Gametocidal genes in wheat and its relatives. IV. Functional relationships between six gametocidal genes.

Gametocidal (Gc) genes in Aegilops species are known to cause gamete abortion and chromosome breakage when they are introduced into the wheat genetic background. Interactions of five Gc genes so far identified were investigated by analysis of wheat hybrids among lines carrying different gametocidal genes. As a result, the genes were classified into three functional groups. The first group includes two Gc genes of Ae. speltoides (Gc1a and Gc1b) and one gene (Gc-Sl3) on chromosome 2S1 of Ae. sharonensis. These genes were hypostatic to the genes (Gc-Sl1, Gc-Sl2) on chromosome 4S1 of Ae. longissima and Ae. sharonensis, which constitute the second group. In addition, plants carrying Gc genes of both the first and the second group produced progeny with higher frequencies of chromosome breakage than those found in the progeny of single gene carriers. It was concluded that there were specific interactions between these genes to enhance chromosome breakage. On the other hand, there was no interaction between the Gc gene (Gc-C) of Ae. triuncialis, the third group, and Gc genes belonging to the former two groups. These functional groups might be a reflection of the mechanisms by which Gc genes induce gamete abortion and chromosome breakage. Based on functional and local relationships, the symbols of the Gc genes were systematically redesignated.     

Characterization of a knock-out mutation at the <Emphasis Type="Italic">Gc2</Emphasis> locus in wheat

Gametocidal (Gc) genes, introduced into common wheat from related Aegilops species, are selfish genetic elements that ensure their preferential transmission by inducing chromosomal breaks. Here we report the production and characterization of a knock-out mutation of the Gc2 gene transferred to wheat as a wheat-Aegilops sharonensis T4B-4S(sh)#1 translocation chromosome. In hemizygous Gc2/- condition, gametophytes lacking Gc2 suffer chromosomal fragmentation and produce non-functional gametes, which leads to sporophytic semisterility and exclusive transmission of the Gc2-carrier chromosome. We have identified one putative ethyl methylsulfonate (EMS)-induced Gc2 mutant that restores spike fertility and shows Mendelian segregation. Progeny screening mapped the mutation to the Gc2-carrier chromosome T4B-4S(sh)#1. C-banding and fluorescence in situ hybridization analyses showed that the loss of Gc2 function in the mutant is not due to a terminal deficiency. Analysis of first and second pollen mitoses in Gc2(mut) /- plants and C-banding analysis of testcross progenies showed that no chromosomal breakage occurs in the mutant. No gametophytic chromosomal breakage was observed in heterozygous Gc2(mut) /Gc2 plants, which had fully fertile spikes. These results suggest that Gc2 encodes two agents, one causing chromosomal breaks in gametophytes lacking Gc2 and another that protects the Gc2 carrier from breakage. The EMS-induced Gc2 mutant appears to be a knock-out of the gene encoding the "breaking" agent. These data are a first crucial step toward the molecular understanding of Gc2 action.     

Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat

We used gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.     

Natural or induced nucleocytoplasmic heterogeneity in Triticum longissimum.

Alien cytoplasms produce a variety of phenotypes in durum wheat (Triticum turgidum) and common wheat (Triticum aestivum) cultivars, which indicate the prevalence of cytoplasmic variability in the subtribe Triticinae. Intraspecific cytoplasmic differences have been demonstrated between the subspecies of Triticum speltoides, Triticum dichasians, and Triticum comosum. In this study, durum wheat lines with cytoplasm from two accessions, B and C, of Triticum longissimum were compared, and meiotic chromosome pairing between the group 4 homoeologues from the same two accessions was examined in common wheat. First, monosomic addition or monosomic substitution lines of common wheat with cytoplasm and one chromosome (designated B) from accession B were crossed with those having cytoplasm and a chromosome designated C-1 or C-2 from accession C. In each substitution line, an alien chromosome substituted for a group 4 homoeologue. Each alien chromosome had a "selfish" (Sf) gene, which remained fixed in the wheat nucleus. The F1s had greatly reduced meiotic pairing between chromosomes B and C-1 and B and C-2, which indicated greatly reduced homology between the group 4 homoeologues from the two accessions. Second, by using Triticum timopheevii as a bridging species, chromosome B in a common wheat line was eliminated and an euploid durum line with cytoplasm from accession B was obtained. This line was fertile. In contrast, a similarly produced durum line with cytoplasm from accession C was male sterile and retained a species cytoplasm specific (scs) nuclear gene from T. timopheevii. In conclusion, nuclear and cytoplasmic heterogeneity pre-existed between accessions B and C and they represent varieties or incipient subspecies in T. longissimum. Alternatively, the Sf genes produced chromosomal heterogeneity and mutated cytoplasmic genes from one or both accessions. Key words : meiotic drive, selfish gene (Sf), gametocidal gene (Gc), Triticum, Aegilops.     

利用中国春-山羊草2C二体附加系与中国春-偃麦草5E二体附加系杂交诱发染色体易位和缺失

通过对中国春－长穗偃麦草（E.elongata 2n=14EE)二体附加系与中国春－柱穗山羊草（Ae.cylindrica 2n=28CCDD)2C二体附加系（杀配子染色体）杂交F1减数分裂的观察,看到F1单价体数超过理论数值,后期出现大量的染色体段片,认为这种异常现象与杀配子染色体的作用有关。对76株中国春－长穗偃麦草5E二体附加系与中国春－柱穗山羊草2C二体附加系杂交、回交一代进行染色体C－分带鉴定,初步认定株系21－5－14、21－5－37、21－5－67、21－5－71为中国春染色体与偃麦草5E染色体易位。经染色体组原位杂交（GISH）进一步确定21－5－14为T5ES.4AS易位系,21－5－37为T5E.2BS易位系,21－5－67为T5E.3AS易位系,21－5－71为T5ES.5BS易位系。易位频率为5.3%。株系21－5－27、21－5－18、21－5－18、21－5－72、21－5－4、21－5－71经染色体C－分带鉴定,分别为6B、4B、5B、4A、4B、2A缺失系。杀配子染色体引起缺失频率为6.5%。证明利用杀配子染色体诱导染色体易位和缺失的频率较高。杀配子染色体引起中国春B组染色体畸变大于A、D组,与Endo观察结果相似。     

Large-scale selection of lines with deletions in chromosome 1 B in wheat and applications for fine deletion mapping.

Terminal deletions of chromosome 1B in common wheat were selected on a large scale. The gametocidal gene of Aegilops cylindrica was used as the inducer of chromosome breakage. First, genes for endosperm storage proteins located on both arms of chromosome 1B were used as the selection markers. However, it was found that the chromosome breakage occurred during female gametogenesis, causing genotypic inconsistency between the embryo and endosperm. Thus, we isolated plants with terminal deletions in chromosome 1B by C-banding. Of 1327 plants examined, 128 showed aberrations in chromosome 1B: 47 in the short arm, 76 in the long arm, and 5 in both arms. The present deletions tended to have the breakpoint at more proximal regions than those produced previously by T.R. Endo and B.S. Gill. Using 33 deletion lines produced in this study and 34 lines previously produced, we mapped 39 RFLP loci and a nucleolar organizer region (NOR) on a specific region of chromosome 1B. The NOR was found to consist of two subregions with different repetitive units, which were termed NOR-Bld and NOR-Blp. Based on this fine deletion map and genotypic inconsistency between embryo and endosperm, the features of the gametocidal gene are discussed.     

来自山羊草的杀配子染色体(Aegilops cylindryca 2C)对小黑麦减数分裂及花粉粒有丝分裂过程的影响

用来自柱穗山羊草的杀配子染色体2C,诱导六倍体、八倍体小黑麦染色体的断裂,观察杂种F1的减数分裂行为,在PMCI及PMCII后期观察到了大量的落后染色体、染色体断片、环状染色体及桥,在二分了解子及四分孢子中有为数甚多的、大小不一的微核,有的还形成多分孢子。在对F1花粉粒的有丝分裂观察中,未见分裂异常。由此推断杀配子染以体诱导染色体断裂可能不发生在配子形成的有丝分裂过程。这与巳有的报道不同。     

Hereditary recombined three-allele variant of the Gc system

A three-allele variant with Gc 2, Gc 1F and Gc 1A2 alleles was detected in both a baby and his mother during paternity testing by isoelectric focusing. His father had a normal Gc phenotype, Gc 2-1F. Further examination of his mother's relatives revealed that his grandfather also had the same three-allele variant, while his grandmother and his aunt had normal Gc 2-1F and Gc 2-2. From these results, it was considered that the Gc 1F and Gc 1A2 alleles were on the same single chromosome. It was suggested that recombination had occurred between two chromosomes that had the Gc 1F and Gc 1A2 allele, respectively, forming the variant allele Gc 1F1A2 on a single chromosome.     

Gene mapping in chicken-Chinese hamster somatic cell hybrids. Serum albumin and phosphoglucomutase-2 structural genes on chicken chromosome 6

Chicken phosphoglucomutase (PGM-2), serum albumin, vitamin D binding protein (Gc) and phosphoribosyl pyrophosphate amidotransferase (PPAT) structural genes have been mapped to chicken chromosome 6 using chicken-Chinese hamster somatic cell hybrids containing this chromosome as the only chicken genetic material. Chicken PGM-2 activity was detected in the hybrids using cellogel electrophoresis and a substrate, ribose-1-phosphate (R-1-P), that allows the detection of PGM-1 activity in mice and PGM-2 activity in humans. Chicken albumin sequences were detected in the hybrids with the use of a labelled chicken serum albumin cloned cDNA. Classical studies have shown linkage of the serum albumin and Gc genes, and the Gc gene also can be localized to chicken chromosome 6. The PPAT gene was localized to this chromosome in previous studies using these hybrids. A homologous linkage group has been identified in mammals and, therefore, a chromosomal linkage group containing at least four genes--Gc, serum albumin, PPAT, and PGM-2--has been conserved over a period of 300 million years, throughout both avian and mammalian evolution.     

Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.)

A single dominant gene Gc controls the trait of high chlorophyll (Chl) content in rice (cultivar (cv.) Zhenshan 97B). The contents of Chl b and total Chl increased 100% and 25%, respectively, when Gc was introduced. In addition, photosynthetic rate, biomass and grain yield also increased by 20%, 17% and 16%, respectively. Three simple sequence repeats (SSR) markers (rm462, rm6340 and rm6464) that are linked to Gc were identified by amplification of DNA samples from near-isogenic lines using two hundred pairs of primers. The genetic distances on the short arm of rice chromosome 1 between Gc and rm6464, rm6340 and rm462 were 0, 0.588 and 1.18 cM, respectively.     

Saturation mapping of a gene-rich recombination hot spot region in wheat

Physical mapping of wheat chromosomes has revealed small chromosome segments of high gene density and frequent recombination interspersed with relatively large regions of low gene density and infrequent recombination. We constructed a detailed genetic and physical map of one highly recombinant region on the long arm of chromosome 5B. This distally located region accounts for 4% of the physical size of the long arm and at least 30% of the recombination along the entire chromosome. Multiple crossovers occurred within this region, and the degree of recombination is at least 11-fold greater than the genomic average. Characteristics of the region such as gene order and frequency of recombination appear to be conserved throughout the evolution of the Triticeae. The region is more prone to chromosome breakage by gametocidal gene action than gene-poor regions, and evidence for genomic instability was implied by loss of gene collinearity for six loci among the homeologous regions. These data suggest that a unique level of chromatin organization exists within gene-rich recombination hot spots. The many agronomically important genes in this region should be accessible by positional cloning.     

中国春—山羊草2C二体附加系同中国春—偃麦草二体附加系杂交后代的细胞遗传学研究

对中国春—长穗偃麦草（E.elongata 2n=14EE）二体附加系（1E-7E）与中国春—柱穗山羊草(Ae.cylindrica 2n=28 CCDD)2C二体附加系杂交后代进行细胞遗传学研究。结果表明,杂交当代（1E-7E）的结实率最高为36.06%,最低为19.43%,平均为29.5%。F₁自交结实率最高为62.09%,最低为30.92%,平均为39.64%。对照实验和方差分析表明,杀配子染色体对不同品系杂交的结实率影响有差异。杂交F₁减数分裂观察,看到F1单价体数超过理论数值,后期出现大量的染色体片段,认为这种异常现象与杀配子染色体的作用有关。     

GC1代换系与小麦杂交减数分裂行为的研究

用来自拟斯卑尔脱山羊草（Ae.speltoides）的杀配子染色体2S(GC基因),诱导普通小麦（Triticum aestivum L.）“宁农”、普通小麦—黑麦（Secale cereale L.）二体代换系（5R/5A）的染色体断裂,观察杂种F₁的减数分裂行为,在减数分裂中期I和中期II均出现较高频率的单价体、多价体,后期I和后期II出现大量的落后染色体、染色体断片和桥等异常现象,在二分孢子和四分孢子中出现较多的微核。在本实验中,GC1代换系与5R/5A代换系杂交F₁代减数分裂行为比GC1代换系与普通小麦“宁农”杂交F₁代减数分裂行为复杂,经统计学分析,含杀配子染色体的代换系间杂交比单一的杀配子染色体作用对单价体、多价体、微核的产生具有显著差异。因此,利用带有杀配子染色体的代换系间杂交来诱导染色体易位是一条有效的途径。     

Inheritance of the Translocation in Chromosome 2D of Common Wheat from <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch with Leaf Rust Resistance Gene

The variety of common spring wheat Chelyaba 75 carries a translocation from Aegilops speltoides Tausch in the chromosome 2D, which contains the leaf rust resistance gene and gametocidal genes. The length of this translocation was determined by molecular-genetic analysis. It is shown that the long arm of chromosome 2D is completely replaced by the long arm of chromosome 2S; it is possible that translocation involves the near-centromere region of the short arm. According to molecular analysis data, the translocation from Ae. speltoides in the Chelyaba 75 variety differs from the 2S chromosome region carrying the Lr35/Sr39 genes. This makes it possible to designate the leaf rust resistance gene of the Chelyaba 75 as LrSp2. The inheritance of LrSp2 in four populations from crossing Chelyaba 75 with different varieties of common wheat was studied. Estimation of leaf rust resistance of F₂ and F₃ hybrids in field conditions (2015–2016) revealed the absence of susceptible plants. The presence of 2DS.2SL translocation in hybrid plants was confirmed by molecular analysis. The results indicate the action of the gametocidal gene localized in the 2DS.2SL translocation and the fact that its tight linkage to the LrSp2 gene is inherited in a series of generations.     

PCR and sequence analysis of barley chromosome 2H subjected to the gametocidal action of chromosome 2C

Gametocidal (Gc) chromosomes induce various types of chromosomal mutations during gametogenesis in the chromosomes of common wheat and alien chromosomes added to common wheat. However, it is not yet known whether the Gc chromosome causes aberrations at the nucleotide level because mutations caused by Gc chromosomes have been studied only by cytological screening. In order to know whether the Gc chromosome induces point mutations, we conducted PCR analysis and sequencing with the progeny of a common wheat line that is disomic for barley chromosome 2H and monosomic for Gc chromosome 2C. We analyzed 18 2H-specific EST sequences using 81 progeny plants carrying a cytologically normal-appearing 2H chromosome and found no nucleotide changes in the analyzed 1,419 sequences (in total 647,075 bp). During this analysis, we found six plants for which some ESTs could not be PCR amplified, suggesting the presence of chromosomal mutations in these plants. The cytological and PCR analyses of the progeny of the six plants confirmed the occurrence of chromosomal mutations in the parental plants. These results suggested that the Gc chromosome mostly induced chromosomal aberrations, not nucleotide changes, and that the Gc-induced chromosomal mutations in the six plants occurred after fertilization.     

Development of Triticum aestivum-Leymus racemosus translocation lines using gametocidal chromosomes

Maan[1] and Endo[2] et al. first reported that some chromosomes from Ae. longgissima, Ae. sharonensis and Ae. triuncialis showed preferential transmission when introduced into wheat background. The mechanism for this phenomenon rests with the fact that contrary to the normal fertility of gametes with these chromosomes, chromosome structural aberrations occur seriously in the gametes without these chromosomes, causing less compatibility in selective fertilization and resulting in semi-sterilit…     

Group-specific component (Gc): subtypes in the Finnish population. Description of a new allele and an apparent mother-child incompatibility

The group-specific component (Gc) subtypes were determined in 575 adult Finns by immunoblotting after isoelectric focusing in agarose gel. The gene frequencies were Gc1S = 0.661, Gc1F = 0.139 and Gc2 = 0.200. This material included one rare allele, a more acidically focusing Gc 2 (named Gc 2A18). The phenotypes of 200 mother-child pairs studied were in accordance with the three-allelic mode of inheritance. An apparent mother-child incompatibility observed during routine paternity testing is reported.     

Frequency of Gc alleles and a variant Gc allele in Iceland

The gene frequency for Gc1 and Gc2 in an Icelandic population was found to be 0.71 and 0.29, respectively. An electrophoretic variant similar to Gc Norway was detected in 5 individuals of the same family. A pedigree of 14 family members, including two spouses, is presented.     

Development of Triticum aestivum-Leymus racemosus translocation lines using gametocidal chromosomes

Specific chromosomes of certain Aegilops species introduced into wheat genome background may often facilitate chromosome breakage and refusion, and finally result in a variety of chromosome restructuring. Such a phenomenon is commonly called gametocidal effect of the chromosomes. The chromosome 2C of Ae. cylindrica is one of such chromosomes. In the present study, scab resistant wheat-L. racemosus addition lines involving chromosomes Lr.2 and Lr.7 were crossed to wheat-Ae. cylindrica disomic addition line Add2C. Then F₁ hybrids were subsequently backcrossed with wheat cv “Chinese Spring”. BC₁ plants with chromosome structural aberration were identified by C-banding. In the self-pollinated progenies of these plants, three translocation lines were developed and characterized by mitotic and meiotic analysis combined with C-banding and fluorescent in situ hybridization (FISH) using biotin-labeled genomic DNA of L. racemosus as probe. Some other putative translocation lines to be further characterized were also found. The practicability and efficiency of the translocation between wheat and alien chromosomes induced by gametocidal chromosomes, as well as the potential use of the developed alien translocation lines were also discussed.