Benju Yan

Alterations and abnormal mitosis of wheat chromosomes induced by wheat-rye monosomic addition lines.

Background Wheat-rye addition lines are an old topic. However, the alterations and abnormal mitotic behaviours of wheat chromosomes caused by wheat-rye monosomic addition lines are seldom reported. Methodology/Principal Findings Octoploid triticale was derived from common wheat T. aestivum L. ‘Mianyang11’×rye S. cereale L. ‘Kustro’ and some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ followed by self-fertilization. Genomic in situ hybridization (GISH) using rye genomic DNA and fluorescence in situ hybridization (FISH) using repetitive sequences pAs1 and pSc119.2 as probes were used to analyze the mitotic chromosomes of these progeny. Strong pSc119.2 FISH signals could be observed at the telomeric regions of 3DS arms in ‘Mianyang11’. However, the pSc119.2 FISH signals were disappeared from the selfed progeny of 4R monosomic addition line and the changed 3D chromosomes could be transmitted to next generation stably. In one of the selfed progeny of 7R monosomic addition line, one 2D chromosome was broken and three 4A chromosomes were observed. In the selfed progeny of 6R monosomic addition line, structural variation and abnormal mitotic behaviour of 3D chromosome were detected. Additionally, 1A and 4B chromosomes were eliminated from some of the progeny of 6R monosomic addition line. Conclusions/Significance These results indicated that single rye chromosome added to wheat might cause alterations and abnormal mitotic behaviours of wheat chromosomes and it is possible that the stress caused by single alien chromosome might be one of the factors that induced karyotype alteration of wheat.

Oligonucleotide Probes for ND-FISH Analysis to Identify Rye and Wheat Chromosomes

Genomic in situ hybridization (GISH) has been widely used to detect rye (Secale cereale L.) chromosomes in wheat (Triticum aestivum L.) introgression lines. The routine procedure of GISH using genomic DNA of rye as a probe is time-consuming and labor-intensive because of the preparation and labeling of genomic DNA of rye and denaturing of chromosomes and probes. In this study, new oligonucleotide probes Oligo-1162, Oligo-pSc200 and Oligo-pSc250 were developed. The three new probes can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) assays and replace genomic DNA of rye as a probe to discriminate rye chromosomes in wheat backgrounds. In addition, previously developed oligonucleotide probes Oligo-pSc119.2-1, Oligo-pSc119.2-2, Oligo-pTa535-1, Oligo-pTa535-2, Oligo-pTa71-2, Oligo-pAWRC.1 and Oligo-CCS1 can also be used for ND-FISH of wheat and rye. These probes have provided an easier, faster and more cost-effective method for the FISH analysis of wheat and hybrids derived from wheat × rye.

Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale.

Eleven weedy or wild species or subspecies of the genus Secale L. were compared with a set of cultivated rye accessions, based on inter-simple sequence repeat (ISSR) markers to analyze their phylogenetic relationships. A total of 846 bands were amplified from reactions using 12 screening primers, including 79 loci with a mean of 10.1 alleles per locus. The number of amplified bands for each primer ranged from 12 to 134, with a mean of 70.5 amplified bands per primer. The presence and distribution of amplified bands in different accessions demonstrate that a rapid evolutionary trend of microsatellite repeats occurred during the speciation process from the perennial wild form to annual cultivated rye. In addition, variation, amplification, and deletion of microsatellites in genomes revealed phylogenetic relationships in the genus Secale. Analysis of the presence, number, and distribution of amplified bands in genomes, as well as the comparison with genetic similarity (GS) indices based on ISSR, indicate that Secale strictum subsp. africanum (Stapf) Hammer, Secale strictum anatolicum (Boiss.) Hammer, Secale sylvestre Host, and Secale strictum subsp. strictum (C. Presl) Hammer emerged in succession from a common ancestor of Secale following geographic separation and genetic differentiation. The annual weedy rye evolved from S. strictum subsp. strictum, which was domesticated as present-day cultivated rye. Data from ISSR analyses separated all investigated accessions of the genus Secale into three distinct groups. These results support the division of the genus Secale into three species: the annual wild species S. sylvestre; the perennial wild species S. strictum, including several differential subspecies forms such as strictum, africanum, and anatolicum; and S. cereale, including cultivated and weedy rye as subspecies forms.

LOSS OF PARENTAL CODING SEQUENCES IN AN EARLY GENERATION OF WHEAT-RYE ALLOPOLYPLOID

During wheat-rye allopolyploidization, the characteristics of the sequences in the bands that appear in both parents and progeny are still unclear. In this study, two different combinations of wheat (Triticum aestivum L.) and rye (Secale cereale L.), including F1 hybrids and the first and second allopolyploid generations, were analyzed by PCR and sequencing using 60 wheat expressed sequence tag (EST)–derived single-sequence repeat markers and EST-derived sequence-tagged site markers. Thirty markers produced the same bands from parental plants, F1 plants, and amphiploids. Seven of the 30 markers amplified identical sequences from wheat and rye parents. Most of these sequences have high similarity between the two parental plants. The variation patterns of sequences in the bands produced by the seven markers were observed. In the F1 hybrids and amphiploids, loss of parental sequences was observed and the frequency of losing rye sequences was higher than that of losing wheat sequences. In addition, a few sequences in these bands exhibited significant differences, indicating that parental sequences changed drastically during allopolyploidization. Therefore, the fact that the parents and progeny contained the same bands should not be regarded as conservation. The results in this study add to the investigations dealing with variation patterns of coding sequences during wheat-rye allopolyploidization.

Genetic and epigenetic variations induced by wheat-rye 2R and 5R monosomic addition lines.

Background Monosomic alien addition lines (MAALs) can easily induce structural variation of chromosomes and have been used in crop breeding; however, it is unclear whether MAALs will induce drastic genetic and epigenetic alterations. Methodology/Principal Findings In the present study, wheat-rye 2R and 5R MAALs together with their selfed progeny and parental common wheat were investigated through amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) analyses. The MAALs in different generations displayed different genetic variations. Some progeny that only contained 42 wheat chromosomes showed great genetic/epigenetic alterations. Cryptic rye chromatin has introgressed into the wheat genome. However, one of the progeny that contained cryptic rye chromatin did not display outstanding genetic/epigenetic variation. 78 and 49 sequences were cloned from changed AFLP and MSAP bands, respectively. Blastn search indicated that almost half of them showed no significant similarity to known sequences. Retrotransposons were mainly involved in genetic and epigenetic variations. Genetic variations basically affected Gypsy-like retrotransposons, whereas epigenetic alterations affected Copia-like and Gypsy-like retrotransposons equally. Genetic and epigenetic variations seldom affected low-copy coding DNA sequences. Conclusions/Significance The results in the present study provided direct evidence to illustrate that monosomic wheat-rye addition lines could induce different and drastic genetic/epigenetic variations and these variations might not be caused by introgression of rye chromatins into wheat. Therefore, MAALs may be directly used as an effective means to broaden the genetic diversity of common wheat.

Isolation of rye-specific DNA fragment and genetic diversity analysis of rye genus Secale L. using wheat SSR markers

Rye (Secale cereale L.) is one of the most widely utilized sources in wheat breeding. Biochemical (Dhaliwal et al. 1988) and cytological (Belyayev et al. 2001) strategies have been used to identify rye chromatins in wheat backgrounds. The major limitation of these techniques is that they are highly technical and time-consuming. Species-specific PCR-based markers are useful and convenient tools for detecting alien chromosome segments incorporated into wheat genomes. Some rye-specific PCR-based markers have been successfully developed by random amplified polymorphic DNA (RAPD) analysis (Iqbal and Rayburn 1995; Katto et al. 2004; Liu et al. 2008; Jia et al. 2009). Simple sequence repeat (SSR) markers are another useful tool to develop species-specific DNA fragments. As previously reported, many wheat SSR markers were successfully applied to the amplification of DNA from several related species, such as triticale (Kuleung et al. 2004), Aegilops (Adonina et al. 2005) and Haynaldia (Zhang et al. 2006). In addition, some reports have already indicated that wheat SSR markers can be amplified in rye (Röder et al. 1995; Kuleung et al. 2004). Thereby, wheat SSR can be used to enrich ryespecific PCR-based markers. However, until now, data were rarely available about using wheat SSR markers to develop rye-specific markers. The objective of this study is to develop rye-specific PCR-based markers using wheat SSR markers. The phenomenon that rye-specific bands amplified by wheat SSR markers could display genetic diversity among genus Secale has also been observed and discussed.

Diversity resistance to Puccinia striiformis f. sp Tritici in rye chromosome arm 1RS expressed in wheat.

The 1BL.1RS wheat-rye translocation contained in the Russian cultivar Aurora has been the most widespread alien translocation in wheat-breeding programs all over the world. However, following the prevalence of new biotypes of the pathogens, disease-resistance genes in this translocation chromosome have been overcome and consequently they have been eliminated in modern wheat-breeding programs. In this paper, we report on 12 new primary 1BL.1RS translocation lines derived from the crosses of a Chinese high yield wheat cv. Mianyang 11 with three rye cultivars collected from China. GISH, C-banding and PCR techniques using the specific primers for 1BS, 1RS and centromeres of wheat and rye were applied to identify the constitution of chromosomes. The results confirmed that all 1BL.1RS chromosomes in the 12 primary translocation lines contained integrated 1RS chromosome arms. In the resistance analysis using five kinds of Pst pathotypes, the 12 primary translocation lines showed diversity resistance to stripe rust, which contained at least five different new genes (alleles), significantly different from the Yr9 gene coming from Russian wheat cultivar Aurora. The results indicated that the chromosome arm 1RS in the rye population carries abundant yet untapped genes (alleles) for resistance to wheat stripe rust, which would originate from the neutral diversity in the natural population of rye. It is suggested that creating more primary translocation lines in genome modification will be extremely important to use the diversity of alien R-genes, which was generated by long-term neutral mutation and maintained in the population of alien species.

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.

A Mutant with Expression Deletion of Gene Sec-1 in a 1RS.1BL Line and Its Effect on Production Quality of Wheat

The chromosome arm 1RS of rye (Secale cereal L.) has been used worldwide as a source of genes for agronomic and resistant improvement. However, the 1RS arm in wheat has end-use quality defects that are partially attributable to the presence of ω-secalins, which are encoded by genes at the Sec-1 locus. Various attempts in removing the Sec-1 genes from the 1RS.1BL translocation chromosome have been made. In the present study, two new primary 1RS.1BL translocation lines, T917-26 and T917-15, were developed from a cross between wheat variety “A42912” and Chinese local rye “Weining.” The lines T917-15 and T917-26 carried a pair of intact and homogeneous 1RS.1BL chromosomes. The line T917-26 also harbored an expression deletion of some genes at the Sec-1 locus, which originated from a mutation that occurred simultaneously with wheat-rye chromosome translocations. These results suggest that the accompanying mutations of the evolutionarily significant translocations are remarkable resources for plant improvement. Comparison of translocation lines with its wheat parent showed improvements in the end-use quality parameters, which included protein content (PC), water absorption (WA), sodium dodecyl sulfate sedimentation (SDSS), wet gluten (WG), dry gluten (DG) and dough stickiness (DS), whereas significant reduction in gluten index (GI) and stability time (ST) were observed. These findings indicate that 1RS in wheat has produced a higher amount of protein, although these comprised worse compositions. However, in the T917-26 line that harbored an expression deletion mutation in the Sec-1 genes, the quality parameters were markedly improved relative to its sister line, T917-15, especially for GI and DS (P < 0.05). These results indicated that expression deletion of Sec-1 genes significantly improves the end-use quality of wheat cultivars harboring the 1RS.1BL translocation. Strategies to remove the Sec-1 genes from the 1RS.1BL translocation in wheat improvement are discussed.

QTL Analysis for Grain Pentosans and Hardness Index in a Chinese 1RS.1BL × non-1RS.1BL Wheat Cross

Pentosans are quantitatively minor constituents of wheat (Triticum aestivum L.), but they influence the end-use and nutritional qualities of grain cereals. Although several studies on the physicochemical properties and genetic basis of pentosans and pentosan-associated traits have been reported, the genetic architecture and determinants of pentosans remained ambiguous. In this research, 107 QTLs for pentosans and pentosan-associated traits were detected based on a population of recombinant inbred lines (RILs) derived from a 1RS/1BL translocation line × non-1RS/1BL translocation line. We identified seven stable expression QTLs for pentosans and one major QTL (Q.HI.scau-7D linked to xwmc634-7D) for hardness index (HI). Q.HI.scau-7D may be the gene responsible for the bimodal HI distribution map. More than one genetic location harbored QTLs for water-soluble pentosan (WSP), and the ratio of WSP content to water-insoluble pentosans (WIP) content (RWW) varied simultaneously, meaning that these loci harbored pleiotropic genes for WSP and RWW. WSP was the main factor in pentosans affecting wheat HI. This study gives a better understanding of the molecular genetics of pentosans and pentosan-related traits, and provides a certain theoretical basis for molecular genetics and breeding for pentosans.