Gametocidal Action Research Articles

RNA sequencing and functional analysis uncover key long non-coding RNAs involved in regulating pollen fertility during the process of gametocidal action in wheat.

Gametocidal (Gc) chromosomes have been widely utilized in genetic breeding due to their ability to induce chromosomal breakage and eliminate gametes that lack them. Long noncoding RNAs (lncRNAs) have various functional mechanisms in regulating pollen and anther development; however, their regulatory contributions to Gc action are still unknown. Here, we identified 2824 differentially expressed lncRNAs (DE-lncRNAs) from the anther tissues of Triticum aestivum cv. Chinese Spring (CS) and Chinese Spring-Gc 3C chromosome monosomic addition line (CS-3C) through sequencing. In this study, we predicted 161 target mRNAs for 145 DE-lncRNAs, including 104 cis-regulatory, 60 trans-regulatory, and three both cis-regulatory and trans-regulatory manner. Combined with our previous miRNA sequencing data, 241 DE-lncRNAs functioned as potential endogenous target mimics (eTMs) for 84 differentially expressed microRNAs (DE-miRNAs, including 12 novel miRNAs). The results of transient transformation in tobacco leaves indicated that L006278 could bind to MTCONS_00006277, which encoded a calcineurin CBL-interacting protein kinase 19-like, and suppress its expression. Furthermore, L117735 could function as an eTM for tae-miR9657b-3p, and L056972 could function as an eTM for gc-m2240-5p. To explore the function of lncRNAs in the process of Gc action, we transformed L006278, an up-regulated lncRNA in CS-3C, into rice to analyze its effect on pollen fertility. Overexpression of L006278 led to a reduction in rice pollen fertility. Overall, our findings indicate that lncRNAs can contribute to the regulation of pollen fertility during the process of Gc action by regulating the expression levels of target mRNAs and acting as eTMs for certain key miRNAs.

Cytological observation of chromosome breakage in wheat male gametophytes caused by gametocidal action of Aegilops triuncialis-derived chromosome 3Ct.

In this study, we investigated the chromosome breakage caused by gametocidal (Gc) chromosome 3Ct and its interaction with the suppressor gene Igc1 (inhibitor of gametocidal gene 1) on wheat chromosome 3B. We demonstrated cytologically that patterns of 3Ct-induced chromosomal fragmentation in microspores differed from patterns observed for other Gc genes. Uninuclear microspores of the monosomic 3Ct addition line had high frequencies of micronuclei, possibly explaining its low fertility. Chromosome fragmentation was observed in prometaphase and metaphase of the first pollen mitosis in the monosomic 3Ct addition line. Patterns of chromosome fragmentation were different from those previously reported for Gc chromosomes 2S of Aegilops speltoides, 4Ssh of Ae. sharonensis and 2Ccy of Ae. cylindrica; many chromosome fragments were observed in prometaphase of the first pollen mitosis in the monosomic 3Ct addition plants. In anthers at the binuclear stage, many microspores at the uninuclear stage coexisted with normally developed microspores.

Uncovering key small RNAs associated with gametocidal action in wheat.

Gametocidal (Gc) chromosomes can kill gametes that lack them by causing chromosomal breakage to ensure their preferential transmission, and they have been exploited in genetic breeding. The present study investigated the possible roles of small RNAs (sRNAs) in Gc action. By sequencing two small RNA libraries from the anthers of Triticum aestivum cv. Chinese Spring (CS) and the Chinese Spring-Gc 3C chromosome monosomic addition line (CS-3C), we identified 239 conserved and 72 putative novel miRNAs, including 135 differentially expressed miRNAs. These miRNAs were predicted to target multiple genes with various molecular functions relevant to the features of Gc action, including sterility and genome instability. The transgenic overexpression of miRNA, which was up-regulated in CS-3C, reduced rice fertility. The CS-3C line exhibited a genome-wide reduction in 24 nt siRNAs compared with that of the CS line, particularly in transposable element (TE) and repetitive DNA sequences. Corresponding to this reduction, the bisulfite sequencing analysis of four retro-TE sequences showed a decrease in CHH methylation, typical of RNA-directed DNA methylation (RdDM). These results demonstrate that both miRNA-directed regulation of gene expression and siRNA-directed DNA methylation of target TE loci could play a role in Gc action.

Gametocidal Factor Transferred from Aegilops geniculata Roth Can Be Adapted for Large-Scale Chromosome Manipulations in Cereals.

Segregation distorters are curious, evolutionarily selfish genetic elements, which distort Mendelian segregation in their favor at the expense of others. Those agents include gametocidal factors (Gc), which ensure their preferential transmission by triggering damages in cells lacking them via chromosome break induction. Hence, we hypothesized that the gametocidal system can be adapted for chromosome manipulations between Triticum and Secale chromosomes in hexaploid triticale (×Triticosecale Wittmack). In this work we studied the little-known gametocidal action of a Gc factor located on Aegilops geniculata Roth chromosome 4Mg. Our results indicate that the initiation of the gametocidal action takes place at anaphase II of meiosis of pollen mother cells. Hence, we induced androgenesis at postmeiotic pollen divisions (via anther cultures) in monosomic 4Mg addition plants of hexaploid triticale (AABBRR) followed by production of doubled haploids, to maintain the chromosome aberrations caused by the gametocidal action. This approach enabled us to obtain a large number of plants with two copies of particular chromosome translocations, which were identified by the use of cytomolecular methods. We obtained 41 doubled haploid triticale lines and 17 of them carried chromosome aberrations that included plants with the following chromosome sets: 40T+Dt2RS+Dt2RL (5 lines), 40T+N2R (1), 38T+D4RS.4BL (3), 38T+D5BS-5BL.5RL (5), and 38T+D7RS.3AL (3). The results show that the application of the Gc mechanism in combination with production of doubled haploid lines provides a sufficiently large population of homozygous doubled haploid individuals with two identical copies of translocation chromosomes. In our opinion, this approach will be a valuable tool for the production of novel plant material, which could be used for gene tracking studies, genetic mapping, and finally to enhance the diversity of cereals.

Naphthaleneacetic acid impairs ovule fertilization and early embryo development in loquat (Eriobotrya japonica Lindl.)

By applying 20mgl−1 of α-naphthaleneacetic acid (NAA) potassium salt to ‘Algerie’ loquat tree flowers prior to or at anthesis pollen grain germination was signficantly inhibited and pollen tube growth was halted, both in vivo and in vitro conditions, thus ovule fertilization and fruit set were prevented. These effects of NAA are direct since stigmas were not damaged or dried off by the treatment. Mature but not fertilized ovule and newly fertilized ovule were also sensitive to the gametocidal action of the NAA, whereas new developing seeds did not, both in vivo and in vitro conditions as well. According to our results, NAA acts as a chemical thinner of loquat by preventing ovule fertilization and fruit set and by causing ovule/embryo abortion, depending on the phenological growth stage at treatment.

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 647,075bp). 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.

Chromosome Aberrations Induced by 5-Azacytine in the Root Tips of Wheat Carrying the Gametocidal Chromosome 3C

When the gametocidal chromosome 3C from Aegilops triuncialis was introduced into common wheat, it was preferentially transmitted and it had severe Gc action in Chinese Spring (CS). In this study, we used the root tips of Chinese Spring and Chinese Spring carrying the gametocidal chromosome 3C to analyze the relationship between gametocidal action and DNA methylation. They were treated with the hypomethylation agent 5-azacytine (5-AC) in 100µM concentration. In the root tips of Chinese Spring we observed abnormal division at a low frequency, which less than 1%. In contrast, in Chinese Spring-Gametocidal chromosome 3C disomic addition line a mass of chromosome aberrations were observed and the major form was chromosome breakage. This suggested that during early seed development chromosome breakage induced by the gametocidal chromosome 3C was repressed by DNA methylation. In addition, after 5-AC treatment the amount of chromosome breakage might be relative to Gc action.

INHERITED SEX-MOSAIC IN MAN

Introgression of agronomically important traits from barley to wheat is important for the improvement of wheat. Also, knowledge about precise location of genes in barley chromosomes is a prerequisite for map based gene isolation. The isolated genes would be introduced into wheat by crop transformation and consequently improve wheat. Many attempts have been conducted to achieve chromosome mediated introgressions from barley to wheat and to construct chromosome maps of barley showing physical locations of genes in the past. In the present study I have developed an alternative chromosome mutation inducing system capable of producing barley/wheat translocations and deletions in barley chromosome. The following is the summary. 1)In the attempt to induce breakage in barley chromosomes, I introduced a gametocidal chromosome 2C into six wheat barley addition lines. Chromosome 2C, from Aegilops cylindrica, which is a related species of wheat, has a gametocidal action causing chromosome breakage in the progeny of the monosomic 2C addition line of Chinese Spring wheat. The critical plants (21″+H″+2C′), disomic for each of barley chromosomes and monosomic for the 2C chromosome, were obtained. 2)The six critical lines were either selfed or backcrossed with the respective wheat barley addition lines. The selfed and backcrossed progeny of these lines were cytologically investigated by N banding and FISH using the barley probe HvT01 that is specific to the subterminal repeats of barley chromosomes. Various types of structural aberrations, most of which were deletions and translocations, were detected for all barley chromosomes with frequencies ranging from 10.8% to 27.9%. 3)Chromosome 7H was chosen to investigate the distribution of the breakpoints in the aberrations. Reciprocal crosses were made between the mutation inducing common wheat line (or critical lines) (21″+7H″+2C′) and the 7H addition line of common wheat (21″+7H″) to obtain more 7H deletions and 7H/wheat translocations. There were various types of aberrations as observed in the previous study. The breakpoints of these deletions and translocations appeared to distribute along the entire length of chromosome 7H.

A gametocidal factor of Oryza glaberrima Steud. in O. sativa L.

Recurrent backcrossing has been carried out with a view to transfer a gene for non-glutinous endosperm from two strains of O. glaberrima (Wxg/Wxg) to glutinous japonica and indica varieties (wx/wx) of Oryza sativa. In the course of backcrosses Wxg/wx segregants were crossed with each of the two glutinous varieties of sativa as the respective recurrent male parent. The wx/wx and Wxg/wx segregants in the successive generations were consistently fully fertile and semi-sterile, respectively. The semi-sterility of Wxg/wx plants was attributable to abortion of most of the pollen grains carrying the gene wx. The nucleus but not cytoplasm was related to the semi-sterility. The Wxg/Wx plants having the gene for non-glutinous endosperm of a glaberrima strain and a japonica variety of sativa were also semi-sterile. Both wx- and Wx-megaspores in the plants heterozygous for the gene Wxgwere deleteriously affected. The results could be explained by assuming that a factor tightly linked with the gene Wxgof glaberrima sterilizes gametes not carrying it in the heterozygotes and that the gametocidal action is exerted when combined with the sativa nucleus by the recurrent backcross method.

Gametocidal chromosomes and their induction of chromosome mutations in wheat.

Certain alien chromosomes have been found to be preferentially transmitted in wheat. They are derived from differnt species of Aegilops, a genus related to wheat (Triticum). Such chromosomes ensure their own selective retention in wheat by causing sterility in gametes not carrying them, hence they are termed gametocidal chromosomes. There are at least three types of the gametocidal chromosomes that have different mode of gametocidal action and homoeology. The gametocidal chromosomes in the same homoeologous group (group 2, 3, or 4) have the same gametocidal action. Some of the gametocidal chromosomes induce chromosome mutations in either gametophytes or zygotes of common wheat with specific genotypes. The chromosome mutations preferentially occur in gametophytes without the gametocidal chromosome or in the zygotes when the gametocidal chromosome is delivered by the pollen. Chromosome mapping using induced deletions is under way.

Two types of gametocidal chromosome of Aegilops sharonensis and Ae. longissima.

Certain chromosomes of Aegilops sharonensis (2n=14, S1S1) and Ae. longissima (2n=14, S1S1), wild species related to wheat (Triticum), have been known to be retained selectively in durum and common wheats by their gametocidal action that is exerted exclusively on gametes lacking the Aegilops chromosome. Three of such gametocidal chromosomes from Ae. sharonensis and two from Ae. longissima, collected from different sources, were studied on their cytological features, homoeology, and interrelation. One of the Ae. sharonensis and one of the Ae. longissima chromosomes had similar N-banding patterns, which resembled that of wheat chromosome 2B, and were homoeologous to the group 2 wheat chromosomes. The other gametocidal chromosomes, which had been proved or speculated to be in homoelogous group 4, had N-banding patterns relatively similar to wheat chromosome 4A; the two Ae. sharonensis chromosomes could not be distinguished by the N-banding. In double monosomic additions of common wheat with the gametocidal chromosomes of the same homoeology, gametes carrying either or both of the alien chromosome became fertile, and in those with non-homoeologous gametocidal chromosomes, only the gametes carrying the alien chromosome of homoeologous group 4 were functional. Thus, it became clear that there were two types of gametocidal chromosomes each in Ae. sharonensis and Ae. longissima.

Transfer of stem rust resistance to wheat from an Agropyron chromosome having a gametocidal effect

Two procedures were used to induce homoeologous recombination between Agropyron elongatum (Host) Beauv. chromosome 7el2 and wheat chromosomes. One procedure involved the use of 'Chinese Spring' nullisomic 5B – tetrasomic 5D, and resulted in plants lacking chromosome 5B. In the second procedure, a line carrying the mutant gene ph1b was used, and plants were produced that had only a 5B chromosome carrying ph1b. Both procedures resulted in the transfer of a gene or genes for stem rust (Puccinia graminis tritici Eriks. and Henn.) resistance from chromosome 7el2 to wheat chromosomes. During the transfer process, it was discovered that both the whole Agropyron chromosome and the recombinant chromosomes showed preferential transmission through the female gametes, but not through the male gametes. On heterozygous plants seed set was greatly reduced. Apparently, the Agropyron chromosome or a gene carried by it had a gametocidal action that resulted in female gametes, which did not carry the gene, failing to function. However, homozygous lines showed normal fertility.

GAMETOCIDAL CHROMOSOMES OF THREE AEGILOPS SPECIES IN COMMON WHEAT

Single chromosomes from Aegilops triuncialis L. (2n = 28, CCCuCu), Ae. sharonensis Eig (2n = 14, S1S1), and Ae. longissima S. &M. (2n = 14, S1S1) were added respectively to common wheat Triticum aestivum L. cv. 'Selkirk' (2n = 42, AABBDD) in monosomic condition and confirmed to have a gametocidal action on common wheat gametes lacking the Agilops chromosome. Three double monosomic addition lines, which had two of the three Aegilops chromosomes, were produced from the monosomic addition lines, and relationships among the three Aegilops chromosomes were studied. The Aegilops chromosomes did not pair with one another in the double monosomic addition lines. In the progeny of the double monosomic addition lines with the triuncialis and sharonensis chromosomes or with the triuncialis and longissima chromosomes, both Aegilops chromosomes were almost always transmitted through male and female gametes. In the progeny of the double monosomic addition line with the sharonensis and longissima chromosomes, only the longissima chromosome was preferentially transmitted through male and female gametes. Thus, the three Aegilops chromosomes were proved to be different from one another in the gametocidal action in common wheat.

Exclusive Preferential Transmission of an Alien Chromosome in Common Wheat1

AbstractAegilops longissima S. & M. and Ae. sharonenis Eig were crossed with Triticum dicoccum Schrank ‘Khapli’ pollen, and F1 hybrids were crossed several times with T. aestivum L. em. Thell. pollen to substitute the T. aestivum nucleus into the cytoplasm of these Aegilops species. Partially male and female‐sterile plants having a monosomic substitution (20″+2′) or a monosomic addition (21″+1′) were obtained from these nuclear‐substitution backcrosses. These plants had normal anther extrusion, abundant pollen, and incomplete scattered seed set on bagged or crossed heads, and the reciprocal cross with euploid T. aestivum as a female gave complete seed set. Euploid, 21” plants were not obtained from crosses in either direction, most offspring having again 20″+2′ or 20″+1′, and having abundant pollen but partial seed set on bagged heads. All the selfed progeny of plants with a monosomic substitution or monosomic addition had a disomic substitution or disomic addition, respectively, and were fertile. Evidently, male and female gametes with one Aegilops chromosome were functional, and those without the Aegilops chromosome did not function.There was thus an apparent gametocidal action of the sporophyte having an Aegilops chromosome, on the gametes lacking this chromosome. Occasionally malesterile euploid T. aestivum plants with Ae. sharonensis cytoplasm were obtained as a result of rare functional female gametes without the critical Ae. sharonensis chromosome.

Effects of Granular Ethephon on Male Sterility in Wheat1

Granular ethephon (2‐chloroethylphosphonic acid) was studied as a gametocide on three winter and six spring wheat (Triticum aestivum L.) cultivars. Applications were made during four stages in ontogeny viz, seeding, primary tiller emergence, initiation of spikelet primordia on the main culm, and during the early‐boot phase, in rate categories of 2 to 8, 26.9 to 109.2, and 164.5 to 822.0 kg a.i./ha. Sterility levels reached mean values of 65 (range 10 to 95), 71 (range 48 to 94) and 89% (range 36 to 100) for the respective rate categories. These values are means of pot/plot data and include different order tillers. Mean sterility levels rose with increase in the rate of gametocide; it was most effective when applied at the highest rate during reproductive development on the main culm. At low rates of application, winter cultivars were more responsive than spring cultivars perhaps because of their increased uptake with a more extensively developed root system.The granular form of ethephon may be more suited to practical use than the liquid form because the latter demands precision in the time of spraying and causes undesirable side effects. No apparent morphological or physiological abnormalities were observed with the granular form even at the very high rates used in these studies. A better understanding of the site and form of uptake may permit the use of lower rates with effective gametocidal action.

A field experiment on the value of plasmoquine in the prophylaxis of malaria

1. 1. Plasmoquine was given in an adult dose of 0·04 g ammes (0·9 mg. per kilogram body weight) twice weekly to a population of about 330 on a Rubber Estate where the incidence of malaria had been high for many years. 2. 2. At regular intervals of three months surveys were carried out to determine the malaria parasite rate (thick film method), the spleen rate, and the average hæmoglobin value of the population on this Experimental Estate. Surveys of anopheline breeding places were also carried out at the same time. 3. 3. Similar surveys were carried out at the same times on two contiguous Control Estates with populations of 405 and 362. 4. 4. The experiment was continued for a period of twelve months. 5. 5. The effect of plasmoquine prophylaxis on the incidence of clinical malaria was marked. In the year preceding the experiment the subtertian and benign tertian cases numbered respectively 30·4 and 52·5 per cent. of the population. Throughout the experimental period of twelve months, the incidence was 7·0 and 8·7 per cent. for subtertian and benign tertian infections respectively. 6. 6. From the parasite rates alone, the results were somewhat inconclusive, since the Control Estates showed a fall in the number of blood infections equal to that found on the Experimental Estate. Towards the end of the experiment, no P. falciparum gametocyte carriers could be found on the Experimental Estate. 7. 7. From the parasite rates only, it appears that the method was perhaps less satisfactory for benign tertian than for sub-tertian infections. This may possibly be due to plasmoquine having a less marked gametocidal action on P. vivax. 8. 8. There was slight improvement in the splenic index and in the average hæmoglobin percentage, but the improvement in the general physical condition of the labourer was most satisfactory. This observation was confirmed by the sickness rate which fell by 51 per cent. on the Experimental Estate, as compared with the previous year. On Control Estate I, the sick rate fell by 11 per cent. during the same period, but there was an increase of 21 per cent. in the sick rate on Control Estate II. 9. 9. The cost of this method of prophylaxis is about $600 (Straits Settlements) or £70 per annum per 100 labourers, including treatment of the average number of dependents.