Rapid Evolution of Simple Sequence Repeat Induced by Allopolyploidization

Microsatellite evolution normally occurs in diploids. Until now, there has been a lack of direct experimental evidence for microsatellite evolution following allopolyploidization. In the present study, F₁ hybrids and newly synthesized allopolyploids were derived from Triticum aestivum Chinese Spring × Secale cereale Jinzhou-heimai. One hundred and sixty-three wheat simple sequence repeat (SSR) markers were used to investigate the variation of wheat microsatellites after allopolyploidization and variation of the PCR products of 29 of the SSR markers was observed. Of these 29 SSR markers, 15 were unable to produce products from amphiploids. The other 14 SSR markers did produce products from parental wheat, F₁ hybrids and amphiploids. However, the length of the products amplified from amphiploids was different from the length of the products amplified from parental wheat and F₁ hybrids. Sequencing indicated that the length variation of the 14 microsatellites stemmed mainly from variation in the number of repeat units. The alteration of repeat units occurred in both perfect and compound repeats. In some compound SSR loci, one motif was observed to expand whereas another to contract. Almost all the microsatellite evolution observed in this study could be explained by the slipped-strand mispairing model. The results of this study seem to indicate that stress caused by allopolyploidization might be one of the factors that induce microsatellite evolution. In addition, the findings of present study provided an instance of how simple sequence repeats evolved after allopolyploidization.     

黑麦特异重复序列pSc119.1在姊妹T1RS·1BL易位系中的变异

根据公布的黑麦特异重复序列pSc119.1设计特异引物, 分别对两套姊妹T1RS·1BL易位系川农12(CN12)、川农17(CN17)、川农18(CN18)和96Ⅰ176-1、96Ⅰ176-3的基因组DNA进行扩增。从CN12、CN17、CN18的基因组中都能扩增出目的片段, 在96Ⅰ176-1的基因组中, 除目的片段外, 还扩增出了一条非目的片段, 但在96Ⅰ176-3的基因组中没有扩增出产物。Southern blot分析表明, pSc119.1序列并未从96Ⅰ176-3的基因组中消失。将CN12、CN17、CN18的目的片段回收克隆后, 对每个片段各随机挑取10个克隆进行测序, pSc119.1序列在3个品种中都发生了变异, 且在CN18中的变异程度最大。所测的序列多数与原序列达94%和95%的相似性, 在这些序列中, 碱基的改变具有一定的规律, 即多数为转换, 少数为颠换, 且变异碱基和变异位置具有较高的一致性。远缘杂交后代的进化可能是一个持续过程, 姊妹系内的株系之间某些性状存在差异, 这些差异很可能与重复序列的变异有关, 这为后生遗传效应机制的研究提供了有用的材料。     

Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

Identification of AFLP markers on the satellite region of chromosome 1BS in wheat.

The satellite region on the short arm of chromosome 1B in wheat (Triticum aestivum L., 2n = 6x = 42) carries many agronomically important genes; i.e., genes conferring fungal disease resistance, seed storage proteins, and fertility restoration. To find molecular markers located on the satellite region, we applied the fluorescent AFLP (amplified fragment length polymorphism) technique to aneuploids and deletion stocks of the cultivar T. aestivum 'Chinese Spring'. Out of 6017 fragments amplified with 80 primer combinations in normal 'Chinese Spring', 24 were assigned to 1BS. Twelve of them clustered within a small region of the satellite known to be rich in RFLP (restriction fragment length polymorphism) markers. AFLPs in 1BS and in the whole genome were calculated between 'Chinese Spring' and T. spelta var. duhamelianum. The polymorphism rates in the satellite region (58.3%) and in the 1BS arm (45.8%) were much higher than the average rate for the whole genome (10.7%). Seven of the 12 AFLP markers in the satellite region were revealed to be specific to 'Chinese Spring' and could potentially be useful for genetic mapping in a segregation population of 'Chinese Spring' x T. spelta.     

Painting the rye genome with genome-specific sequences

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.     

Comparative analysis of the nucleosomal structure of rye,wheat and their relatives

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175–185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.     

Development and application of EST-based markers specific for chromosome arms of rye (Secale cereale L.)

To develop a set of molecular markers specific for the chromosome arms of rye, a total of 1,098 and 93 primer pairs derived from the expressed sequence tag (EST) sequences distributed on all 21 wheat chromosomes and 7 rye chromosomes, respectively, were initially screened on common wheat 'Chinese Spring' and rye cultivar 'Imperial'. Four hundred and fourteen EST-based markers were specific for the rye genome. Seven disomic chromosome addition lines, 10 telosomic addition lines and 1 translocation line of 'Chinese Spring-Imperial' were confirmed by genomic in situ hybridization and fluorescencein situ hybridization, and used to screen the rye-specific markers. Thirty-one of the 414 markers produced stable specific amplicons in 'Imperial', as well as individual addition lines and were assigned to 13 chromosome arms of rye except for 6RS. Six rye cultivars, wheat cultivar 'Xiaoyan 6' and accessions of 4 wheat relatives were then used to test the specificity of the 31 EST-based markers. To confirm the specificity, 4 wheat-rye derivatives of 'Xiaoyan 6 × German White', with chromosomes 1RS, 2R and 4R, were amplified by some of the EST-based markers. The results indicated that they can effectively be used to detect corresponding rye chromosomes or chromosome arms introgressed into a wheat background, and hence to accelerate the utilization of rye genes in wheat breeding.     

Rapid and repeatable elimination of a parental genome-specific DNA repeat (pGc1R-1a) in newly synthesized wheat allopolyploids

Recent work in the Triticum-Aegilops complex demonstrates that allopolyploidization is associated with an array of changes in low-copy coding and noncoding sequences. Nevertheless, the behavior and fate of repetitive DNA elements that constitute the bulk of nuclear DNA of these plant species is less clear following allopolyploidy. To gain further insight into the genomic events that accompany allopolyploid formation, we investigated fluorescence in situ hybridization (FISH) patterns of a parental-specific, tandem DNA repeat (pGc1R-1) on three sets of newly synthesized amphiploids with different parental species. It was found that drastic physical elimination of pGc1R-1 copies occurred in all three amphiploids in early generations. DNA gel-blot analysis confirmed the FISH data and estimates indicated that approximately 70-90% of the copies of the pGc1R-1 repeat family were eliminated from the amphiploids by the second to third selfed generations. Thus, allopolyploidy in Triticum-Aegilops can be accompanied by rapid and extensive elimination of parental-specific repetitive DNA sequences, which presumably play a role in the initial stabilization of the nascent amphiploid plants.     

Random chromosome elimination in synthetic Triticum-Aegilops amphiploids leads to development of a stable partial amphiploid with high grain micro- and macronutrient content and powdery mildew resistance

Synthetic amphiploids are the immortal sources for studies on crop evolution, genome dissection, and introgression of useful variability from related species. Cytological analysis of synthetic decaploid wheat (Triticum aestivum L.) - Aegilops kotschyi Boiss. amphiploids (AABBDDUkUkSkSk) showed some univalents from the C1 generation onward followed by chromosome elimination. Most of the univalents came to metaphase I plate after the reductional division of paired chromosomes and underwent equational division leading to their elimination through laggards and micronuclei. Substantial variation in the chromosome number of pollen mother cells from different tillers, spikelets, and anthers of some plants also indicated somatic chromosome elimination. Genomic in situ hybridization, fluorescence in situ hybridization, and simple sequence repeat markers analysis of two amphiploids with reduced chromosomes indicated random chromosome elimination of various genomes with higher sensitivity of D followed by the Sk and Uk genomes to elimination, whereas 1D chromosome was preferentially eliminated in both the amphiploids investigated. One of the partial amphiploids, C4 T. aestivum 'Chinese Spring' - Ae. kotschyi 396 (2n = 58), with 34 T. aestivum, 14 Uk, and 10 Sk had stable meiosis and high fertility. The partial amphiploids with white glumes, bold seeds, and tough rachis with high grain macro- and micronutrients and resistance to powdery mildew could be used for T. aestivum biofortification and transfer of powdery mildew resistance.     

Inter- and intra-genomic transfer of small chromosomal segments in wheat-rye allopolyploids

Newly synthesized wheat-rye allopolyploids, derived from Triticum aestivum Mianyang11 × S. cereale Kustro, were investigated by sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) using rye tandem repeat pSc200 and rye genomic DNA as probes, respectively, over the first, second and third allopolyploid generations. FISH signals of pSc200 could be observed at both telomeres/subtelomeres of all 14 chromosomes of the parental rye. In the first allopolyploid generation, there were ten rye chromosomes bearing FISH signals at both telomeres/subtelomeres and four rye chromosomes bearing FISH signals at only one telomere/subtelomere. However, in the second and the third allopolyploid generations, there were 12 rye chromosomes bearing FISH signals at both telomeres/subtelomeres and 2 rye chromosomes bearing FISH signals at only one telomere/subtelomere. Rye telomeric segments were transferred to the centromeric region of wheat chromosomes in some cells and small segments derived from non-telomeric regions of rye chromosome were transferred to the telomeric region of wheat chromosomes in some other cells. These observations indicated that the rye telomeric/subtelomeric region was unstable in newly synthesized wheat-rye allopolyploids and allopolyploidization was accompanied by rapid inter/intra-genomic exchange. The inter-genomic exchange may have occurred in somatic cells.     

小麦体细胞无性系SSR位点的遗传变异特性分析

研究结果表明:(1)小麦体细胞无性系SSR位点变异类型有:扩增片段迁移率的变大或变小、扩增片段缺失以及新的扩增片段;(2)变异特点为:变异频率与基因型有关,不同染色体组上的SSR位点变异频率不同,而不同无性系后代的SSR位点变异频率也不同;(3)同一SSR位点的变异类型在同一基因型的无性系后代中变异表现一致,在不同基因型无性系后代中的变异表现不同,有的SSR位点在无性系后代中表现出一致的变异,而有的则不一致。     

Increasing the physical markers of wheat chromosomes using SSRs as FISH probes

In plants the marker sequences used to identify chromosomes are mainly repetitive DNA probes. Simple sequence repeats (SSRs) are major components of many plant genomes and could be good markers for chromosome identification. In a previous work, we reported the physical distribution of 4 oligonucleotides, (AG)12, (CAT)5, (AAC)5, and (AAG)5, on Triticum aestivum L. chromosomes. The distinctive distribution pattern found suggested that SSR in situ hybridization is useful as a diagnostic tool in wheat cytogenetics. To check whether that finding is generally applicable, we analyzed the chromosomal distribution of the rest of the 14 possible classes of di- and tri-nucleotide repeats by FISH. A detailed knowledge of the sequence content of hexaploid wheat chromatin was acquired based on the hybridization signals, which also provide a rich set of chromosome markers for chromosome identification. Except for (AT)10 and (GC)10, for which the chromosomal distribution could not be accurately determined, and (AC)8 and (GCC)5, which were found dispersed throughout the chromosomes, the remaining repeats were observed as clusters on specific chromosome sites. (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 exhibited a preferential distribution in the pericentromeric regions of the B genome chromosomes. The richest patterns of intercalary signals on several A and B genome chromosomes were produced by (ACT)5. A karyotype based on the SSR probes providing the best FISH patterns was constructed for T. aestivum 'Chinese Spring'.     

Characteristics and a comparison of three classes of microsatellite-based markers and their application in plants

Microsatellites (SSR--simple sequence repeats, STR--short tandem repeats, SSLP--simple sequence length polymorphism, VNTR--variable number of tandem repeats) are the class of repetitive DNA sequences present in all living organisms. Particular characteristics of microsatellites, such as their presence in the genomes of all living organisms, high level of allelic variation, co-dominant mode of inheritance and potential for automated analysis make them an excellent tool for a number of approaches like genotyping, mapping and positional cloning of genes. The three most popular types of markers containing microsatellite sequences that are presently used are: (1) SSR (simple sequence repeats), generated by amplifying in a PCR reaction with the use of primers complementary to flanking regions; (2) ISSR (inter-simple sequence repeats), based on the amplification of regions between inversely oriented closely spaced microsatellites; and (3) SAMPL (selective amplification of microsatellite polymorphic loci), which utilises AFLP (amplified fragment-length polymorphism) methodology, with one exception--for the second amplification, one of the starters is complementary to the microsatellite sequence. The usefulness of the three above-mentioned markers for numerous purposes has been well documented for plants.     

Diverse patterns of the tandem repeats organization in rye chromosomes

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.     

Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.     

New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat

Subcloning of a clone of the 120-bp family of rye, pSc119, has produced two extremely useful probes. pSc119.1 assays rye-specific dispersed repetitive sequence families. It is present on all seven rye chromosomes and hybridizes to the entire length of each chromosome, with the exception of some telomeres and the nucleolar organiser region. pSc119.2, in contrast, hybridizes predominantly to the telomeric regions of rye chromosomes, with some interstitial sites. Unlike pSc119.1, it assays similar repetitive sequence families in both wheat and rye chromosomes.     

Isolation and mapping of microsatellite markers specific for the D genome of bread wheat.

The potential of Aegilops tauschii, the diploid progenitor of the D genome of wheat, as a source of microsatellite markers for hexaploid bread wheat was investigated. By screening lambda phage and plasmid libraries of Ae. tauschii genomic DNA, dinucleotide microsatellites containing GA and GT motifs were isolated and a total of 65 functional microsatellite markers were developed. All primer pairs that were functional in Ae. tauschii amplified well in hexaploid wheat. Fifty-five loci amplified by 48 primer sets were placed onto a genetic framework map of the reference population of the International Triticeae Mapping Initiative (ITMI) 'Opata 85' x 'W7984'. The majority of microsatellite markers could be assigned to the chromosomes of the D genome of wheat. The distribution of the markers along the chromosomes is random. Chromosomal location of 22 loci nonpolymorphic in the reference population was determined using nullitetrasomic lines of Triticum aestivum 'Chinese Spring'. The results of this study demonstrate the value of microsatellite markers isolated from Ae. tauschii for the study of bread wheat. The microsatellite markers developed improve the existing wheat microsatellite map and can be used in a wide range of genetic studies and breeding programs.     

Characterization of a new 4BS.7HL wheat-barley translocation line using GISH, FISH, and SSR markers and its effect on the β-glucan content of wheat

A spontaneous interspecific Robertsonian translocation was revealed by genomic in situ hybridization (GISH) in the progenies of a monosomic 7H addition line originating from a new wheat 'Asakaze komugi' × barley 'Manas' hybrid. Fluorescence in situ hybridization (FISH) with repetitive DNA sequences (Afa family, pSc119.2, and pTa71) allowed identification of all wheat chromosomes, including wheat chromosome arm 4BS involved in the translocation. FISH using barley telomere- and centromere-specific repetitive DNA probes (HvT01 and (AGGGAG)(n)) confirmed that one of the arms of barley chromosome 7H was involved in the translocation. Simple sequence repeat (SSR) markers specific to the long (L) and short (S) arms of barley chromosome 7H identified the translocated chromosome segment as 7HL. Further analysis of the translocation chromosome clarified the physical position of genetically mapped SSRs within 7H, with a special focus on its centromeric region. The presence of the HvCslF6 gene, responsible for (1,3;1,4)-β-D-glucan production, was revealed in the centromeric region of 7HL. An increased (1,3;1,4)-β-D-glucan level was also detected in the translocation line, demonstrating that the HvCslF6 gene is of potential relevance for the manipulation of wheat (1,3;1,4)-β-D-glucan levels.     

Characterization of a 5HS-7DS.7DL wheat-barley translocation line and physical mapping of the 7D chromosome using SSR markers

A spontaneous wheat-barley translocation line was previously detected in the progenies of the Mv9kr1?×?‘Igri’ wheat-barley hybrid and the translocation was identified as 5HS-7DS.7DL. Multicolor genomic in situ hybridization (mcGISH) with D and H genomic DNA probes and three-color fluorescence in situ hybridization (FISH) with repetitive DNA probes (Afa-family, pSc119.2, and pTa71) were performed to characterize the rearranged chromosome. The effect of 5HS and the deleted 7DS fragment on the morphological traits (plant height, fertility, yield, and spike characteristics) of wheat was assessed. Despite the non-compensating nature of the translocation, the plants showed good viability. The aim of the study was to physically localize SSR markers to the telomeric and subtelomeric regions of the 7DS chromosome arm. Of the 45 microsatellite markers analyzed, ten (Xbarc0184, Xwmc0506, Xgdm0130, Xgwm0735, Xgwm1258, Xgwm1123, Xgwm1250, Xgwm1055, Xgwm1220, and Xgwm0635) failed to amplify any 7DS-specific fragments, signaling the elimination of a short chromosome segment in the telomeric region. The breakpoint of the 5HS-7DS.7DL translocation appeared to be more distal than that of reported deletion lines, which provides a new physical landmark for future deletion mapping studies.