Physical Location of New PCR-Based Markers and Powdery Mildew Resistance Gene(s) on Rye (Secale cereale L.) Chromosome 4 Using 4R Dissection Lines.

Rye (Secale cereale L.) 4R chromosome contains elite genes that are applicable for wheat (Triticum aestivum L.) cultivar improvement. PCR-based 4R-specific markers can benefit the detection of elite genes on 4R in wheat backgrounds. In this study, a new fluorescence in situ hybridization (FISH) map of the 4RKu chromosome of rye Kustro has been constructed. A set of 4RKu dissection lines was obtained and 301 new 4RKu-specific markers were developed using specific length amplified fragment sequencing (SLAF-seq) technology. These markers were combined with the 99 4RKu-specific markers previously developed, and were physically mapped to 4RKu chromosome using the new FISH map and the 4RKu dissection lines. A total of 338 of the 400 markers have been successfully mapped to six regions of 4RKu chromosome. Additionally, the powdery mildew resistance gene(s) on the 4RLKu arm was located to the segment between L.4 and L.8, the same region where 115 4RLKu-specific markers were mapped. The markers developed in this study can be used to identify a specific segment of 4R chromatin in wheat backgrounds, help construct a high-density physical map of 4R chromosome, and facilitate the utilization of elite genes on 4R chromosome in wheat breeding programs.

Development of new PCR-based markers specific for chromosome arms of rye (Secale cereale L.).

PCR-based rye (Secale cereale L.) chromosome-specific markers can contribute to the effective utilization of elite genes of rye in wheat (Triticum aestivum L.) breeding programs. In the present study, 578 new PCR-based rye-specific markers have been developed by using specific length amplified fragment sequencing (SLAF-seq) technology, and 76 markers displayed different polymorphism among rye Kustro, Imperial, and King II. A total of 427 and 387 markers were, respectively, located on individual chromosomes and chromosome arms of Kustro by using a set of wheat-rye monosomic addition lines and 13 monotelosomic addition lines, which were derived from T. aestivum L. 'Mianyang11' × S. cereale L. 'Kustro'. In addition, two sets of wheat-rye disomic addition lines, which were derived from T. aestivum L. var. Chinese Spring × S. cereale L. var. Imperial and T. aestivum L. 'Holdfast' × S. cereale L. var. King II, were used to test the chromosomal specificity of the 427 markers. The chromosomal locations of 281 markers were consistent among the three sets of wheat-rye addition lines. The markers developed in this study can be used to identify a given segment of rye chromosomes in wheat background and accelerate the utilization of elite genes on rye chromosomes in wheat breeding programs.

[Identification of the 1RS-7DS.7DL wheat-rye small segment translocation lines].

Rye (Secale cereale L., RR) is a valuable genetic resource for the improvement of common wheat (Triticum aestivum L., AABBDD). Transferring alien rye genes into wheat by distant hybridization and automatic chromosome doubling is an important and efficient method to boost agronomic traits, disease resistance and widening the gene pool in wheat. In this study, an octoploid triticale CD-13 (AABBDDRR) was obtained via automatic chromosome doubling by crossing landrace Penganbaimaizi (T. aestivum L., AABBDD) and rye "Qinling rye" (S. cereale cv. Qinling, RR). GISH and FISH analyses indicated that CD-13 contained a 1RS-7DS.7DL wheat-rye small segment translocation chromosome. In order to transfer the 1RS-7DS small segment translocation into hexaploid wheat, 58 lines of the F5 inbred population from the cross CD-13 x Chuanmai 42 were screened for rye chromosome segments by GISH and FISH analyses. The results showed that 13 lines contained the 1RS-7DS.7DL small segment translocation chromosome by reciprocal translocation between 1RS and 7DS. These translocation lines carrying 1RS small rye alien segment were tested for the translocation breakpoints and the presence of a storage protein locus Sec-1. The Sec-1 locus was absent in the line 811, a stable 1RS-7DS.7DL small segment translocation line. The translocation breakpoint of 1RS-7DS.7DL of this line was located in the interval of IB267-IAG95 around the telomere of 1RS chromosome. Thousand-kernel weight of the line 811 was much higher than the parent CD-13, but not significantly different from Chuanmai 42. This indicated that 1RS-7DS.7DL small segment translocation had no negative effect on thousand-kernel weight in the genetic background of Chuanmai 42. The line with 1RS-7DS.7DL translocation chromosomes can be used as a new genetic material for further studies of valuable genes and their genetic effect on 1RS small segment.

Abnormal mitosis induced by wheat-rye 1R monosomic addition lines.

Octoploid triticale were derived from common wheat (Triticum aestivum L. 'Mianyang11') × rye (Secale cereale L. 'Kustro'), and some progeny were obtained by the backcrossing of triticale with 'Mianyang11' followed by self-fertilization. In situ hybridization using rye genomic DNA and repetitive sequences pAs1 and pSc119.2 as probes was used to analyze the mitotic chromosomes of these progeny. Three wheat-rye 1R monosomic addition lines and a wheat line (12FT-1685) containing a 1R and a 1BL.1RS translocation chromosome were identified. Abnormal mitosis was observed in the two lines. During mitosis of a 1R monosomic addition line (3-8-20-1R-2), lagging chromosomes, micronuclei, chromosomal bridges, and the one pole segregation of 1R chromosome were observed. Abnormal mitotic behaviour of chromosomes was also observed in some of the self-progeny plants of lines 12FT-1685 and 3-8-20-1R-2. These progeny contained 1R chromosome or 1R chromosome arm. In addition, 4B chromosomes were absent from one of the progeny of 3-8-20-1R-2. This abnormal mitotic behaviour of chromosomes was not observed in two other 1R monosomic addition lines. These results indicate that a single 1R chromosome added to wheat might cause abnormal mitotic behaviour of both wheat and rye chromosomes and different genetic variations might occurr among the sibling 1R monosomic addition lines.

[Establishment of wheat-rye addition lines and De Novo powdery mildew resistance gene from chromosome 5R].

The F5 plants derived from octoploid triticale × common wheat were investigated by FISH methods using repetitive DNA sequences pAS1 and pSc119.2 as probes. The disease resistance of these plants was also screened and evaluated in the field. The 1R, 2R, 3R, 4R, 5R, 6R and 7R monosomic addition lines and 1R and 2R disomic addition lines were found. The occurrence frequencies of chromosomes 1R and 4R addition lines were higher than that of chromosomes 2R, 3R, 5R, 6R, and 7R addition lines in the high generation screened. The 5R and 6R monosomic addition lines were immune to powdery mildew. The chromosome 5R in this study might carry new powdery mildew resistance gene(s). In addition, the preferential elimination of chromosome 4B was observed in several plants.

Diversity and evolution of four dispersed repetitive DNA sequences in the genus Secale.

We present the first characterization of 360 sequences in six species of the genus Secale of both cultivated and wild accessions. These include four distinct kinds of dispersed repetitive DNA sequences named pSc20H, pSc119.1, pSaO5(411), and pSaD15(940) belonging to the Revolver family. During the evolution of the genus Secale from wild to cultivated accessions, the pSaO5(411)-like sequences became shorter mainly because of the deletion of a trinucleotide tandem repeating unit, the pSc20H-like sequences displayed apparent homogenization in cultivated rye, and the second intron of Revolver became longer. In addition, the pSc20H-, pSc119.1-, and pSaO5(411)-like sequences cloned from wild rye and cultivated rye could be divided into two large clades. No single case of the four kinds of repetitive elements has been inherited by each Secale accession from a lone ancestor. It is reasonable to consider the vertical transmission of the four repetitive elements during the evolution of the genus Secale. The pSc20H- and pSaO5(411)-like sequences showed evolutionary elimination at specific chromosomal locations from wild species to cultivated species. These cases imply that different repetitive DNA sequences have played different roles in the chromosome development and genomic evolution of rye. The present study adds important information to the investigations dealing with characterization of dispersed repetitive elements in wild and cultivated rye.

Variations of tandem repeat, regulatory element, and promoter regions revealed by wheat-rye amphiploids.

To better understand the evolution of allopolyploids, 4 different combinations between wheat (Triticum aestivum L.) and rye (Secale cereale L.) including 12 F1 hybrids and 12 derived amphiploids were analyzed and compared with their direct parental plants by PCR analysis using 150 wheat SSR (single sequence repeat) markers and by FISH analysis using a rye-specific repetitive sequence (pSc200) as a probe. Nine SSR markers amplified rye-specific fragments whose sizes ranged from 471 bp to 1089 bp. These fragments contain regulatory elements and (or) promoters. Some of these fragments were amplified from all 24 progenies, while others were amplified from a subset of the progenies. The disappearance of rye-specific fragments from some progenies was caused by sequence elimination or DNA modification. Marker Xgwm320 amplified a new fragment (403 bp), a rye-specific tandem repeat, from some of the progenies. Twenty-eight SSR markers displayed microsatellite variation in progenies derived from 'Chinese Spring' x 'Jinzhou-heimai', but none of the 150 SSR markers displayed microsatellite variation in the progenies derived from the other three combinations. FISH signals of pSc200 were eliminated from one telomere/subtelomere of 4 chromosomes of 'Kustro' during allopolyploidization and expanded in amphiploids derived from 'Chinese Spring' x 'AR106BONE'. Thus, allopolyploidization in wheat-rye can be accompanied by rapid variation of tandem repeats, regulatory elements, and promoter regions. The alterations of repetitive sequence pSc200 indicate coordination between the constituent genomes of the newly formed amphiploids. Different genetic backgrounds of parents appear to affect genome changes during allopolyploidization.

[Variation of Rye-Specific repetitive DNA pSc119.1 among sister T1RS.1BL translocations].

Specific primers were designed according to the rye-specific repetitive sequence pSc119.1 and were used for polymerase chain reaction (PCR) analysis using the genomic DNA of two sets of sister T1RS.1BL translocations, CN12, CN17, CN18, 96 I 176-1 and 96 I 176-3 as templates. The results indicated that the target fragments were amplified from CN12, CN17, and CN18. A target and a non-target fragment were produced from 96 I 176-1. However, no products were obtained from 96 I 176-3. Southern blot analysis indicated that the elimination of pSc119.1 did not occur in line 96 I 176-3. Three target fragments were cloned from CN12, CN17, and CN18 respectively through recovering. For each target fragment, ten clones were selected randomly for sequencing. Variation of the sequence pSc119.1 was observed in all of the three wheat lines and line CN18 had the most obvious variation. Most of the 30 sequences had 94% or 95% similarity with the sequence pSc119.1 published and the variation of bases of these sequences. Most variations of most bases arose from transition, and a few of them were transversion. Furthermore, there was great coherence among these changed bases in type and site. The evolution process of progenies of wide hybrids may be continuous. For each set of sister 1RS.1BL translocation, difference of some traits was observed among the wheat lines or cultivars. The difference was probably related to the variation of the repetitive DNA. This research provides some useful information for studying on mechanism of epigenetics.

[Transfer of a rye small chromosomal segment with powdery mildew-resistant gene(s) into common wheat (Triticum aestivum L.)].

The advanced progeny lines (BC1F5) from the monosomic addition lines between common wheat cultivar Mianyang 11, which is highly susceptible to powdery mildew, and an inbred rye line R12 were analyzed for selection of wheat-rye translocations. Based on a rye-specific repetitive sequence of pSc20H, which spread over all chromosomes of rye but did not existed in wheat, a set of PCR primer was designed and used to identify the rye chromosome segments in wheat. From 300 of the BC1F5 progeny lines 70 were found to contain chromosome composition of rye. An advanced line, 96II691-830-98, originated from 6R monosomic addition line was observed to be immune to powdery mildew, different from its wheat parent Mianyang 11. A small segment of rye chromosome at telomere in a pair of wheat chromosome in the line was found by means of GISH. The results indicated that a small segment of rye chromosome 6R carrying the gene(s) for resistance to powdery mildew has been transferred into common wheat. In the progeny of monosomic addition lines a high frequency of wheat-alien species translocation with various segments of chromosomes could be found by application of both PCR and GISH technique.