RL Kumi119 with powdery mildew resistance was produced. Sixteen 6RL Kumi119 specific markers were developed, and 11 S. cereale L. Lo7 scaffolds were located on the small rye segment 6RL Kumi119 . The small segment translocation chromosome 6DL/6RL Kumi119 makes the practical utilization of the powdery mildew resistance gene on 6RL Ku more likely. The 6RL Kumi119 -specific markers and the S. cereale L. Lo7 scaffolds that were located on the 6RL Kumi119 segment may find application in future detailed studies of the structure and function of this small segment.