Production of wheat-rye substitution lines based on winter rye cultivars with karyotype identification by means of C-banding,GISH, and SSR markers

The study presents a continuation of the research aimed at producing of wheat-rye substitution lines (2n = 42) based on the cross (Triticum aestivum L. × Secale sereale L.) × Triticum aestivum L., and using winter rye cultivars Vyatka and Vietnamskaya Mestnaya. In BC 1 F 5 two lines were identified, having karyotypes in which a pair of homologous wheat chromosomes was substituted by a homeologous pair of rye chromosomes. The chromosome composition of these lines was analyzed using C-banding, GISH, and SSR markers. It was demonstrated that karyotype of each line included a single pair of rye chromosomes and lacked wheat-rye translocations. The rye chromosomes were identified, and the chromosomes of wheat, at which the substitutions occurred, were determined. The lines generated by crosses with rye of Vyatka and Vietnamskaya Mestnaya cultivars were designated 1Rv(1A) and 5Rviet(5A), respectively. Chromosome identification and classification of the lines makes it possible to use them in breeding programs and genetic studies.     

Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers

Based on the cross (Triticum aestivum L. × Secale cereale L.) × T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat-rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)₂, 2R(2D)₃, 3R(3B), 6R(6A)₂. The chromosome composition of lines 1R(1A), 2R(W)₁, 5R(W), 5R(5A), and 6R(W)₁, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)₁, 5R(5D), 5R(5A), and 6R(6A)₁, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The “combined” long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising “secondary gene pools” for the purpose of plant breeding.     

Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers

Based on the cross (Triticum aestivum L. x Secale cereale L.) x T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat--rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)2, 2R(2D)3, 3R(3B), 6R(6A)2. The chromosome composition of lines IR(1A), 2R(W)1, 5R(W), 5R(5A), and 6R(W)1, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)1, 5R(5D), 5R(5A), and 6R(6A)1, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The "combined" long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising "secondary gene pools" for the purpose of plant breeding.     

小麦-黑麦小片段易位系的分子细胞遗传学分析

为选育具有经济价值的带有黑麦R染色体组小片段的小麦-黑麦育种基础材料,对小麦-黑麦5R/5A×6R/6A代换系杂交后代的8份高代材料6-30、6-31、7-1、7-9、7-13、7-21、7-22和7-28进行形态学、细胞学观察,及SSR分析和GISH检测。结果表明,8个品系田间生长整齐、育性正常,具有大穗、多小穗,抗白粉病、叶锈病等优良性状;对其中2个品系7-1和7-9进行花粉母细胞减数分裂观察,发现大多数细胞染色体构型为2n=21Ⅱ,具有良好的遗传稳定性;选择黑麦R染色体通用引物及5R、6R染色体上的微卫星引物共8对,对8个品系进行SSR分析,结果表明8个品系都有黑麦5R或6R染色体片段的导入,进一步进行GISH检测,发现5个品系6-31、7-1、7-13、7-21、7-22都存在黑麦杂交信号,为小麦-黑麦小片段易位系。本研究综合多种手段鉴定的8份材料皆为小麦-黑麦小片段易位系,在育种上具有利用价值。     

Identification of a complete set of isogenic wheat/rye D-genome substitution lines by means of Giemsa C-banding

Summary A complete set of isogenic wheat/rye D-genome substitutions were produced by crossing an inbred line of spring rye Secale cereale L. cv. Prolific to a tetraploid wheat, the A-and B-genomes of which had previously been extracted from hexaploid wheat, Triticum aestivum L. em Thell. cv. Thatcher. After chromosome doubling, the derived hexaploid triticale (x Triticosecale Wittmack) was backcrossed to 6x Thatcher and selection for wheat/rye substitution lines was carried out in BCF₃ to BCF₆ families by using Giemsa C-banding. Five fertile disomic wheat/rye D-genome substitution lines were obtained and their chromosomal constitution was determined to be 1D/1R, 2D/2R, 7D/4R, 6D/6R, 7D/7R. The two remaining 3R and 5R substitutions are at the moment in a monosomic condition. Another 1D/7R substitution was detected but this plant was very weak and sterile, indicating that only substitutions between homoeologous chromosomes result in fertile, vigorous plants. Furthermore, many rye telocentrics as well as rye-rye and rye-wheat translocations were selected. Since all lines selected in this program share the same genetic background of Thatcher wheat, genetic heterogeneity is excluded. The material is very useful, therefore, for analyzing the effects of different rye chromosomes or chromosome segments in an otherwise homozygous background.Contribution No. 797     

染色体C-带在小麦—黑麦易位系和代换系鉴定中的作用

用改良的Giemsa C-带技术对10个经辐射处理的带有黑麦某些性状的普通小麦品系进行鉴定。结果鉴定出1个小麦—黑麦易位系及2个小麦—黑麦异代换系,探索出冬季和夏季染色体分带的不同条件。因此,只要条件控制得当,C-分带不失为一种快速、精确而经济的鉴定外源染色体的方法。     

Genetic identification of 91 poplar cultivars based on SSR markers

Accurate genetic identification and relationship analysis of poplar cultivars is necessary to establish commercial poplar plantations and select suitable breeding strategies. In this study, 91 poplar cultivars belonging to four sections (Aigeiros, Tacamahaca, Populus and Turanga) and inter/intra-sectional hybrids were genotyped using 18 polymorphic simple sequence repeat (SSR) markers. In total, 222 alleles were amplified with an average of 12.3 alleles per marker. The mean polymorphic information content and power of discrimination were 0.706 and 0.813, respectively. Five SSR markers (ORPM_103, ORPM_247, GCPM_1048, GCPM_1255 and LG_X_19) constituted a core fingerprint and were sufficient to identify all the tested cultivars. With some notable exceptions, cultivars of the same species generally clustered together in cluster (UPGMA) and ordination (PCO) analyses. Flow cytometry indicated that 11 poplar cultivars were triploid. Among these, seven had three alleles at some loci, suggesting that SSR markers could indicate the ploidy level to some extent. This study provides useful genetic information for the identification and protection of poplar cultivars in China and offers a guideline for the selection of poplar crossing parents based on ploidy level and genetic relationships.     

Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.     

Transferability of SSR markers among wheat,rye, and triticale

Simple sequence repeat (SSR) markers are a valuable tool for many purposes, such as mapping, fingerprinting, and breeding. However, they are only available in some economically important crops because of the high cost and labor intensity involved in their development. Comparative mapping reveals a high degree of colinearity between closely related species, which allows the exchange of markers between them. Our objective was to examine the transferability of SSR markers among wheat (Triticum aestivum L.), rye (Secale cereale L.), and triticale (X Triticosecale Wittmack). One hundred forty-eight wheat and 28 rye SSR markers were used to amplify genomic DNA extracted from five lines each of wheat, rye, and triticale. Transferability of wheat SSR markers to rye was 17%, whereas 25% of rye markers were amplifiable in wheat. In triticale, 58% and 39% transferability was achieved for wheat and rye markers, respectively. Wheat markers gave an average of 2.6, 2.7, and 2.4 polymorphic bands in wheat, rye, and triticale, respectively, while rye markers gave an average of 2.0 in rye and none in wheat and triticale. These transferable markers can now be exploited for further genetic and breeding studies in these species.Nebraska Agricultural Research Division, Journal Series No. 14243Communicated by B. Friebe     

Cytological markers of rRNA gene expression during microsporogenesis in rye, wheat and wheat-rye hybrids

The quantity of nucleolei during microsporogenesis of wheat, rye and F1 of wheat-rye hybrids has been investigated. Dependence of nucleoli quantity from microsporogenesis stage and number of nucleolar organizer regions in chromosomes have been shown. The volumes of nuclei and nucleoli as well as nucleus-nucleolus ratio have been calculated. The changes of these indices during microsporogenesis were regular. The possibility of using cariometrical indices as cytological markers of rRNA gene expression in the process of microsporogenesis has been substantiated.     

Comparative effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat-rye substitution lines dependending on the line origin

The effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat--rye substitution lines was studied as dependent on the cultivar origin of the rye chromosomes and on the wheat genome (A or D) subjected to substitution. Chromosome 1R stimulated embryogenesis in anther cultures, while chromosome 5R suppressed it regardless of whether the corresponding wheat chromosomes were substituted in the A or D genome. The effect of chromosome 1R on embryogenesis proved to depend on its cultivar origin. Along with rye chromosome 1R, wheat chromosome 1A was shown to substantially affect total seedling regeneration. Regeneration of green seedlings was dramatically affected both by rye chromosome 1R and by wheat chromosome 1D. The results supported the published data that individual androgenesis parameters (embryogenesis, total plant regeneration, green plant regeneration) are controlled by different genetic mechanisms.     

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from 'Petkus' and SrR from 'Imperial' rye. RFLP markers previously mapped to chromosome 1HS of barley - flanking the complex Mla powdery mildew resistance gene locus - and chromosome 1DS of Aegilops tauschii - flanking the Sr33 stem rust resistance gene - were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in 'Imperial' and 'Petkus' rye and which have potential for use in marker-assisted breeding.     

中国香菇主栽品种遗传多样性的SSR分析及指纹图谱构建

【目的】香菇(Lentinulaedodes)是世界第二大食用菌,研究我国现有栽培种群体的遗传多样性和遗传构成以及准确鉴定品种是新品种开发和产业健康发展的基础。【方法】采用多态性的SSR(Simple sequence repeat)分子标记对中国历年来使用主栽品种进行遗传多样性及群体结构分析,比较其谱系来源,解析中国主栽品种的群体多样性构成,并构建指纹图谱用于品种鉴定。【结果】24对多态性SSR引物对51份香菇菌株都具有多态性。聚类分析在相似系数0.69处可将栽培种群体分为4个类群,野生种驯化或参与杂交获得的菌株位于类群Ⅲ和Ⅳ,其他菌株位于另外两个类群Ⅰ和Ⅱ。群体结构分析可将栽培群体分为6个遗传构成,显示L808、L135等代表性菌株在各自的构成中参与了其他菌株的选育过程,解释了以其为亲本的部分品种的谱系来源。依据筛选出的9对条带清晰的SSR引物组合构建了多位点SSR指纹图谱,可对45个香菇商业菌种进行辨识。【结论】我国香菇主栽品种亲缘关系较近,育种多围绕L808、L135、9015等核心代表性品种进行,本研究可为选育具有自主知识产权、适应不同栽培模式的新品种提供依据;指纹图谱的构建也能为香菇品种的准确鉴定提供保证。     

Comparison of rapeseed cultivars and resynthesized lines based on allozyme and RFLP markers

It has frequently been suggested to use the resynthesis of rapeseed (Brassica napus) from B. campestris and B. oleracea to broaden its genetic base. The objective of the present study is twofold: (1) to compare the genetic variation within resynthesized rapeseed with a world-wide collection of oilseed rape cultivars, and (2) to compare genetic distances estimated from RFLP markers with distances estimated from a relatively small number of allozyme markers. We investigated 17 resynthesized lines and 24 rapeseed cultivars. Genetic distances were estimated either based on the electrophoresis of seven allozymes, with a total of 38 different bands, or based on RFLP data of 51 probe/enzyme combinations, with a total of 355 different bands. The results of allozyme and RFLP analyses agreed reasonably well. Genetic distances, estimated from two independent sets of RFLP data with 25 and 26 probe/enzyme combinations respectively, were highly correlated; hence about 50 RFLP markers are sufficient to characterize rapeseed material with a large genetic diversity. The cultivars were clustered into three groups: (1) spring rapeseed of European and Northern American origin, (2) winter rapeseed of European and Northern American origin, and (3) rapeseed of Asian origin. Several of the resynthesized rapeseed lines were similar to European winter rapeseed cultivars, whereas others had quite unique patterns. It is concluded, that resynthesized rapeseed is a valuable source for broadening the genetic variation in present breeding material of Brassica napus. However, different lines differ widely in their suitability for this purpose.     

Chromosome composition of wheat-rye lines and the influence of rye chromosomes on disease resistance and agronomic traits

Identification of the chromosomal composition of common wheat lines with rye chromosomes was carried out using genomic in situ hybridization and 1RS- and 5P-specific PCR markers. It was demonstrated that wheat chromosomes 5A or 5D were substituted by rye chromosome 5R in the wheat-rye lines. It was established that one of the lines with complex disease resistance contained rye chromosome 5R and T1RS.1BL, while another line was found to contain, in addition to T1RS.1BL, a new Robertsonian translocation, T5AS.5RL. Substitution of the wheat chromosome 5A with the dominant Vrn-A1 gene for the Onokhoiskaya rye chromosome 5R led to lengthening of the germination-heading period or to a change in the type of development. A negative influence of T1RS.1BL on SDS sedimentation volume and grain hardness was demonstrated, along with a positive effect of the combination of T1RS.1BL and 5R(5D) substitution on grain protein content. Quantitative traits of the 5R(5A) and 5R(5D) substitution lines were at the level of recipient cultivars. A line with two translocations, T1RS.1BL + T5AS.5Rl, appeared to be more productive as compared to the line carrying T1RS.1BL in combination with the 5R(5D) substitution.     

Genetic variability of introduced and local Spanish peach cultivars determined by SSR markers

A set of 94 peach cultivars including Spanish native peach and foreign commercial cultivars were analyzed using 15 SSR markers, selected for their high level of polymorphism. The number of alleles obtained varied from two to 11 with an average of 6.73 giving 185 different genotypes. All the cultivars showed a unique genetic profile, each one using different genotypic combination of all loci. BPPCT001 was the most informative locus showing also the highest discrimination power. Only six loci allowed the unambiguous separation of all the Spanish native cultivars studied, and the genotypic combination of only eight loci permitted the total differentiation of the 94 peach cultivars analyzed. The six selected loci (BPPCT001, BPPCT006, BPPCT008, PS9f8, UDP98-022, and UDP98-412) seem to be very useful for future Spanish peach identification works, and they will help to establish a molecular data base for native peach cultivars. UPGMA analysis was performed from the genetic distance matrix, and allowed the arrangement of all genotypes according to their genetic diversity. The genetic diversity among cultivars, observed in this work, led to their separation according to their regional origin, their morphological characteristics, and especially according to their fruit traits. Analysis of molecular variance was performed for seven populations from different regions of Spain and USA to examine the distribution of genetic variation of the studied accessions, showing that the major variation occurred within populations in each geographic site. The results reveal the existence of two diversity regions in Spain for peach germplasm.     

Identification of wheat-barley translocations by sequential GISH and two-colour FISH in combination with the use of genetically mapped barley SSR markers.

Five wheat-barley translocations in a wheat background were characterized through the combination of cytogenetic and molecular genetic approaches. The wheat chromosome segments involved in the translocations were identified using sequential GISH and two-colour FISH with the probes pSc119.2 and pAs1. The barley chromatin in these lines was identified using SSR markers. A total of 45 markers distributed over the total barley genome were selected from a recently published linkage map of barley and tested on the translocation lines. The following translocations were identified: 2DS.2DL-1HS, 3HS.3BL, 6BS.6BL-4HL, 4D-5HS, and 7DL.7DS-5HS. Wheat-barley disomic and ditelosomic addition lines for the chromosomes 3HS, 4H, 4HL, 5H, 5HL, and 6HS were used to determine the correct location of 21 markers and the position of the centromere. An intragenomic translocation breakpoint was detected on the short arm of the barley chromosome 5H with the help of SSR marker analysis. Physical mapping of the SSR markers on chromosomes 1H and 5H was carried out using the intragenomic and the interspecific translocation breakpoints, as well as the centromere, as physical landmarks.     

基于SSR标记的荷花品种遗传多样性及群体结构分析

采用SSR标记技术对42个荷花品种( Nelumbo spp.)的基因组DNA进行扩增,在此基础上,对供试品种进行UPGMA聚类分析、群体结构分析和主坐标分析( PCoA)。结果表明：采用17对SSR引物从42个荷花品种的基因组DNA中扩增出77个位点,多态性位点百分率为88.31%;每对引物可扩增出1~9个多态性位点。根据Nei's遗传距离,供试的42个荷花品种可被分成Ⅰ和Ⅱ两组,分别包含3和39个品种;在Nei's遗传距离0.150处,Ⅱ组被进一步分成Ⅱa、Ⅱb和Ⅱc 3个亚组,分别包含3、16和20个品种。群体结构分析结果表明：组分概率高于等于0.80时,供试的42个荷花品种被分成Pop1、Pop2和混合群3个亚群,分别包含17、16和9个品种。 PCoA分析结果表明：在F1水平上,供试的42个荷花品种被分成2个部分;其中,Pop1亚群的品种均分布在第二和第三象限,而Pop2亚群的品种则分布在第一和第四象限。总体来看,聚类分析、群体结构分析和PCoA分析的结果基本一致。综合分析结果表明：玉组包含美洲黄莲( N. lutea Pers.)品种‘艾江南',且与传统中国莲( N. nucifera Gaertn.)品种的亲缘关系最远,故认为该组为美洲黄莲;Ⅱ组为中国莲,其中,Ⅱc亚组以传统中国莲品种为主,而Ⅱb亚组则偏重于美洲黄莲。总体上看,供试的42个荷花品种主要被分为中国莲和美洲黄莲两组,而中美杂交莲并没有独立成组,其成因有待进一步研究。     

Effect of crop improvement on genetic diversity in oilseedBrassica rapa (turnip-rape) cultivars, detected by SSR markers

With the improvement of seed quality, Brassica rapa oilseed germplasm went through 2 major breeding bottlenecks during the introgression of genes for zero erucic acid content and low glucosinolate content, respectively. This study investigates the impact of these bottlenecks on the genetic diversity in European winter B. rapa by comparing 3 open-pollinated cultivars, each representing a different breeding period. Diversity was estimated on 32 plants per cultivar, with 16 simple sequence repeat (SSR) markers covering each of the B. rapa linkage groups. There was no significant loss of genetic diversity over the 3 cultivars as indicated by allele number (ranging from 59 to 55), mean allele number (from 3.68 to 3.50), Shannon information index (from 0.94 to 0.87) and expected heterozygosity (from 0.53 to 0.48). About 83% of the total variation was attributed to within-cultivar variation, and the remaining 17% to between-cultivar variation by analysis of molecular variance (AMOVA). Individual plants were separated into the 3 cultivars by principal coordinate analysis (PCoA). In conclusion, genetic diversity within cultivars was high and quality breeding in B. rapa did not significantly reduce the genetic diversity of B. rapa winter cultivars, so there is no risk of decline in performance due to quality improvement.     

Phylogenetic relationships among Portuguese rye based on isozyme, RAPD and ISSR markers

The phylogenetic relationships of 10 rye landraces and cultivars from the north of Portugal and from Brazil were analysed using 20 isozyme loci, and a total of 511 PCR markers (342 ISSRs and 169 RAPDs). The isozymes were analysed in at least 100 plants of each population/cultivar and, therefore, we have data about intra and inter population/cultivar genetic variability. However, the analyses with ISSRs and RAPDs were obtained using a mix of 25 plants of each population. Therefore, each population/cultivar was reduced to one tube and we have no data about intra genetic variability. As expected in a cross pollinated crop we found genetic diversity and a larger variation within than among the populations using isozymes. Somewhat unexpectedly, however, we found that the breeding cultivars have the same level of heterozygosity as the landraces. The phylogenetic relationships obtained using isozymes among the landraces, synthetic cultivar and the cultivars from breeding programs do not reflect their origin. Moreover, the cultivar from Brazil is not separated from the remaining populations/cultivars studied. However, the data observed using RAPDs and ISSRs are in agreement with their known origin. The populations maintained by the farmers in the north of Portugal are grouped in a cluster in the phenogram and the C902591 (from Brazil), the Alv?o (synthetic variety) and Larouco (a hybrid between Montalegre and Brazil) are in a different cluster. The ISSRs and RAPDs provide a rapid method for the production of polymorphic markers, which appear to correspond to known pedigree information.