抗白粉病小偃麦异代换系的细胞学和RAPD鉴定

利用细胞学和RAPD方法,对从长穗偃麦草与普通小麦复合杂交后代中选育的抗白粉病小麦种质系山农87074－526和山农87074－551进行了鉴定。结果表明,两种质系的根尖细胞染色体数目均为2n=42,花粉母细胞减数分裂中期I（PMC MI）染色体构型为2n=21Ⅱ;二者杂交F1 PMC MI染色体构型亦为2n=21Ⅱ,两种质系分别与小麦中国春的杂种F1 PMC MI染色体构型均为2n=20Ⅱ 2I,说明两种质系为相同的双体异代系。在苗期和成株期两种质系对白粉病15号菌种均表现免疫,其白粉病抗性为显性,并且来自长穗偃麦草,抗白粉病基因位于它们所含的偃麦草染色体上。从80个随机引物中,筛选出2个引物OPE13和OPH15能在两种质系中稳定地扩增出长穗偃麦草亲本的特异DNA片段。     

抗白粉病耐盐小麦-中间偃麦草附加系‘山农120211’的鉴定

以中间偃麦草(Thinopyrum intermedium,2n=42)与普通小麦‘烟农15’杂交,从其杂种后代中选育出一个细胞学稳定的二体异附加系‘山农120211’,该研究对其细胞学和主要性状特点进行了鉴定。白粉病抗性鉴定结果表明,‘山农120211’成株期对白粉病的田间抗性为免疫,苗期对白粉病菌种E09表现为免疫。以耐盐品种‘山融3号’为对照进行苗期耐盐性鉴定表明,‘山农120211’耐盐级别为2级(较强)。细胞学鉴定表明:‘山农120211’根尖细胞染色体数目为2n=44,PMC MI染色体构型为2n=22Ⅱ,具有高度的细胞学稳定性。以拟鹅观草基因组DNA为探针,‘烟农15’DNA为封阻,在‘山农120211’的根尖有丝分裂细胞中检测到2条染色体具有明显的杂交信号,确定其为二体异附加系。利用该实验室筛选的71对E组染色体特异分子标记,对‘山农120211’分析显示,标记BE494262在中间偃麦草和‘山农120211’中可以稳定扩增出1条440bp特异带,而‘烟农15’中缺少此带,BE494262可作为‘山农120211’中附加中间偃麦草染色体的特异标记。利用二倍体长穗偃麦草和一套中国春-长穗偃麦草异附加系(1Ee~7Ee),进一步将BE494262定位在2Ee染色体,确定‘山农120211’所附加的中间偃麦草染色体为2Ee染色体。     

兼抗白粉和条锈病小滨麦种质系山农6343的细胞学和SSR鉴定

利用抗性接种鉴定、细胞学和SSR分子标记技术相结合的方法,对从八倍体小滨麦和普通小麦烟农15杂种后代选育出的兼抗白粉病和条锈病的小滨麦种质系山农6343进行了鉴定.结果表明,山农6343的根尖细胞染色体数目2n=42,花粉母细胞减数分裂中期I(PMC MI)绝大多数细胞内可观察到21个二价体,平均染色体构型为2n=21Ⅱ,与普通小麦烟农15杂种F1的花粉母细胞内观察到2n=19Ⅱ+1Ⅳ的染色体构型,四价体出现频率为24.1%.利用SSR分子标记技术,在1283对SSR和EST-SSR引物中筛选出两对特异引物BARC236-4A和KSUM134,均能稳定地在山农6343中扩增出滨麦草的特异标记BARC236255和KSUM134245,且两个标记在小滨麦易位系山农0096中得到了验证.初步确定山农6343是一个小滨麦易位系.由于在目前已命名的小麦白粉病和条锈病抗性基因中尚未有来自滨麦草的,推测山农6343可能为新的白粉病和条锈病抗源,对小麦白粉病和条锈病的抗性遗传改良将具有重要的利用价值.     

普通小麦-中间偃麦草TAI-27中附加染色体的显微切割及特异性探针的筛选

试验以长穗偃麦草基因组DNA为探针 ,与普通小麦 中间偃麦草TAI 2 7进行染色体原位杂交 ,表明有 4条与长穗偃麦草同源的染色体 ;以P .stipifolia (St)基因组DNA为探针 ,有 4条与St同源的染色体 .这说明TAI 2 7中有 4条St染色体 .TAI 2 7是异代换 附加系 .对TAI 2 7中附加的中间偃麦草染色体进行显微切割 ,并建立其微克隆库 ,从中筛选获得了中间偃麦草的特异性探针 ,同源性分析表明该序列为一新序列 .这为进一步筛选抗病、抗逆和优质基因打下基础 .     

普通小麦与长穗偃麦草杂种及其衍生材料的细胞遗传学特性

对十倍体长穗偃麦草(Thinopyrum ponticum)与普通小麦杂交F1及其与普通小麦回交BC1F1的形态学和细胞学特性进行了分析。结果表明,长穗偃麦草与普通小麦‘兰考矮早八’衍生F1(‘兰考小偃麦’)的根尖细胞染色体数为56条;花粉母细胞减数分裂中期Ⅰ染色体构型平均值为19.81Ⅰ+15.78Ⅱ+0.75Ⅲ+0.59Ⅳ;基因组荧光原位杂交(GISH)显示,兰考小偃麦中含有35条完整的长穗偃麦草和21条小麦染色体。‘兰考小偃麦’/‘科育818’和‘兰考小偃麦’/‘Cp02-3-5-5’杂交F1的根尖细胞染色体数及其所遗传的长穗偃麦草染色体数分别为50~52和16~22条,且存在染色体易位;花粉母细胞减数分裂中期Ⅰ平均染色体构型为14.54Ⅰ+17.40Ⅱ+0.55Ⅲ+0.14Ⅳ,平均49.4%的细胞出现多价体(三价体或四价体)。这些材料为创造小麦-长穗偃麦草新种质奠定了基础。     

一个异源胞质单体附加系小麦的遗传分析

以长穗偃麦草(Elytrigia elongata=Agropyron elongatum,2n=70)为母本,普通小麦为父本,进行核代换回交,在第九次回交的F_1(BC,F_1)代里,发现了一个异源胞质单体附加系小麦,代号E617。以E617做母本,普通小麦做父本进行杂交,杂种F_1出现正常株和弱株两种类型。正常株具有43条染色体,弱株具有42条染色体。反交,以普通小麦做母本,E617做父本,杂种F_1,无论是具有43条染色体还是具有42条染色体的植株,都是正常的。正反杂交结果表明,长穗偃麦草细胞质对普通小麦的细胞核产生了抑制作用,使核质杂种生长不正常,而附加一条特异的长穗偃麦草染色体(或端体),就能消除长穗偃麦草细胞质的抑制作用,使核质杂种恢复正常生长。     

小偃麦异代换系山农0095辐照花粉后代的细胞学及SSR标记分析

本研究采用^60Co-γ射线辐照小偃麦异代换系山农0095花粉,对其M1、M2代的细胞遗传学特点进行分析,结果表明：M1、M2代均出现频率不同和染色体数目不等（2n=41、2n=40和2n：39）的染色体数目变异类型;在两个世代的花粉母细胞减数分裂过程中．普遍观察到单价体、多价体、染色体片段、落后染色体、染色体桥及微核等现象,说明辐射有效地促进了染色体数目和结构的改变,有可能导致染色体易位重组。利用已建立的山农0095中中间偃麦草染色体的特异SSR标记BARC159240对M2代进行检测,结果发现大部分M2单株仍含有该标记位点,说明这些植株仍然具有标记位点的中间偃麦草染色体特异区段：少部分单株虽然保留了该位点．但缺少普通小麦中的条带．这些单株可能发生了染色体重组。     

一个小麦-中间偃麦草异代换系的形态学和细胞学鉴定

中间偃麦草含有丰富的优良基因,在小麦的遗传改良中具有重要利用价值。对从中间偃麦草与小麦品种烟农15杂种后代(BC2F4)中选育的小麦种质系山农0095进行形态学和细胞学鉴定,结果表明：山农0095株高78cm,穗长17．3cm,旗叶长36．3cm,旗叶宽3．03cm,茎杆粗壮,繁茂性好,既长又宽的旗叶、长圆锥型穗是其显著的形态学特征;其根尖细胞染色体数日为2n=42,花粉母细胞减数分裂中期Ⅰ(PMC M Ⅰ)染色体构型为2n=21Ⅱ;它与普通小麦的杂种FⅠPMC M Ⅰ绝大多数细胞出现2个单价体,没有观察到多价体,平均染色体构型为2n=20．08Ⅱ 1．84Ⅰ。以上结果表明,山农0095是一个小麦-中间偃麦草的双体异代换系。     

兼抗白粉、条锈病小偃麦渗入系CH7124抗性遗传及细胞学鉴定

CH7124是通过八倍体小偃麦TAI8335与感病小麦杂交、回交育成的兼抗白粉病、条锈病的小偃麦种质系。利用抗性接种鉴定、细胞学和基因组原位杂交(GISH)技术相结合的方法,对CH7124的抗性来源、遗传方式及细胞学特征进行了分析和鉴定。结果表明,CH7124在苗期和成株期对条锈菌系CYR29、CYR31、CYR32、CYR33和白粉菌系E09、E20、E21、E26表现为免疫或近免疫,其抗性来自中间偃麦草,受1对显性核基因控制;CH7124的根尖细胞染色体数目为2n=42,花粉母细胞减数分裂中期I(PMC MI)绝大多数细胞内可观察到21个二价体,平均配对构型为2n=0.30 I+20.79 II+0.04 III;与普通小麦中国春、绵阳11的杂种F1中,有80%以上的花粉母细胞可观察到2n=21Ⅱ的染色体构型,其平均配对构型均为2n=21II。说明CH7124具有与普通小麦相似的染色体结构和规则的配对构型。由于利用以中间偃麦草总DNA为标记探针的原位杂交未观察到可见的外源DNA杂交信号,进一步证明CH7124是一个小麦-中间偃麦草的隐形异源渗入系。     

拟鹅观草St基因组ISSR特异标记的建立

以拟鹅观草(Pseudoroegneria spicata)、中间偃麦草(Thinopyrum intermedium)、长穗偃麦草(Th.elonga-tum)、二倍体簇毛麦(Dasypyrum villosum)、澳冰草(Australopyrum retrofractum)等10份小麦族物种为材料,对100条ISSR引物进行分析,结果显示引物811可以在拟鹅观草(GenBank登录号为EU368859)和中间偃麦草中扩增出一条长441 bp的特异DNA片段命名为St441,而其它供试物种均未扩出.经序列比对、软件分析结合原位杂交结果认为St441为一类新的低拷贝重复序列.利用ISSR引物811对10份不同居群的中间偃麦草、20份披碱草属物种、4份小麦-偃麦草部分双二倍体、6份小麦-茸毛偃麦草后代和12份小麦对照进行扩增,结果发现除对照小麦外均能扩增出St441;进而对小麦-中间偃麦草两套附加系进行扩增,将St441初步定位于包括第四同源群在内的8条St染色体上.同时,发现只含有整条St染色体和St染色体片段的材料能扩增出St441,而仅有Js染色体的材料未扩增出St441.因此,该标记St441可以作为检测不同背景下St染色质的分子标记.     

A FISH karyotype to study chromosome polymorphisms for the Elytrigia elongata E genome

Elytrigia elongata (Host) Nevski(= Agropyron elongatum, Thinopyrum elongatum, 2n = 2x = 14, EE) has long been used as a source of various types of resistance for wheat improvement, and numerous transfers have been made. However, despite heavy use, no high-resolution karyotype exists. We characterized the E. elongata karyotype of several accessions applying highly repetitive DNA sequences as mcFISH probes for chromosome identification. The complete E. elongata disomic chromosome addition series and 11 ditelosomic addition lines in Chinese Spring wheat were exposed to sequential GISH-mcFISH. Based on the mcFISH results, each complete chromosome and each telocentric studied was unambiguously identified. The validation of the karyotype in 4 E. elongata accessions with different geographical origins showed extensive variations in the probe hybridization patterns, but this did not prevent chromosome identification. The established karyotype will be useful for the rapid identification of potential donor chromosomes in wheat improvement programs, allowing appropriate alien transfer.     

小麦—中间偃麦草抗条锈衍生系的分子细胞遗传学研究

应用缺体回交法,以部分阿勃缺体为母本,中4为父本,培育出1个对目前条锈病优势小种和新小种高抗至免疫的小麦－－中间偃麦草衍生系N9025－3－3－2－1－1。研究表明,该选系在形态学和细胞学上已经基本稳定,染色体构型为2n=42=21“,抗病性来自中间偃麦草（Thinopyron intermedium）。以中间偃麦草DNA为探针,对N9025－3－3－2－1－1进行基因组原位杂交分析结果证明,它为小麦－中间偃麦草异代换－易位系。     

Physical mapping of the blue-grained gene(s) from Thinopyrum ponticum by GISH and FISH in a set of translocation lines with different seed colors in wheat.

The original blue-grained wheat, Blue 58, was a substitution line derived from hybridization between common wheat (Triticum aestivum L., 2n=6x=42, ABD) and tall wheatgrass (Thinopyrum ponticum Liu & Wang=Agropyron elongatum, 2n=10x=70, StStEeEbEx), in which one pair of 4D chromosomes was replaced by a pair of alien 4Ag chromosomes (unknown group 4 chromosome from A. ponticum). Blue aleurone might be a useful cytological marker in chromosome engineering and wheat breeding. Cytogenetic analysis showed that blue aleurone was controlled by chromosome 4Ag. GISH analysis proved that the 4Ag was a recombination chromosome; its centromeric and pericentromeric regions were from an E-genome chromosome, but the distal regions of its two arms were from an St-genome chromosome. On its short arm, there was a major pAs1 hybridization band, which was very close to the centromere. GISH and FISH analysis in a set of translocation lines with different seed colors revealed that the gene(s) controlling the blue pigment was located on the long arm of 4Ag. It was physically mapped to the 0.71-0.80 regions (distance measured from the centromere of 4Ag). The blue color is a consequence of dosage of this small chromosome region derived from the St genome. We speculate that the blue-grained gene(s) could activate the anthocyanin biosynthetic pathway of wheat.     

The 3Ns chromosome of Psathyrostachys huashanica carries the gene(s) underlying wheat stripe rust resistance

Stripe rust (Puccinia striiformis tritici (Pst)) is one of the most destructive diseases of wheat in the world. Exploiting and utilizing stripe rust resistance genes of wild species has become an essential strategy for resistance breeding. Psathyrostachyshuashanica Keng ex Kuo is a wild species in Triticeae that has been used for wheat improvement because of its high resistance or immunity to stripe rust. In this study, 9 wheat-P. huashanica addition lines were characterized by Giemsa C-banding, genomic in situ hybridization (GISH), and disease resistance evaluation. Giemsa C-banding and GISH demonstrated that lines 163-5, 165-1, 183-5, 240-3, and 240-4 are P. huashanica 3Ns chromosome monosomic addition lines; lines 183-1 and 183-20 are P. huashanica 3Ns chromosome disomic addition lines; line 165-20 is a P. huashanica 3Ns and 4Ns chromosomes double disomic addition line, and line 219-1 is a P. huashanica 1Ns and 3Ns/5A chromosomes double disomic addition-substitution line. All these addition lines with P. huashanica 3Ns chromosome(s) expressed high resistance or immunity to stripe rust. By comparing the series of wheat-P. huashanica chromosome addition lines, we concluded that the P. huashanica 3Ns chromosome carries the gene(s) for resistance or immunity to stripe rust. These addition lines can be used as a donor source of novel stripe rust resistance to wheat breeding programs.     

携带抗黄矮病基因的中间偃麦草染色体2Ai—2特异的分子标记

小麦－中间偃麦草二体异附加系Ｚ１、Ｚ２具有一对携带抗黄矮病基因的中间偃麦草染色体２Ａｉ－２。利用中间偃麦草（Ｔｈｉｎｏｐｙｒｕｍ ｉｎｔｅｒｍｅｄｉｕｍ （Ｈｏｓｔ） Ｂａｋｗｏｔｈ ａｎｄ Ｄｅｗｅｙ）和拟鹅冠草（Ｐｓｅｕｄｏｒｏｅｇｎｅｉａ ｓｔｒｉｇｏｓａ）基因组ＤＮＡ作探针,对Ｚ１、Ｚ２进行基因组原位杂交分析。结果表明,Ｚ１、Ｚ２附加的一对中间偃麦草染色体２Ａｉ－２为Ｓｔ－Ｅ染色体,Ｅ组染     

Molecular cytogenetic characterization of two high protein wheat-<Emphasis Type="Italic">Thinopyrum intermedium</Emphasis> partial amphiploids

Fluorescence and genomic in situ hybridization (FISH and GISH) were used to establish the cytogenetic constitution of two wheat × Thinopyrum intermedium partial amphiploids H95 and 55(1-57). Both partial amphiploids are high-protein lines having resistance to leaf rust, yellow rust and powdery mildew and have in total 56 chromosomes per cell. Repetitive DNA probes (pTa71, Afa family and pSc119.2) were used to identify the individual wheat chromosomes and to reveal the distribution of these probes within the alien chromosomes. FISH detected 6B tetrasomy in H95 and a null (1D)-tetrasomy (1B) in 55(1-57). GISH was carried out using biotin labeled Th. intermedium DNA and digoxigenin labeled Pseudoroegneria spicata DNA as probes, subsequently. GISH results revealed 44 wheat chromosomes and four Thinopyrum chromosome pairs, including three S and one J chromosome pairs in line H95. Line 55(1-57), contained 42 wheat chromosomes and six Th. intermedium pairs, including two S and one J^S pairs. Additionally, two identical translocated chromosome pairs with diminished affinity to the alien chromatin were detected in both amphiploids. Another two translocations were found in 55(1-57), with satellite sections from the Thinopyrum J genome.     

Characterization of a partial wheat-Thinopyrum intermedium amphiploid and its reaction to fungal diseases of wheat

Partial amphiploids between wheat (Triticum aestivum L.) and Thinopyrum species play an important role in the transfer and use of traits from alien species. A wheat-Thinopyrum intermedium partial amphiploid, TAI8335, and its alien parent were characterized by a combination of genomic in situ hybridization (GISH) and cytological observations. Evidence from GISH indicated that the donor parent Th. intermedium possessed seven pairs of S, seven J(s) and 21 J chromosomes. Mitotic observation showed that the majority of TAI8335 plants had 56 chromosomes, but a few had 54 to 55, in some cases with two to three additional telochromosomes. The chromosomes in most pollen mother cells of plants with 2n = 56 formed 28 bivalents, averaging 27.12 in 223 cells, suggesting a basic cytological stability. Sequential GISH patterns using genomic Pseudoroegneria spicata and genomic Th. intermedium DNA as probes revealed that TAI8335 had fourteen chromosomes derived from Th. intermedium and its alien genome consisted of one pair of S-, three pairs of J(s) - and one pair of J-genome chromosomes as well as two translocated chromosome pairs, one being a Robertsonian translocation and another an intercalary translocation, both of which involved J and S genome. Two of the telochromosomes in the aneuploid plants originated from the J genome and one from wheat. Disease screening demonstrated this line was highly resistant to leaf rust, stem rust, stripe rust and powdery mildew. This study showed that the partial amphiploid TAI8335 appears to serve as a novel source for the transfer of resistance genes for multiple fungal pathogens to wheat.     

Molecular characterization of a wheat--Thinopyrum ponticum partial amphiploid and its derivatives for resistance to leaf rust.

Leaf rust (caused by Puccinia triticina Eriks.) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum (Podp.) Z.-W. Liu & R.-C. Wang has provided several leaf rust resistance genes to protect wheat from this fungal disease. Three chromosome substitution lines, Ji806, Ji807, and Ji859, and two chromosome addition lines, Ji791 and Ji924, with a winter growing habit were developed from crosses between wheat (Triticum aestivum L. em Thell.) and the wheat - Th. ponticum partial amphiploid line 693. These lines were resistant to leaf rust isolates from China. Sequence-tagged site (STS) analysis with the J09-STS marker, which is linked to the gene Lr24, revealed that the partial amphiploid line 693 and all of the substitution and addition lines carried gene Lr24. Genomic in situ hybridization (GISH) analysis was carried out on chromosome preparations using total genomic DNA from Pseudoroegneria strigosa (M. Bieb) A. L?ve (St genome, 2n = 14) as a probe in the presence of total genomic DNA from T. aestivum 'Chinese Spring' wheat (ABD genomes, 2n = 42). The GISH analysis demonstrated that these lines had a pair of chromosomes displaying the typical pattern of a Js genome chromosome. This indicates that the chromosome that carries gene Lr24 belonged to the Js genome of Th. ponticum. In addition to 40 wheat chromosomes, eight Js and eight J genome chromosomes were also differentiated by GISH in the partial amphiploid line 693. Since most sources of Lr24 have a red grain color, the white-colored seeds in all of these substitution and addition lines, together with high protein content in some of the lines, make them very useful as a donor source for winter wheat breeding programs.     

携带抗黄矮病基因的中间偃麦草染色体2Ai_2特异的分子标记

The wheat-Thinopyrum intermedium addition lines Z1,Z2 contain a pair of Th. intermedium chromosomes 2Ai-2 carrying the gene with resistance to barley yellow dwarf virus (BYDV). Genomic in situ hybridization (GISH) was used to analyze the chromosome constitution of Z1,Z2 by using genomic DNA probes from Th. intermedium and Pseudoroegneria strigosa. The results showed that the chromosome constitution of either Z1 or Z2 composes of 42 wheat chromosomes and two Th. intermedium chromosomes (2Ai-2). The 2Ai-2 chromosome is St-E intercalary translocation, in which the E genomic chromosome segment translocated into the middle region of the long arm of chromosome belonging to St genome. With the genomic DNA probe of Ps. strigosa, the GISH pattern specific to the 2Ai-2 chromosome may be used as a molecular cytogenetic marker. A detailed RFLP analysis on Z1, Z2 and their parents was carried out by using 12 probes on the wheat group 2 chromosomes. Twenty RFLP markers specific to the 2Ai-2 chromosome were identified. Two RAPD markers of OPR16 –350 and OPH09 -1580, specific to the 2Ai-2 chromosome, were identified from 280 RAPD primers. These molecular markers could be used to assisted-select translocation lines with small segment of the 2Ai-2 chromosome and provide tools to localize the BYDV resistance.