簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。     

簇毛麦染色体组特异性RAPD标记的筛选、定位和应用

以普通小麦中国春、中国春－簇毛麦二体附加系以及不同来源的簇毛麦为材料,用100个10碱基随机引物进行RAPD扩增。引物OPF02能在不同来源的簇毛麦及所有中国春－簇毛麦二体附加系中扩增出一条长约750bp的片段OPF02 750。普通小麦和硬粒小麦不能扩增出该片段。因此,OPF02 750为分布于簇毛麦所有染色体上的一个簇毛麦染色体组特异片段。用引物OPF02对普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体以及几个普通小麦的簇毛麦二体代换系、二体附加系进行检测,发现NAU302已经丢失了其所附加的簇毛麦3V染色体。     

普通小麦各染色体组有效利用土壤磷基因的遗传分析

以部分中国春双端体为材料用土培盆栽法对小麦各染色体组有效利用土壤潜在磷基因的遗传分析表明：小麦不同染色体臂上所携基因对小麦有效利用土壤潜在磷特性具有不同效应,在供试材料中,Ｂ组染色体所缺失的臂在缺磷下对籽粒产量的贡献较大,其中以４Ｂｓ、５Ｂｓ,效应最强,而Ｄ组所有缺失的染色体臂及大部分Ａ组所缺失的染色体臂在缺磷下则对籽粒产量有较强的抑制效应,其中以５Ｄｓ、３ＤＬ及２ＡＬ,１Ａｓ的效应最强。     

VE161小麦的外源染色体鉴定

通过染色体原位杂交、ＲＦＬＰ分析和染色体重双端体分析,对在小麦遗传育种研究中具有重要理论和应用价值的ＶＥ１６１小麦不育异代换系及其附加系进行了染色体鉴定。结果表明,ＶＥ１６１小麦代换的或附加的外源染色体为来自长穗偃麦草的４Ｅ染色体,被代换的小麦染色体为４Ｂ。过去曾鉴定为７Ｂ,可能是由于ＶＥ１６１早代染色体易位较多所致。同时发现,ＶＥ１６１小麦在５Ｂ,７Ｂ和１Ｄ所在的３个部分同源群中仍有染色体相互易     

小麦耐盐突变体的RAPD分析

以冀麦２４和其耐盐的一代８９０１－１７为材料,用ＲＡＰＤ技术对其进行比较分析研究。在的用的２１５个引物中有５１个扩增出多态性,既有量的差异又有质的差异,初步断定此５１个引物扩增出的多态性与突变有关,为进一步用ＢＳＡ方法细筛出与耐盐突变体紧密连锁的ＲＡＰＤ分子标记下打下基础,同时,还分析了影响ＲＡＰＤ扩增结果的因素。     

抗白粉病小麦——中间偃麦草异附加系的细胞学和RAPD鉴定

利用细胞学和ＲＡＰＤ技术,对从小麦与中间偃麦草杂种后代中选育的抗白粉病异附加系ＤＡＬ６６进行了鉴定。结果证明ＤＡＬ６６根尖细胞染色体数为４４,花粉母细胞减数第一分裂中期（ＰＭＣ ＭＩ）杂色体模型为２ｎ＝２２Ⅱ。对ＤＡＬ６６及其双亲进行ＲＡＰＤ分析,从４０个随机引物中筛选出１个特异引物（ＯＰＥ－０２）能够稳定地扩增出特异带型。     

控制小麦种、属旗叶水分利用效率的染色体背景分析

利用ＬＣＡ－３便携式光合仪对不同染色体背景的材料测定表明,就旗叶水分利用效率（ＷＵＥ）而言,不同染色体组的上排序为ＡＡ＞ＢＢ＞ＤＤ＞ＲＲ,说明Ａ组染色体上载有控制高ＷＵＥ的基因,对中国春双端体系列的研究表明,Ａ组染色体无论在缺失长臂和短臂时,其端体也保持较高的旗叶ＷＵＥ,在１ＡＬ、２ＡＬ、２ＡＳ、７ＡＳ染色体臂上载有控制高ＷＵＥ的基因。对小黑麦附加系的研究表明,４Ｒ染色体上载有控制高ＷＵＥ的基因,     

大麦6H染色体特异性标记的筛选和鉴定

从大麦、小麦和小麦－大麦６Ｈ染色体附加系ＲＡＰＤ分析筛选出对６Ｈ染色体特异的２个ＲＡＰＤ标记,转换为特异性ＰＣＲ标记,利用标记对不同植物材料进行ＰＣＲ扩增鉴定。表明凡含有大麦６Ｈ染色体的材料（Ｂｅｔｚｅｓ、Ｉｇｒｉ、ＣＳ６Ｈ附加系）均能扩增出特异带;而不含６Ｈ染色体的材料,包括小科、黑麦、长穗偃麦草、中间偃麦草、簇毛麦以及含有其他大麦染色体的小麦附加系均不主增出特异带。可见,２对ＰＣＲ引物具有大麦     

一种小麦蓝粒标记单体代换系4E（3D）的创制

以中国春3D单体和小麦-长穗偃麦草4E二体异附加系为材料,通过杂交、回交结合染色体鉴定等方法,培育出了一种具有蓝粒标记的小麦4E(3D)单体代换系.该小麦4E(3D)单体代换系籽粒为浅蓝色,能够正常生长,自交结实率为36.1%,其自交后代可分离出深蓝籽粒小麦4E(3D)二体代换系、浅蓝籽粒小麦4E(3D)单体代换系和白粒小麦3D缺体.结果表明,长穗偃麦草4E染色体对小麦3D染色体缺失有一定的补偿功能,对以染色体定向代换方式快速创制蓝粒标记小麦单体系统具有一定的参考价值.     

山羊草属五个基本基因组系统发育的RAPD分析

利用ＲＡＰＤ技术,从ＯＰＥ、ＯＰＦ、ＯＰＧ、ＯＰＵ、ＯＰＸ、ＯＰＹ和ＯＰＺ共７组随机引物中筛选出２８个能产生基因组特异带的稳定引物,对山羊草属（ＡｅｇｉｌｏｐｓＬ．）的５个基本基因组及普通小麦“中国春”的ＤＮＡ进行随机扩增,根据扩增的４８８条ＤＮＡ片段绘制出系统发育图。普通小麦ＡＢＤ基因组与Ｓ基因组亲缘关系最近,Ｃ与Ｕ基因组具有比较近的亲缘关系,Ｄ基因组与其它基因组的亲缘关系比较远     

Chromosomal location of a K/Na discrimination character in the D genome of wheat

Summary K/Na ratios have been determined in the leaves of salt-treated plants of 14 disomic substitution lines in which each of the D-genome chromosomes replaces the homoeologous A- or B-genome chromosome in the tetraploid wheat variety Langdon (AABB genome). Aneuploid lines of hexaploid bread wheat (cv Chinese Spring) having a reduced or an enhanced complement of chromosome 4D have also been examined. These investigations show that the gene(s) determining K/Na ratios in the leaves of wheat plants grown in the presence of salt is located on the long arm of chromosome 4D.     

Effect of individual Sumai 3 chromosomes on resistance to scab spread within spikes and deoxynivalenol accumulation within kernels in wheat

Two sets of substitution lines were developed by crossing individual monosomic lines of Chinese Spring (recipient) with scab (Fusarium graminearum) resistant cultivar Sumai 3 (donor) and then using the monosomics as the recurrent male parent for four backcrosses (without selfing after each backcross). The disomic substitution lines were separated from selfed BC4F2 plants. Chromosome specific SSR markers were analyzed for polymorphism between Sumai 3 and Chinese Spring. Polymorphic markers were used to identify substitution lines for specific chromosomes. Based on the specific SSR markers, chromosome substitutions occurred in thirty-six lines, and six lines segregated alleles from the two parents or were homozygous for the allele from Chinese Spring. These substitution lines were used to evaluate Type II (spread within the head) and Type V (deoxynivalenol accumulation within kernels) scab resistance. The objective was to use the substitution lines to evaluate the effect of individual chromosomes of Sumai 3 on Type 11 and Type V scab resistance in the greenhouse. Significant differences in Type II scab resistance and deoxynivalenol (DON) levels among different Chinese Spring (Sumai 3) substitution lines were detected. Positive chromosome substitution effects on Type II scab resistance were found on chromosomes 2B, 3B. 6B, and 7A from Sumai 3. Chromosomes 3B and 7A also reduced DON accumulation within the kernels, while chromosomes IB, 2D, and 4D from Sumai 3 increased DON concentration. Chromosome 7A from Sumai 3 had the largest effect on resistance to scab spread and DON accumulation. Additional research is in progress on the scab resistance conferred by chromosome 7A.     

Comparison of genetic and physical maps of group 7 chromosomes from Triticum aestivum L.

We present a high density physical map of homoeologous group 7 chromosomes from Triticum aestivum L. using a series of 54 deletion lines, 6 random amplified polymorphic DNA (RAPD) markers and 91 cDNA or genomic DNA clones from wheat, barley and oat. So far, 51 chromosome segments have been distinguished by molecular markers, and 54 homoeoloci have been allocated among chromosomes 7A, 7B and 7D. The linear order of molecular markers along the chromosomes is almost identical in the A- B- and D-genome of wheat. In addition, there is colinearity between the physical and genetic maps of chromosomes 7A, 7B and 7D from T. aestivum, indicating gene synteny among the Triticeae. However, comparison of the physical map of chromosome 7D from T. aestivum with the genetic map from Triticum tauschii some markers have been shown to be physically allocated with distortion in more distal chromosome regions. The integration of genetic and physical maps could assist in estimating the frequency and distribution of recombination in defined regions along the chromosome. Physical distance did not correlate with genetic distance. A dense map facilitates the detection of multiple rearrangements. We present the first evidence for an interstitial inversion either on chromosome arm 7AS or 7DS of Chinese Spring. Molecularly tagged chromosome regions (MTCRs) provide landmarks for long-range mapping of DNA fragments.     

Molecular characterization and chromosome-specific TRAP-marker development for Langdon durum D-genome disomic substitution lines.

The aneuploid stocks of durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husnot) and common wheat (T. aestivum L.) have been developed mainly in 'Langdon' (LDN) and 'Chinese Spring' (CS) cultivars, respectively. The LDN-CS D-genome chromosome disomic substitution (LDN-DS) lines, where a pair of CS D-genome chromosomes substitute for a corresponding homoeologous A- or B-genome chromosome pair of LDN, have been widely used to determine the chromosomal locations of genes in tetraploid wheat. The LDN-DS lines were originally developed by crossing CS nulli-tetrasomics with LDN, followed by 6 backcrosses with LDN. They have subsequently been improved with 5 additional backcrosses with LDN. The objectives of this study were to characterize a set of the 14 most recent LDN-DS lines and to develop chromosome-specific markers, using the newly developed TRAP (target region amplification polymorphism)-marker technique. A total of 307 polymorphic DNA fragments were amplified from LDN and CS, and 302 of them were assigned to individual chromosomes. Most of the markers (95.5%) were present on a single chromosome as chromosome-specific markers, but 4.5% of the markers mapped to 2 or more chromosomes. The number of markers per chromosome varied, from a low of 10 (chromosomes 1A and 6D) to a high of 24 (chromosome 3A). There was an average of 16.6, 16.6, and 15.9 markers per chromosome assigned to the A-, B-, and D-genome chromosomes, respectively, suggesting that TRAP markers were detected at a nearly equal frequency on the 3 genomes. A comparison of the source of the expressed sequence tags (ESTs), used to derive the fixed primers, with the chromosomal location of markers revealed that 15.5% of the TRAP markers were located on the same chromosomes as the ESTs used to generate the fixed primers. A fixed primer designed from an EST mapped on a chromosome or a homoeologous group amplified at least 1 fragment specific to that chromosome or group, suggesting that the fixed primers might generate markers from target regions. TRAP-marker analysis verified the retention of at least 13 pairs of A- or B-genome chromosomes from LDN and 1 pair of D-genome chromosomes from CS in each of the LDN-DS lines. The chromosome-specific markers developed in this study provide an identity for each of the chromosomes, and they will facilitate molecular and genetic characterization of the individual chromosomes, including genetic mapping and gene identification.     

杀配子染色体特异RAPD 标记及山羊草属与普通小麦间分子多态性的研究

以普通小麦"中国春"、三个"中国春"具杀配子染色体的二体异附加系及作为三个杀配子基因种源的三种山羊草为材料, 用46 个10 nt 随机引物对基因组DNA 进行扩增, 以筛选杀配子染色体特有的RAPD 标记, 并检测普通小麦与三种山羊草之间的RAPD 多态性。结果表明:46 个引物中有35 个扩增出比较稳定的RAPD 产物。其中引物OPF-14 和OPQ-09 分别在普通小麦"中国春"-山羊草附加系间扩增出多态性产物;因而认定OPF-14₁₃₀₀ 与OPQ-09₈₀₀、OPF-14₁₁₆₀ 与OPQ-09₇₇₀、OPF-14₁₂₈₀分别为三个杀配子染色体3C、Gcl 和2C 的特异性RAPD 标记, 可以用于快速跟踪鉴定3C、Gcl、2C 杀配子染色体。对三种山羊草与普通小麦"中国春"进行了基因组DNA多态性分析, 结果表明, 35 个引物在"中国春"中共扩增出162 个产物, 在离果山羊草、拟斯卑尔脱山羊草、柱穗山羊草中分别扩增出140、154 和155 个产物;三种山羊草与"中国春"之间的共有扩增产物分别为69、87、96 个, 占总扩增产物的29.61%、37.70%和43.44%。上述结果, 从分子水平揭示了山羊草属与普通小麦之间存在着较近的亲缘关系。     

Identification of wheat (Triticum aestivum L.) chromosomes with genes controlling the level of nitrate reductase,nitrite reductase,and acid proteinase using the Chinese Spring-Hope substitution lines

The levels of nitrate reductase, nitrite reductase, and acid proteinase were compared in the primary leaves of 8-day-old wheat seedlings of Chinese Spring, Hope, and the 21 disomic substitution lines of Hope in Chinese Spring. Two chromosomes, 7B and 7D, were considered to contain genes controlling the level of nitrate reductase. Substitution of Hope chromosome 7B caused a highly significant increase in the in vitro stability of nitrate reductase. Nitrite reductase appeared to be controlled by two major genes, located on chromosomes 4D and 7D, and two minor genes, located on chromosomes 3D and 5A. In the case of acid proteinase, substitution of chromosome 1D caused a significant reduction in enzyme activity.     

RAPDs as molecular markers for the detection of Aegilops markgrafii chromatin in addition and euploid introgression lines of hexaploid wheat

 Aegilops markgrafii contains resistance genes to powdery mildew, leaf rust and stripe rust, and also has high crude protein and lysine contents, which can be useful for wheat improvement. These important traits are localized on different chromosomes. Disomic Triticum aestivum-Ae. markgrafii addition lines and euploid introgression lines showing leaf-rust and powdery mildew resistance were screened with RAPDs to detect chromosome-specific markers which can accelerate the breeding process. RAPD markers for all six available disomic addition lines were obtained. The additional chromosomes B, C, D, E, F and G were identified by three, three, three, two, one and seven primers, respectively. All three chromosome-B-specific RAPD markers demonstrated the presence of alien chromatin in the leaf-rust-resistant 42-chromosome introgression lines as well as in the segregating progeny. The three chromosome-C-identifying primers also demonstrated the presence of that chromosome in powdery mildew-resistant euploid introgression lines. The substitution lines (5A)5C and (5D)5C with different genetic backgrounds for both parents, in comparison to the lines mentioned above, showed the chromosome C-specific band with only two of the three primers. The chromosome F-specific primer and a primer evident on all the Ae. markgrafii chromosomes analysed did not generate the expected fragments on the chromosome F_del addition line, indicating that the markers are located on the deleted part of chromosome F. Received: 20 August 1996 / Accepted 17 January 1997     

Production of durum wheat substitution haploids from durum x maize crosses and their cytological characterization.

The objective of this study was to investigate the effect of individual durum wheat (Triticum turgidum L.) chromosomes on crossability with maize (Zea mays L.) and to cytologically characterize the haploids recovered. Fourteen 'Langdon' (LDN) D-genome disomic substitution lines, a LDN Ph mutant (Ph1b ph1b), and normal 'Langdon' were pollinated with maize pollen. After pollination, hormonal treatment was given daily for up to 14 days. Haploid embryos were obtained from all lines and were aseptically cultured. From a total of 55,358 pollinated florets, 895 embryos were obtained. Only 14 of the embryos germinated and developed into healthy plants. Different substitution lines showed varying degrees of success. The most successful was the substitution 5D(5B) for both embryo formation and haploid plantlet production. These results indicate that the substitution of 5D for 5B confers on durum wheat a greater ability to produce haploids. Fluorescent genomic in situ hybridization (GISH) showed that the substitution haploids consisted of 7 A-genome chromosomes, 6 B-genome chromosomes, and 1 D-genome chromosome. Triticum urartu Turn. genomic DNA was efficient in probing the 7 A-genome chromosomes, although the D-genome chromosome also showed intermediate hybridization. This shows a close affinity between the A genome and D genome. We also elucidated the evolutionary translocation involving the chromosomes 4A and 7B that occurred at the time of evolution of durum wheat. We found that the distal segment translocated from chromosome 7B constitutes about 24% of the long arm of 4A.     

Development of T. aestivum L.eH. californicum Alien Chromosome Lines and Assignment of Homoeologous Groups of Hordeum californicum Chromosomes

Hordeum californicum(2n=2x=14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring(CS)eH. californicum amphidiploid(2n=6x=56, AABBDDHH) was established. By genomic in situ hybridization(GISH) and multicolor fluorescent in situ hybridization(FISH) using repetitive DNA clones(pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheatealien chromosome lines, including four disomic addition lines(DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines(MtH7L,MtH1 S, MtH1 L, DtH6 S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line(DSH4) and one translocation line(TH7S/1BL), were identified from the progenies derived from the crosses of CSeH. californicum amphidiploid with common wheat varieties. A total of 482 EST(expressed sequence tag) or SSR(simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2,H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5Hc, 2Hc, 6Hc, 3Hcand 1Hc, respectively. The chromosomes H1 and H6 were designated as 7Hcand 4Hc, respectively, by referring to SSR markers located on rye chromosomes.     

小麦-鹅观草第一部分同源群染色体渗入系鉴定与基因组归属分析

鹅观草（Roegneria kamoji,2n=42,SSHHYY）是小麦异源六倍体野生近缘种,对小麦赤霉病具有良好抗性,是改良小麦赤霉病抗性的重要遗传资源。通过远缘杂交,将鹅观草第一部分同源群染色体上的抗赤霉病基因Fhb6导入普通小麦。由于第一部分同源群染色体包含1S、1H和1Y三条染色体,为研究这些同源染色体对小麦赤霉病抗性的影响,筛选出4个鹅观草第一部分同源群染色体特异分子标记,通过PCR扩增鹅观草属不同野生种的基因组DNA,明确了抗赤霉病Fhb6基因位于鹅观草1Y#1染色体。进一步利用分子细胞遗传学技术从中国春与鹅观草的后代中选育出5份涉及鹅观草1Y#2和1S#2染色体的渗入系材料。其中：21RK?1为二体异代换系DS1Y#2(1A),21RK?2为二体异代换系DS1S#2（1D）,21RK?3为二体异附加系DA1S#2,21RK?4为1S#2和TW·1S#2S的双单体附加系,21RK?5为纯合TW·1S#2S易位系。这些新种质为小麦抗赤霉病基因的发掘及遗传改良奠定了基础。