phKL基因和Ph2基因缺失重组体的创制及其与小麦外源物种杂种的染色体减数分裂观察

以小麦(Triticum aestivum L.)双端体3DL为细胞学标记,用具有phKL基因的小麦地方品种"开县罗汉麦"为受体连续回交,将促进小麦外源部分同源染色体配对的phKL基因和Ph2基因缺失重组在一起获得了重组体phKL+Ph2.这种重组体有正常的育性.与只有phKL基因的小麦材料相比,重组体与外源物种AegilopsvariabilisEig.或黑麦(Secale cereale L.)杂种的部分同源染色体配对水平显著增加,表明Ph2基因的缺失体与phKL基因可能存在加性效应.部分同源染色体配对水平的增加表现在棒状二价体、环状二价体和三价体的数量变多而单价体数量减少.单价体的减少主要是由于棒状二价体的增加所造成的.在小麦外源遗传转移中,运用重组体pHKL +Ph2可能比单纯应用Ph2缺失或phKL基因材料更理想.当与具有ph1b基因的材料比较时发现,重组体phKL+Ph2与 Ae.variabilis(或黑麦)杂种的部分同源染色体配对水平显著降低,这主要是由环状二价体和多价体的减少造成的,但是棒状二价体数量表现增加(与Ae.variabilis杂种)或达到类似水平(与黑麦杂种),这一有趣的发现从表现型上证明了Ph1基因与Ph2或phKL基因在诱导部分同源染色体配对时的遗传作用机制存在差异.     

phKL基因和Ph2基因缺失重组体的创制及其与小麦外源物种杂种的染色体减数分裂观察

以小麦(Triticum aestivum L.)双端体3DL为细胞学标记,用具有phKL基因的小麦地方品种“开县罗汉麦”为受体连续回交,将促进小麦外源部分同源染色体西己对的phKL基因和Ph2基因缺失重组在一起获得了重组体phKL Ph2^-。这种重组体有正常的育性。与只有phKL基因的小麦材料相比,重组体与外源物种Aegilops variabilis Eig．或黑麦(Secale cereale L.)杂种的部分同源染色体配对水平显著增加,表明Ph2基因的缺失体与phKL基因可能存在加性效应。部分同源染色体配对水平的增加农现在棒状二价体、环状二价体和三价体的数量变多而单价体数量减少。单价体的减少主要足由于棒状二价体的增加所造成的。在小麦外源遗传转移中,运用重组体phKL Ph2^-可能比单纯应用Ph2缺铁或phKL基因材料更理想。当与具有phlb基因的材料比较时发现,重组体phKL Ph2^-与Ae,variabilis(或黑麦)杂种的部分同源染色体配对水平显著降低,这丰要是由环状二价体和多价体的减少造成的,但是棒状二价体数量表现增加(与Ae.variabilis杂种)或达到类似水平(与黑麦杂种),这一有趣的发现从表现型上证明了Ph1基因与Ph2或phKL基因在诱导部分同源染色体配对时的遗传作用机制存在差异。     

ph1b基因在普通小麦与Ae.crassa、Ae.crassassp杂种中的作用

通过对(中国春ph1b突变体×Ae. crassa)F_1、(中国春×Ae.crassa)F_1;(中国春ph1b突变体×Ae.crassassp)F_1、(中国春×Ae.crassassp)F_4花粉母细胞减数分裂中期Ⅰ染色体配对的研究。第一次证明了中国春ph1b突变体在诱导Ae.crassa、Ae.crassassp与普通小麦部分同源染色体配对方面有显著作用。可以利用ph1b基因通过诱导部分同源染色体配对交换的方法以染色体易位的方式把Ae.crassa和Ae.crassassp的有益基因导入普通小麦中。Ae.crassa和Ae.crassassp中不含有拟ph1基因。Ae.crassa和Ae.crassassp的D染色体组与普通小麦的D染色体组有明显区别。     

小麦远缘杂交的珍贵种质沙丘小麦

在用普通小麦(Triticum aestivum L.)与兰州黑麦(Secale cereale cv lanzhou)杂交筛选具有可交配性基因(kr)品种中,发现普通小麦品种沙丘小麦(原产于日本)不仅具有kr基因和诱导部分同源染色体配对基因(ph),而且与兰州黑麦杂交的第一代自然结实。这样的种质资源在小麦远缘杂交和品种改良中,具有十分重要的作用。     

利用分子标记将2Ai-2染色体转移到小麦ph1b遗传背景的研究

小麦ph1b突变体可诱导部分同源染色体配对和交换,产生遗传上较为稳定、补偿性较好的重组体。将外源染色体引入ph1b的小麦遗传背景是产生目标染色体重组体的基础,但ph1b植株没有明显而稳定的表型性状,难以从表型上进行选择。本研究利用CSph1b缺失区中的分子标记Mads及外源染色体特异的分子标记P4和P68,对小麦-中间偃麦草2Ai-2(2B)异代换系N420与CSph1b的杂种F2群体及其衍生的F5株系进行ph1b-2Ai-2染色体综合体的选择,高效地获得了目标基因型。     

小麦的Ph基因及其应用

约州·年前,Oka motor e’首先发现在普通小麦T.ae- 5trvuln的511染色体上携带有抑制部分同源染色体配 对的基因,接着1958年Sears和Okamotot'", Riley和 Chapman"'同时证明抑制部分同源染色体配对的基因 位于5B染色休的长臂上。1971年Wall["]等人命名 为Ph基因,即取用Pairing homoeologous的第一个字 母为名。Ph基因为一显性基因,当它发生突变或缺失 时则表现为．犯性（即Ph}Ph)。为排除Ph的抑制效应, Okamoto (1'966）用X射线处理小麦去雄穗子,并授以 黑麦花粉,fit得的杂种后代有3.佗％具高水平部分同 源配对的个沐,经分析证明是Ph基因缺失的效果。但 当时Okamoto未能诱导染色体加倍以获得这个缺失突 变体。1971 _=乒Wall和Riley利用FMS处理小麦种子, 再以黑麦授粉,也获得约1.5％突变率的“部分Ph突变 体”。Searsc"'l 0 从1966年就试用X射线处理中国春小麦 (Chinese Spring）的德子,然后将它的花粉授于一个有 颈毛标志的5B单体植株上（即小黑麦易位系HN-2, Sears, 1967a),再以处理的M,后代用作父本,与粘 果小麦(T. kotschyi）杂交,在杂种后代中仅发现 一种具中间0!d对水平的突变体,其每个细胞大约包含 5个二价体,有证据表明,该突变体乃由于一个较弱的 抑制配对基因的缺失,或即3DS上的抑制基因缺失。     

小麦-黑麦染色体配对的遗传控制

关于普通小麦减数分裂二倍化的遗传控制,国外已有不少报道。近来,一些学者对小麦染色体配对进行人工调控,以期诱导小麦与近缘属种间的染色体交换,培育栽培小麦优良新品种。笔者曾报道过普通小麦Ph基因缺失对于小麦-黑麦杂种染色体配对的遗传     

中国春ph2b突变体×华山新麦草F1自交和回交一代细胞遗传学研究

用中国春ph2b突变体(Triticumaestivum L.cv.Chinese Spring)与华山新麦草(Psathyrostachys huashani-caKeng)杂种F1的部分进行自交,而另一部分与3个不同的普通小麦品种以及中国春ph2b突变体进行回交,并对自交和回交一代的形态学和细胞学进行研究。结果表明:(1)含有AABBDDNs的自交F2的花粉母细胞减数分裂中期Ⅰ平均单价体数都在7以上,说明来自华山新麦草的Ns基因组可引起普通小麦ABD基因组中某一个(几个)同源组发生不联会或联会消失;(2)在回交一代(F1×CS、F1×CSph2b)中,平均单价体数都在7以下,说明其中的ph2b基因起到了促进染色体配对的作用;而在回交一代(F1×J-11、F1×郑麦-9023)中,则表现正常,与理论一致;(3)在所有自交和回交一代中,减数分裂期间均出现染色体桥、落后染色体、三分孢子体、多分孢子体以及微核等各种染色体异常行为,说明Ns基因组引起了所得材料细胞学上的不稳定性。从总体上讲,回交比自交能更快地恢复ph2b基因促进部分同源染色体配对的作用,为染色体重组提供更多的机会,期望在后代中获得新种质。     

小麦ph1b突变体与小黑麦杂交的细胞遗传学研究

本文利用普通小麦品系"中国春"(对照)、中国春ph1b突变体分别与八倍体小黑麦、六倍体小黑麦杂交,杂种F1的减数分裂前期Ⅰ染色体行为表现异常,中期Ⅰ出现较多的单价体、棒状二价体和多价体,在后期和末期出现落后染色体、染色体片断和微核。原因是ph1b基因的存在造成染色体联会机制紊乱,致使一些部分同源染色体配对并发生互换,有可能在以后的世代产生染色体易位与基因重组。     

一个柱穗山羊草（AegilposCylindrica）系统中的类Ph1基因（Ph1—like）…

第一次在一个Ａｅ．ｃｙｌｉｎｄｒｉｃａ系统中发现了Ｐｈ１－ｌｉｋｅ基因,但它的作用略小于Ｐｈ１,同时证明了Ａｅ．ｃｙｌｉｎｄｒｉｃａ在控制染色体配对基因方面存在多态现象,Ｐｈ１ｂ基因可以诱导普通小麦与Ａｅ．ｃｙｌｉｎｄｒｉｃａ间的部分同源染色体配对,同时用普通小麦对（中国春ｐｈ１ｂ突变体ＸＡｅ．ｃｙｌｉｎｄｒｉｃａ）Ｆ１回交获得了成功,表明利用Ｐｈ１ｂ基因通过诱导部分同源染色体配对可以把Ａｅ．ｃｙ     

A DNA Fragment Mapped within the Submicroscopic Deletion of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

Bread wheat is an allohexaploid consisting of three genetically related (homoeologous) genomes. The homoeologous chromosomes are capable of pairing but strict homologous pairing is observed at metaphase 1. The diploid-like pairing is regulated predominantly by Ph1, a gene mapped on long arm of chromosome 5B. We report direct evidence that a mutant of the gene (ph1b) arose from a submicroscopic deletion. A probe (XksuS1-5) detects the same missing fragment in two independent mutants ph1b and ph1c and a higher intensity fragment in a duplication of the Ph1 gene. It is likely that XksuS1-5 lies adjacent to Ph1 on the same chromosome fragment that is deleted in ph1b and ph1c. XksuS1-5 can be used to tag Ph1 gene to facilitate incorporation of genetic material from homoeologous genomes of the Triticeae. It may also be a useful marker in cloning Ph1 gene by chromosome walking.     

Synthesis and cytological characterization of trigeneric hybrids of durum wheat with and without Ph1.

Wild grasses in the tribe Triticeae, some in the primary or secondary gene pool of wheat, are excellent reservoirs of genes for superior agronomic traits, including resistance to various diseases. Thus, the diploid wheatgrasses Thinopyrum bessarabicum (Savul. and Rayss) A. Love (2n = 2x = 14; JJ genome) and Lophopyrum elongatum (Host) A. Love (2n = 2x = 14; EE genome) are important sources of genes for disease resistance, e.g., Fusarium head blight resistance that may be transferred to wheat. By crossing fertile amphidiploids (2n = 4x = 28; JJEE) developed from F1 hybrids of the 2 diploid species with appropriate genetic stocks of durum wheat, we synthesized trigeneric hybrids (2n = 4x = 28; ABJE) incorporating both the J and E genomes of the grass species with the durum genomes A and B. Trigeneric hybrids with and without the homoeologous-pairing suppressor gene, Ph1, were produced. In the absence of Ph1, the chances of genetic recombination between chromosomes of the 2 useful grass genomes (JE) and those of the durum genomes (AB) would be enhanced. Meiotic chromosome pairing was studied using both conventional staining and fluorescent genomic in situ hybridization (fl-GISH). As expected, the Ph1-intergeneric hybrids showed low chromosome pairing (23.86% of the complement), whereas the trigenerics with ph1b (49.49%) and those with their chromosome 5B replaced by 5D (49.09%) showed much higher pairing. The absence of Ph1 allowed pairing and, hence, genetic recombination between homoeologous chromosomes. Fl-GISH analysis afforded an excellent tool for studying the specificity of chromosome pairing: wheat with grass, wheat with wheat, or grass with grass. In the trigeneric hybrids that lacked chromosome 5B, and hence lacked the Ph1 gene, the wheat-grass pairing was elevated, i.e., 2.6 chiasmata per cell, a welcome feature from the breeding standpoint. Using Langdon 5D(5B) disomic substitution for making trigeneric hybrids should promote homoeologous pairing between durum and grass chromosomes and hence accelerate alien gene transfer into the durum genomes.     

Molecular Markers of Ph1 Gene in Chinese Spring and Development of Winter Wheat New Line Harboring ph1b Gene

he genomic DNA of common wheat (Triticum aestivum L.) “Chinese Spring” (CS) and its ph1b mutant were analyzed by using 19 sequence tagged site PCR (STS-PCR) primers, which derived from RFLP probes from barley (Hordeum vulgare L.) chromosome 5H. One marker was identified on wheat chromosome 5BL, which is 5.7 cM (centiMorgan) proximal to Ph1 gene, using the CS homoeologous group 5 nullisomic-tetrasomic, ditelosomic 5BL line and an F2 population from CS×ph1b mutant. This linked PCR marker was converted into a more specific sequence characterized amplified region (SCAR) marker. To obtain a new winter wheat line containing ph1b gene, the authors used a nullisomic 5B line of “Abbodanza”as a bridge parent and crossed respectively with the CS ph1b mutant (donor) and a winter wheat variety, “Jing 411” (recipient). The meiotic chromosome pairing was checked in the progeny of each cross, as well as using the marker-assistant selection of the SCAR marker identified for ph1b gene. After three inter-crossing and one selfing, a relatively stable ph1b substitution line of winter wheat with “Jing 411” background was obtained.     

Fine Physical Mapping of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

The diploid-like chromosome pairing in polyploid wheat is controlled by the Ph1 (pairing homoeologous) gene that is located on chromosome arm 5BL. By using a combination of cytogenetic and molecular techniques, we report the physical location of the Ph1 gene to a submicroscopic chromosome region (Ph1 gene region) that is flanked by the breakpoints of two deletions (5BL-1 and ph1c) and is marked by a DNA probe (XksuS1). The Ph1 gene region is present distal to the breakpoint of deletion 5BL-1 but proximal to the C-band 5BL2.1. Two other DNA probes (Xpsr128 and Xksu75) flank the region-Xpsr128 being proximal and Xksu75 being distal. The estimated size of the region is less than 3 Mb. The chromosome region around the Ph1 gene is high in recombination as the genetic distance of the region between 5BL-1 breakpoint and C-band 5BL2.1 (not resolved by the microscope) is at least 9.3 cM.     

Comparison of homoeologous chromosome pairing between hybrids of wheat genotypes Chinese Spring ph1b and Kaixian-luohanmai with rye

The ph-like genes in the Chinese common wheat landrace Kaixian-luohanmai (KL) induce homoeologous pairing in hybrids with alien species. In the present study, meiotic phenotypic differences on homoeologous chromosome pairing at metaphase I between hybrids of wheat genotypes Chinese Spring ph1b (CSph1b) and KL with rye were studied by genomic in situ hybridization (GISH). The frequency of wheat-wheat associations was higher in CSph1b×rye than in KL×rye. However, frequencies of wheat-rye and rye-rye associations were higher in KL×rye than in CSph1b×rye. These differences may be the result of different mechanisms of control between the ph-like gene(s) controlling homoeologous chromosome pairing in KL and CSph1b. Wheat-wheat associations were much more frequent than wheat-rye pairing in both hybriods. This may be caused by lower overall affinity, or homoeology, between wheat and rye chromosomes than between wheat chromosomes.     

Comparative analysis of the meiotic effects of wheat ph1b and ph2b mutations in wheat×rye hybrids

 Wheat-wheat and wheat-rye homoeologous pairing at metaphase I and wheat-rye recombination at anaphase I were examined by genomic in situ hybridization (GISH) in wild-type (Ph1Ph2) and mutant ph1b and ph2b wheat×rye hybrids. The metaphase-I analysis revealed that the relative contribution of wheat-rye chromosome associations in ph2b wheat×rye was similar to that of the wild-type hybrid genotype but differed from the effect of the ph1b mutation. The greater pairing promotion effect of the ph1b mutation appears to be relatively more on distant homoeologous partner metaphase-I associations, whereas the lower promoting effect of ph2b is evenly distributed among all types of homoeologous associations. This finding reveals that distinct mechanisms are involved in the control of wheat homoeologous pairing by the two Ph genes. The frequency of wheat-rye recombination calculated from anaphase-I analysis was lower than expected from the metaphase-I data. A greater discrepancy was found in ph2b than in ph1b wheat×rye hybrids, which may suggest a more distal chiasma localization in the former hybrid genotype. Received: 20 June 1997 / Accepted: 9 December 1997     

Cytogenetic processes in the course of Triticum aestivum and Haynatricum hybridization

Meiosis in hybrids obtained in direct and return crossings between Haynatricum and wheat was studied. In F1 hybrids the possibility of stimulation of homoeologous pairing between the chromosomes of T. aestivum and T. dicoccum and, probably, H. villosum, if Haynatricum was used as pollinator, has been shown. This process is considerably intensified when the genes regulating chromosome pairing, in particular ph1b mutation, are used. In reciprocal crosses it was shown that wheat genotypes can differently influence on homoeologous chromosome pairing. In BC1 and F2 generations the chromosome compositions are determining which arise as a result of stochastic processes in premeiotic mitoses and in meiosis in F1 hybrids.     

Effect of the pairing gene Ph1 on centromere misdivision in common wheat.

The cytologically diploid-like meiotic behavior of hexaploid wheat (i.e., exclusive bivalent pairing of homologues) is largely controlled by the pairing homoeologous gene Ph1. This gene suppresses pairing between homoeologous (partially homologous) chromosomes of the three closely related genomes that compose the hexaploid wheat complement. It has been previously proposed that Ph1 regulates meiotic pairing by determining the pattern of premeiotic arrangement of homologous and homoeologous chromosomes. We therefore assume that Ph1 action may be targeted at the interaction of centromeres with spindle microtubules--an interaction that is critical for movement of chromosomes to their specific interphase positions. Using monosomic lines of common wheat, we studied the effect of this gene on types and rates of centromere division of univalents at meiosis. In the presence of the normal two doses of Ph1, the frequency of transverse breakage (misdivision) of the centromere of univalent chromosomes was high in both first and second meiotic divisions; whereas with zero dose of the gene, this frequency was drastically reduced. The results suggest that Ph1 is a trans-acting gene affecting centromere-microtubules interaction. The findings are discussed in the context of the effect of Ph1 on interphase chromosome arrangement.