Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance

Rye (Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt) pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.     

New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance

Powdery mildew is one of the serious diseases of wheat (Triticum aestivum L., 2n = 6 × = 42, genomes AABBDD). Rye (Secale cereale L., 2n = 2 × = 14, genome RR) offers a rich reservoir of powdery mildew resistant genes for wheat breeding program. However, extensive use of these resistant genes may render them susceptible to new pathogen races because of co-evolution of host and pathogen. Therefore, the continuous exploration of new powdery mildew resistant genes is important to wheat breeding program. In the present study, we identified several wheat-rye addition lines from the progeny of T. aestivum L. Mianyang11 × S. cereale L. Kustro, i.e., monosomic addition lines of the rye chromosomes 4R and 6R; a disomic addition line of 6R; and monotelosomic or ditelosomic addition lines of the long arms of rye chromosomes 4R (4RL) and 6R (6RL). All these lines displayed immunity to powdery mildew. Thus, we concluded that both the 4RL and 6RL arms of Kustro contain powdery mildew resistant genes. It is the first time to discover that 4RL arm carries powdery mildew resistant gene. Additionally, wheat lines containing new wheat-rye translocation chromosomes were also obtained: these lines retained a short arm of wheat chromosome 5D (5DS) on which rye chromosome 4R was fused through the short arm 4RS (designated 5DS-4RS·4RL; 4RL stands for the long arm of rye chromosome 4R); or they had an extra short arm of rye chromosome 4R (4RS) that was attached to the short arm of wheat chromosome 5D (5DS) (designated 4RS-5DS·5DL; 5DL stands for the long arm of wheat chromosome 5D). These two translocation chromosomes could be transmitted to next generation stably, and the wheat lines containing 5DS-4RS·4RL chromosome also displayed immunity to powdery mildew. The materials obtained in this study can be used for wheat powdery mildew resistant breeding program.     

含有抗白粉病基因的黑麦染色体小片段向小麦的转移

利用感白粉病的小麦品种绵阳11的纯系和黑麦自交系R12杂交, 在其单体附加系自交后代的BC₁F₅株系中选择小麦－黑麦异源易位系。根据已报道的黑麦特异重复序列pSc20H设计了一对特异引物, 用PCR方法鉴定了300个单体附加系的自交BC₁F₅株系,发现其中70个株系含有黑麦染色体成分。一个来源于6R单体附加系的小麦株系96Ⅱ691-830-98表现了对白粉病的高度抗性, PCR方法鉴定证明其含有黑麦染色体成分。对该株系作进一步的基因组原位杂交(GISH)鉴定, 证明它的一对染色体的端部含有黑麦染色体的小片段。这一结果指出, 含有抗白粉病基因的黑麦染色体6R小片段被引入了小麦。研究表明利用单体附加诱导染色体小片段易位是一种有效的方法。利用PCR和GISH原位杂交相结合的方法可提高检测外源染色体小片段的准确性和选择效率。     

小偃6号背景下黑麦遗传物质的荧光原位杂交分析

利用普通小麦(Triticum aestivum L.)“小偃6号”与黑麦(Secale cereale L.)品种“德国白粒”杂交,选育出“小偃6号”类型带有黑麦性状的种质材料。应用总基因组原位杂交(GISH)进行检测,在8份材料中探测到黑麦染色质的存在,其中附加系3个,代换系1个,易位系4个;进一步用荧光绿标记探针pSc119.2及荧光红标记探针pAs1的双色荧光原位杂交(FISH)技术,对其中部分品系的染色体组成进行分析鉴定,结果表明:易位系BC116-1是1RS/1BL小麦/黑麦易位系,BC152-1是涉及一条1B染色体的1RS/1BL易位系, 代换系BC97-2是2R(2D)二体代换系;附加系BC122-3附加了一条6R黑麦染色体,一条6B染色体的长臂缺失。同时,对连续的总基因组原位杂交和双色荧光原位杂交技术在小麦育种中的应用进行了讨论。     

三个小黑麦花粉株系的染色体组成分析与抗白粉病鉴定

对来自小黑麦与小麦杂种的３个花粉株系,ＤＨ２２０－４,ＤＨ２２０－５和ＤＨ２２０－１４进行了形态性状观察,染色体组成分析和抗白粉病鉴定。经过染色体形态和数目观察、原位杂交、Ｃ－分带、同工酶等电聚焦和贮藏蛋白的ＳＤＳ－聚丙烯酰胺凝胶电泳分析,证明其中两个株系,ＤＨ２２０－４和ＤＨ２２０－５是６Ｒ／６Ｄ代换系,另一个株系ＤＨ２２０－１４是１Ｒ／１Ｄ代换系。经人工接种鉴定,两个６Ｒ／６Ｄ代换系高抗白粉病。从而进一步证明黑麦的６Ｒ染色体上存在抗白粉病的基因。同时还对小麦遗传背景下异源染色体的识别及６Ｒ染色体的利用价值等问题进行了讨论。     

Role of rye chromosome 2R from wheat-rye substitution line 2R(2D)1 (Triticum aestivum L. cv. Saratovskaya 29-Secale cereale L. cv. Onokhoiskaya) in genetic regulation of meiotic restitution in wheat-rye polyhaploids

A study was made of the role of rye chromosome 2R from the wheat-rye substitution line 2R(2D)1 (Triticum aestivum L. cv. Saratovskaya 29-Secale cereale L. cv. Onokhoiskaya) in genetic regulation of meiotic restitution in wheat-rye polyhaploids 2R(2D)1 x S. cereale L. cv. Onokhoiskaya. Rye chromosome 2R proved to affect the completeness of the meiotic program, suppressing the formation of restitution gametes. This was evident from the reductional division of univalent chromosomes in AI and the occurrence of the second meiotic division. The interrelationships between the type of chromosome division in AI and the two-step character of meiosis are discussed. The structural and functional organization of the centromeric regions of chromosomes undergoing reductional division is assumed to determine the two-step character of division.     

The effect of rye chromosomes on callus induction and regeneration in callus cultures of immature embryos of wheat-rye substitution lines, Triticum aestivum L. cultivar Saratovskaia 29/Secale cereale L. cultivar Onokhoiskaia

The effect of individual rye chromosomes on the induction of callus and the character of its regenerating capacity was studied with cultured immature embryos of wheat-rye (Triticum aestivum L. cv. Saratovskaya 29-Secale cereale L. cv. Onokhoiskaya) substitution lines. The genotypic diversity of the substitution lines proved to significantly affect variation of parameters characterizing the major types of callus cultures, that is, frequencies of embryogenic calli, which are capable of shoot regeneration, and of morphogenic calli, which produce root structures. Functioning in the genotypic background of common wheat cultivar Saratovskaya, chromosomes 2R and 3R of rye cultivar Onokhoiskaya stimulated significantly the induction of embryogenic callus highly capable of shoot regeneration. Rye chromosome 2R present in place of chromosome 2D in the common wheat genome suppressed the induction of callus producing root structures. Rye chromosomes 1R and 6R suppressed the induction of embryogenic callus capable of shoot regeneration.     

Identification and development of diagnostic markers for a powdery mildew resistance gene on chromosome 2R of Chinese rye cultivar Jingzhouheimai

Chinese rye cultivar Jingzhouheimai (Secale cereale L.) shows a high level of resistance to powdery mildew. Identification, location, and mapping of the resistance gene would be helpful for developing a highly resistant germplasm or cultivar in wheat. Using sequential C-banding, GISH, and marker analysis, an addition chromosome with powdery mildew resistance was identified in a line derived from a cross between Chinese wheat landrace Huixianhong and rye cultivar Jingzhouheimai. The line, designated H-J DA2RDS1R(1D), had 44 chromosomes including two pairs of rye chromosomes, 1R and 2R, and lacked a pair of wheat chromosomes 1D, that is, it is a double disomic addition disomic substitution line. According to its reaction to different isolates of the powdery mildew pathogen, the resistance gene in H-J DA2RDS1R(1D) differed from the Pm8 and Pm7 genes located earlier on rye chromosomes 1R and 2R, respectively. In order to determine the location of the resistance gene, line H-J DA2RDS1R(1D) was crossed with wheat landrace Huixianhong and the F2 population and corresponding F2:3 families were tested for disease reaction and assessed with molecular markers. The results showed that a resistance gene, designated PmJZHM2RL, is located in rye chromosome arm 2RL.     

黑麦6R抗白粉病基因向小麦的渗进与鉴定

为了将黑麦６Ｒ染色体上抗小麦白粉病的基因导入小麦,选用了一个６Ｒ／６Ｄ代换系Ｍ２４为亲本之一,分别与小麦栽培品种和第６部分同源群缺体系杂交,杂种出现６Ｒ或／或６Ａ,６Ｂ,６Ｄ单,双或三单体等各种情况,取其花药进行培养,共获得２４１个再生植株,对其中３２个抗白粉病的花粉植株经染色体计数,Ｃ－分带,基因组原位杂交,同工酶等电聚焦电泳和或／ＲＦＬＰ分子标记检测,发现有６株仍保持为６Ｒ／６Ｄ代换系,有１０     

威岭栽培黑麦抗白粉病特性导入小麦的研究

威岭黑麦(Weiling rye)是一个高抗白粉病(Erysiphe gramininis f．sp．tritici)的中国矮杆栽培黑麦。以Weiling rye作为白粉病抗源,高感白粉病小麦栽培品种My8443为母本,从Weiling rye与小麦My8443远缘杂交的BC_2F_6后代中鉴定出一个新的小麦-黑麦易位系No．147,以实现威岭黑麦白粉病抗性向普通栽培小麦的转移。No．147及其亲本的抗白粉病特性通过苗期和成株期优势生理小种混合接种和室内单生理小种接种鉴定,改良的染色体C-分带和基因组原位杂交技术(GISH。Ge- nomic in situ hybridization)被用于鉴定小麦和黑麦的染色质,酸性聚丙烯酰胺凝胶电泳(APAGE)被用于鉴定黑麦醇溶蛋白1RS特异条带,11个黑麦种属特异性标记SCM(Secale cereale marker)引物被用于扩增分析黑麦特异性简单重复序列(SSR)。研究结果证实No．147是一个新的高抗白粉病的1BL/1RS小麦-黑麦染色体易位系,并对其产生的细胞学机制进行了分析。论文对中国栽培黑麦抗性基因资源的利用和该易位系在小麦遗传育种改良中的利用价值进行了讨论。     

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.     

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.     

The chromosomal location of rye AFLP bands

23 AFLP bands were assigned to different rye chromosomes by means of two different sets of wheat-rye addition lines. Only one AFLP band could be assigned to 4R, and no specific AFLPs were found on the 5R chromosome. Only one AFLP band was explicitly assigned to 4R, and no specific AFLPs were found on the 5R chromosome. At least seven co-migrating AFLPs showed the same chromosomal location in both sets of addition lines. A total of 22 AFLPs were assigned to chromosome 1R using wheat-rye substitution lines. Six of them have counterparts in one of the addition lines analyzed, but only four have the same chromosomal location. Six and four of the total AFLPs located using addition (23) and substitution (22) lines segregated in the mapping population DS2 x RXL10, but only six were simultaneously assigned to the same chromosome by both approaches. Although co-migrating AFLPs could be located on different rye chromosomes using addition and substitution lines, we believe that AFLPs can be useful as rye chromosome markers.     

Features of crossability, haploidy and polyembryony in hybrid combinations between common barley Hordeum vulgare L. (2n = 14) and wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya

The role of individual chromosomes of rye in the manifestation of crossability and seedling development in hybrid combinations between common barley Hordeum vulgare L., cultivar Nepolegayushchii (2n = 14) and five wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya (2n = 40 wheat + 2 rye chromosomes). Crossability, which was measured by two parameters--frequency of set grains and frequency of grains with embryos--was shown to be significantly affected by each of the five rye chromosomes examined: 1R, 2R, 3R, 5R, and 6R; the development of barley haploids was affected by rye chromosomes 1 R, 3R, and 5R. We were the first to demonstrate that polyembryony could be induced by mutual effects of barley cytoplasm and rye chromosome 1R. Possible mechanisms controlling the development of haploids and twins in hybrid combinations H. vulgare x T. aestivum/S. cereale are discussed. The conclusion is drawn that hybrid combinations between common barley and wheat-rye substitution lines can serve as new models for studying incompatibility mechanisms in distant crosses and genetic control of parthenogenesis.     

Effect of rye Secale cereale L. chromosomes 1R and 3R on polyembryony expression in hybrid combinations between (Hordeum vulgare L.)-Triticum aestivum L. alloplasmic recombinant lines and wheat T. aestivum L.-rye S. cereale L. substitution lines

The effect of rye chromosomes on polyembryony was studied for reciprocal hybrid combinations between (Hordeum vulgare L.)-Triticum aestivum L. alloplasmic recombinant lines and five wheat T. aestivum L. (cultivar Saratovskaya 29)-rye Secale cereale L. (cultivar Onokhoiskaya) substitution lines: IR(1D), 2R(2D), 3R(3B), 5R(5A), and 6R(6A), and for direct hybrid combinations between the [H. marinum ssp. gussoneanum (H. geniculatum All.)]-T. aestivum alloplasmic recombinant line and the wheat-rye substitution lines 1R (1A), 1R (1D), and 3R(3B). Chromosomes 1R and 3R of rye cultivar Onokhoiskaya proved to affect the expression of polyembryony in the hybrid combinations that involved the alloplasmic recombinant lines of common wheat as maternal genotypes. Based on this finding, polyembryony was regarded as a phenotypic expression of nuclear-cytoplasmic interactions where an important role is played by rye chromosomes 1R and 3R and the H. vulgare cytoplasm. Consideration is given to the association between the effect of rye chromosomes 1R and 3R on polyembryony in the [(Hordeum)-T. aestivum x wheat-rye substitution lines] hybrid combinations and their stimulating effect on the development on angrogenic embryoids in isolated anther cultures of the wheat-rye substitution lines.     

Role of rye chromosome 2R from wheat-rye substitution line 2R(2D)1 ( Triticum aestivum L. cv. Saratovskaya 29- Secale cereale L. cv. Onokhoiskaya) in genetic regulation of meiotic restitution in wheat-rye polyhaploids

A study was made of the role of rye chromosome 2R from the wheat-rye substitution line 2R(2D)₁ (Triticum aestivum L. cv. Saratovskaya 29-Secale cereale L. cv. Onokhoiskaya) in genetic regulation of meiotic restitution in wheat-rye polyhaploids 2R(2D)₁ × S. cereale L. cv. Onokhoiskaya. Rye chromosome 2R proved to affect the completeness of the meiotic program, suppressing the formation of restitution gametes. This was evident from the reductional division of univalent chromosomes in AI and the occurrence of the second meiotic division. The interrelationships between the type of chromosome division in AI and the two-step character of meiosis are discussed. The structural and functional organization of the centromeric regions of chromosomes undergoing reductional division is assumed to determine the two-step character of division. Original Russian Text ? O.G. Silkova, A.I. Shchapova, V.K. Shumny, 2007, published in Genetika, 2007, Vol. 43, No. 7, pp. 971–981.     

利用杀配子染色体2C诱导中国春-黑麦二体附加系染色体畸变的研究

将中国春-黑麦(1R-7R)二体附加系与中国春-2C(Aegilops cylindrica)二体附加系杂交,获得F1,对F1体细胞染色体进行C分带鉴定和花粉母细胞减数分裂行为的观察与分析,发现减数分裂行为异常。对自交获得的430株F2进行单株染色体C分带和荧光原位分子杂交鉴定,检测到易位、缺失、等臂染色体、双着丝点染色体等染色体畸变类型。此外还检测到2C与小麦2A、2B、2D染色体的二体或单体自发代换系。杂交F。染色体畸变的规律与频率如下：研究共得到含黑麦染色体的变异22株,变异频率为5,1％。其中含黑麦染色体的易位系为10株,占2,3％;缺失12株,占2．79％;黑麦的等臂染色体3株,占O．7％。易位染色体既有含小麦着丝点的(大部分),也含有黑麦着丝点的(仅1例)。黑麦的染色体畸变中,发生于不同同祖群的频率不同,1R为5个,2R为3个;3R为1个;4R为3个;5R为6个;6R为4个。易位多为端部易位。共鉴定出小麦的缺失系54株,其中A基因组有27个,占6.27％;B基因组有20个,占4,65％;D基因组有7个,占1.66％。对杀配子染色体对小麦及黑麦不同同祖群染色体作用的差异性及作用特点进行了探讨。     

小麦-黑麦代换系5A/5R与6A/6R杂交诱导同祖染色体配对与易位的研究

利用两个小麦-黑麦异源双代换系DS 5A/5R与DS 6A/6R杂交,探讨同祖染色体配对的可能性与创制小麦黑麦异源易位系.在方法上对杂种F1的减数分裂行为进行研究,观察5R与5A、6R与6A配对频率,探讨同祖染色体配对规律.实验结果看到杂交F1减数分裂中有22.91%的花粉母细胞有小麦染色体(ABD组)与黑麦染色体(R组)发生同祖配对.在F2及以后世代,通过染色体C分带、原位杂交检测,选择小麦-黑麦易位系.在F2代的45株中检测到9株有易位,易位频率为20%,是目前小麦-黑麦染色体易位频率最高的.染色体易位有的来源于同祖配对的交换,有的来源于单价体错分裂或断裂的重建.     

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.     

小麦主栽品种中的1RS分布和兰考90(6)系列白粉病新抗源

利用黑麦染色体臂1RS的特异性PCR标记,对黄淮麦区138个小麦主栽品种、系进行了PCR扩增,结果表明：有42．0%的小麦品种、系携带1RS染色体臂。以六倍体小黑麦Mzalenod Beer为黑麦染色体供体,培育的兰考90(6)系列小麦品系是新的小麦-黑麦1BL／1RS易位系。这些品系对小麦白粉病具有很高的抗性,是小麦抗白粉病育种的新抗源。对兰考90(6)系列品系白粉病抗性进行了研究,结果表明,兰考90(6)系列品系的抗谱与许多已经知道的小麦抗白粉病基因的抗谱不同,并具有数量抗性特点。