Male Sterile Strain Research Articles

PiggyBac-based transgenic RNAi of serine protease 2 results in male sterility in Hyphantria cunea

Fall webworm, Hyphantria cunea, is a global invasive forest pest that causes serious damage to the economy and ecosystem of agriculture and forestry. Due to the extent of the problem and the difficulty of conventional chemical control, new technologies must be pursued, such as genetic-based inheritable insect sterile technology (gSIT), which exhibits promise for pest control. In the present study, we established a piggyBac-based transgenic system in fall webworm and generated a dominant male-sterile strain by targeting the seminal fluid protein serine protease 2 (Hcser2), displaying an outstanding trait of gSIT. First, an RNA polymerase type III (Pol III) promoter, the HcU62 small nuclear RNA (snRNA) gene promoter, was identified and characterized through direct injection of RNAi plasmids in vivo. Quantitative real-time PCR revealed that HcU62 had the greatest knockdown efficiency of the Hcyellow gene among five short hairpin RNA (shRNA) plasmids tested, designated HcU61–HcU65. Second, subsequent application of piggyBac-based transgenic RNAi (HcU62: shHcyellow, Ysh2) significantly reduced the expression level of the Hcyellow gene, resulting in a stable yellow observable phenotype from the larval to pupal stages in Ysh2 transgenic mutants. Finally, an HcU62-driven transgenic RNAi strain targeting the Hcser2 gene was obtained, resulting in a dominant male-sterile phenotype. Significantly, this process did not affect the growth, development, mating behavior or egg laying of the mutants, and the dominant sterile trait could be inherited in the next generation through female Hcser2 mutants. Furthermore, CRISPR/Cas9-mediated disruption of the Hcser2 gene further confirmed the dominant sterile phenotype, supporting it as a generalized target for genetic control of H. cunea. This study reports the first piggyBac-mediated transgenic system in H. cunea, providing a promising genetic method for controlling this pest by targeting Hcser2 gene.

Generating Novel Male Sterile Tomatoes by Editing Respiratory Burst Oxidase Homolog Genes

Hybrid breeding of tomatoes (Solanum lycopersicum), an important vegetable crop, is an effective way to improve yield and enhance disease and stress resistance. However, the efficiency of tomato hybridization is hindered by self-fertilization, which can be overcome using male sterile lines. It has been reported that reactive oxygen species (ROS) act as a key regulator for anther development, mediated by RBOH (Respiratory Burst Oxidase Homolog) genes. Here, two tomato anther-expressed genes, LeRBOH (Solyc01g099620) and LeRBOHE (Solyc07g042460), were selected to cultivate novel tomato male sterile strains. By using a CRISPR/Cas9 system with a two-sgRNA module, the lerboh, lerbohe, and lerboh lerbohe mutant lines were generated, among which the lerbohe and lerboh lerbohe mutants displayed complete male sterility but could accept wild-type pollens and produce fruits normally. Further analysis uncovered significantly decreased ROS levels and abnormal programmed cell death in lerboh lerbohe anthers, indicating a key role of ROS metabolism in tomato pollen development. Taken together, our work demonstrates a successful application of gene editing via CRISPR/Cas9 in generating male sterile tomatoes and afforded helpful information for understanding how RBOH genes regulating tomato reproduction process.

Mutation of the seminal protease gene, serine protease 2, results in male sterility in diverse lepidopterans

Sterile insect technology (SIT) is an environmentally friendly method for pest control. As part of our efforts to develop a strategy that results in engineered male-sterile strains with minimum effects on viability and mating competition, we used CRISPR/Cas9 technology to disrupt Ser2, which encodes a seminal fluid protein, in the model lepidopteran insect, Bombyx mori, and an important agricultural pest, Plutella xylostella. Disruption of Ser2 resulted in dominant heritable male sterility. Wild-type females mated with Ser2-deficient males laid eggs normally, but the eggs did not hatch. We detected no differences in other reproductive behaviors in the mutant males. These results support the conclusion that Ser2 gene is necessary for male reproductive success in diverse lepidopterans. Targeting Ser2 gene has the potential to form the basis for a new strategy for pest control.

A novel Rf gene controlling fertility restoration of Ogura male sterility by RNA processing of orf138 found in Japanese wild radish and its STS markers

To reveal the molecular and genetic mechanism of fertility restoration in Ogura male sterility in Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides), we investigated fertility restoration of a plant that lacks the dominant type of orf687, a previously identified fertility restorer gene. A total of 100 F2 plants were made from the cross between a male-sterile strain with the Ogura cytoplasm, 'MS-Gensuke', and a Japanese wild radish plant. Segregation of pollen fertility in the F2 plants led us to assume that 2 dominant complementary genes controlled the fertility restoration of the plants. However, the fertility of 27 of 59 male-fertile plants was not completely restored, resulting in a group of plants with partial male fertility. Northern blot analysis of the CMS-associated gene orf138 indicated that one restorer allele (termed Rft) was involved in the processing of orf138 RNA. Rapid amplification of cDNA ends (RACE) and subsequent Northern blot analysis confirmed that the orf138 transcript lost a 5' part of the coding region of the orf138 gene in the restored plants. The accumulation of ORF138 protein was significantly reduced by Rft, but trace amounts of the protein were recognized in both partially male-fertile and male-sterile plants with Rft. The relationship of pollen fertility and segregation of co-dominant sequence tagged site (STS) markers in the F2 generation suggested that the penetrance of Rft was so low that Rft needs suitable conditions to function sufficiently for the complete restoration of fertility.

Fertility response to photoperiod and temperature in indica photoperiod-sensitive male sterile rice

The aim of this work was to develop a photoperiod-sensitive male sterile rice with stable sterility. We developed Changguang S, an indica rice strain, by using a short critical day length. Differences in the fertility responses of Changguang S strain pollen to temperature and photoperiod under natural and controlled conditions were studied. The results showed that Changguang S strain exhibited stable sterility under long-day and low-temperature conditions (22°C, 15 days). The stability of sterility was significantly higher than that of other such rice strains, Nongken 58S and 7001S. The critical photoperiod for inducing male sterility in Changguang S was 13 h or shorter, and its duration was significantly shorter than that required for rice strains Nongken 58S and 7001S. It is suggested that Changguang S is a typical photoperiod-sensitive male sterile rice strain with a shorter critical day length and a lower critical temperature. It is promising to apply this strain to two-line hybrid rice production.

Karyotypes and meiotic behavior of chromosomes in two male sterile strains of Brassica campestris L.

Male sterility was examined in backcross families of the hybrid Diplotaxis muralis×B. campestris (murcytoplasm). Segregation of sterile and fertile plants did not fit to any of the Mendelian ratios. Since an extra chromosome is known to occur in sterile plants having a similar combination of mwr-cytoplasm and B. napus nucleus, the mitotic and meiotic chromosomes were analysed. Similarly, the chromosomes were examined in another male sterility system, in backcross families of B. napus x B. campestris (pol-cytoplasm). The normal chromosome number (2n = 20) was detected in all the sterile as well as the fertile plants. After improvements of the staining technique, all the chromosomes were identified in some cells. Ten bivalents were formed in meiosis and the segregation of chromosomes appeared to be normal. The visibility of satellites was examined at diakinesis. Variation among plants was observed but no correlation with male sterility was found.

Neurochondrin Negatively Regulates CaMKII Phosphorylation, and Nervous System-specific Gene Disruption Results in Epileptic Seizure

Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca(2+)/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.

Characterization of genetic relationships of Rosa roxburghii Tratt and its relatives using morphological traits, RAPD and AFLP markers

SummaryCili (Rosa roxburghii Tratt) is of great interest due to its high content of vitamin C, very high superoxide dismutase (SOD) activity, attractive senescence-retarding and cancer-preventing effects, together with immunity to rose blackspot disease. Thirty-six morphological traits, 251 RAPD markers amplified with 29 arbitrary primers, and 1685 AFLP markers generated from 54 primer combinations were employed to discriminate between 15 accessions, and to evaluate the relatedness of cili with a sample set of its relatives. The genotypes could be efficiently identified by genotype-specific RAPD or AFLP markers, yielded from the four most polymorphic primers: OPAN-07, OPB-11, OPAF-16 and OPW-02, or the six most polymorphic primer combinations: EA01/MC04, EA01/MC05, EA01/MC06, E02/MC01, EA05/MC04 and EA06/MC03, respectively. Compared with the relatives, very close relationships were found among the genotypes of cili. Furthermore, a high relatedness was strikingly obtained between R. sterilis S. D. Shi and R. hiveichowensis Yu and Ku with the three kinds of data. Using the Mantel correspondence test, significant correlations between the three kinds of data were observed, among which matrices of RAPD and AFLP were the most highly correlated: r= 0.7154 (P<0.01 ) . Based on the assessments of the three kinds of data, multi-corolla cili and white-flower cili are certainly variants from R. roxburghii Tratt, while seedless cili presumably originates from a completely male-sterile strain of R. kweichonensis.

Evaluation of a new culture method for mass-propagation of a photoperiod-temperature sensitive genic male sterile rice strain N19S

A newly established culture method was evaluated for application in mass-propagation of a photoperiod-temperature sensitive genic male sterile rice strain. It was found that the new culture method was very efficient for the regeneration of plants from callus of this strain, and a high concentration of 6-benzyladenine necessary for highly efficient plant regeneration culture did not seem to have negative effects on some agronomic traits investigated of the regenerated plants and their progenies. By the new culture method, a large number of plants could be regenerated in a relatively short time (approximately 8×1012 plants within 300 days) from one naked seed-explant. Because of this, prominent drift of the PTGMS rice strain, which accumulates gradually during the course of propagation of the seeds through generations, in the response to photoperiod-temperature conditions could be avoided when this in vitro culture protocol is employed instead of seed propagation. Differences in rates of pollen fertility and seed setting were found between regenerated plants and seed-grown plants, and between their respective first and second progenies. These differences are also of important application value in two-line rice production: Higher pollen fertility of the regenerated plants can be applied with higher efficiency for the propagation of the strain seeds because they have much higher seed setting by self-fertilization; lower pollen fertility of the regenerated plant progenies will result in much lower seed setting by self-fertilization and is, therefore, conducive to the production of hybrid seeds of higher purity by natural crossing with the other line in the two-line system.

Ami1, an orthologue of the Aspergillus nidulans apsA gene, is involved in nuclear migration events throughout the life cycle of Podospora anserina.

The Podospora anserina ami1-1 mutant was identified as a male-sterile strain. Microconidia (which act as male gametes) form, but are anucleate. Paraphysae from the perithecium beaks are also anucleate when ami1-1 is used as the female partner in a cross. Furthermore, in crosses heterozygous for ami1-1, some crozier cells are uninucleate rather than binucleate. In addition to these nuclear migration defects, which occur at the transition between syncytial and cellular states, ami1-1 causes abnormal distribution of the nuclei in both mycelial filaments and asci. Finally, an ami1-1 strain bearing information for both mating types is unable to self-fertilize. The ami1 gene is an orthologue of the Aspergillus nidulans apsA gene, which controls nuclear positioning in filaments and during conidiogenesis (at the syncytial/cellular transition). The ApsA and AMI1 proteins display 42% identity and share structural features. The apsA gene complements some ami1-1 defects: it increases the percentage of nucleate microconidia and restores self-fertility in an ami1-1 mat+ (mat-) strain. The latter effect is puzzling, since in apsA null mutants sexual reproduction is quite normal. The functional differences between the two genes are discussed with respect to their possible history in these two fungi, which are very distant in terms of evolution.

Distribution and allelism of restorer genes for Ogura cytoplasmic male sterility in wild and cultivated radishes.

The distribution of the fertility restorer genes for Ogura cytoplasmic male sterility in Raphanus raphanistrum, Japanese wild radish, and cultivated radishes (R. sativus) was studied by observing the pollen fertility of the F1's from crosses with a male sterile strain having the Ogura cytoplasm. The restorer gene is widely distributed in the wild species and wild radishes irrespective of cytoplasm type. Among the cultivated radishes, the European and Chinese varieties had the restorer gene, while most of the Japanese cultivars did not. The allelism of three restorer genes from R. raphanistrum, Japanese wild radish, and the European cultivar was estimated by the segregation of the fertility in the progenies produced by crosses between strains with different restorer genes. The results suggested that the restorer genes from these three sources were allelic.

Selective DNA amplification regulates transcript levels in plant mitochondria

Most plant mitochondrial genomes exist as subgenomic-size fragments apparently due to recombination between repetitive sequences. This leads to the possibility that independently replicating subgenomic domains could result in mitochondrial gene copy number variation. We show, through Southern-blot analysis of both restricted and intact mtDNA, that there are gene-specific copy number differences in the monocot Zea mays. Comparison of two different maize genotypes, B37(N) and B37(T), a cytoplasmic male-sterile strain, reveal fewer gene copy number differences for B37(T) than for B37(N). In contrast to maize, significant gene copy number differences are not detected in the dicot Brassica hirta. We also demonstrate that mitochondrial transcriptional rates in both species are apparently dependent on gene copy number since relative rates determined by run-on analysis are proportional to relative gene copy numbers. Thus a direct relationship exists between plant mitochondrial gene copy number and transcriptional rate.

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.

Sequence analysis of a mitochondrial DNA fragment isolated from cultured cells of carrot cytoplasmic male-sterile strain.

2.0 kb Hind III fragment isolated from cytoplasmic male-sterile carrot mitochondria, designated PKT5, was hybridized to ORF13 which is the coding region of a unique polypeptide in maize CMS (Dewey et al., 1986). Sequence analysis indicated that PKT5 is consisted of 3 domains. Domain 1 was identical to the 5'-flanking region of atp6 in maize CMS-TURF2H3 sequence (Dewey et al., 1986). Domain 2 contained a novel ORF encoding 72 amino acids, which was extremely homologous to the amino-terminal 67 amino acids of the unique ORF13 in maize CMS. Domain 3 except an amino acid change (Ile87 = ATT for Asn87 = AAT), was identical to ORF25 polypeptide in maize CMS. Connective sequences of these 3 domains were also highly homologous to the maize CMS-TURF2H3 sequence. Out of 7 recombination points in maize CMS-TURF2H3 sequence, at least 4 points were conserved in PKT5 sequence.

Analysis of mitochondrial DNAs from Oryza glaberrima and its cytoplasmic substituted line for Oryza sativa associated with cytoplasmic male sterility.

It was described previously that the combination of the cytoplasm of a Japanese variety of Oryza sativa and the nucleus of O. glaberrima produced by successive crossings led to male sterility. In the present study, the mitochondrial DNAs (mtDNAs) were isolated from this cytoplasmic male-sterile (CMS) strain and its maintainer strain, and were analyzed with restriction enzymes and by agarose gel electrophoresis. Differences in the restriction pattern were observed between the CMS strain having sativa cytoplasm and its maintainer strain having glaberrima cytoplasm. The plasmid-like DNAs (B1 and B2), which are often observed in mitochondria of CMS strains derived from O. sativa, could not be detected by Southern hybridization analysis as well as gel electrophoresis. It was suggested that the plasmid-like DNAs were not associated with the cytoplasmic male sterility. However, the homologous sequences to B1 and B2 were detected in the nuclear genomes of all species tested.

Analyis sof homology of small plasmid-like mitochondrial DNAs in the diferent cytoplasmic male sterile strains in rice.

Mitochondrial DNAs (mtDNAs) were isolated from several rice strains of Chinsurah boro II (fertile), Zhen Shan 97A (male sterile), Zhen Shan 97 (fertile), and Akihikari (normal japonica). Small plasmid-like DNAs, B1 and B2, which were found firstly in BT line, were cloned into plasmid vectors and mapped in terms of some of restriction enzymes. Plasmid-like DNAs were also observed in the mitochondria of Zhen Shan 97A and Zhen Shan 97 by agarose gel electrophoresis. To examine whether these plasmids were identical or not, hybridization analyses were carried out with B1 and B2 as probes. Consequently, it was suggested that two plasmids of Zhen Shan 97A were identical to B1 and B2, and one of the plasmids of Zhen Shan 97 was identical to B2. The smaller plasmid of Zhen Shan 97 had no homology to either B1 or B2. Co-existence of B1 and B2, while common, is not an obligatory feature. Because B2 was found in the fertile cytoplasm of Zhen Shan 97, an involvement of B2 with cytoplasmic male sterility (cms) might be denied.

Anther culture application to breeding of a restorer for a male-sterile cytoplasm of wild rice (ms-CW).

Breeding of a restorer line for the male-sterile cytoplasm [ms-CW] of wild rice (W 1 ; Oryza rufipogon Griff.), identified by KATSUO and MIZUSHIMA (1958), was demon-strated by transferring the restoring gene(s) of W 1 to a cultivar through anther culture. Anthers of the F1 hybrid between W I (=) and O. sativa L. cv. Somewake (=) were cultured and two regenerated plants with high pollen stainability were obtained. Of these two plants, one was sterile in seed set, but the other (A1) was fertile and its selfed progenies (A2) also showed a high seed fertility. In a test cross of one of A 2 plants (=) with a cytoplasmic male-sterile strain carrying the cytoplasm of W1 and the nucleus of cv. Reimei, the resulting F1 gave a high seed fertility, demonstrating that the regenerated plant (A1) possessed restorer gene(s). The F2 and BIFI progenies of W1 (=)/Somewake in the conventional crossing showed a wide variation in pollen stainability and seed fertility, that might be due to not only cytoplasmic but also genic hybrid sterility. These results suggest that anther culture is effectively utilized for breeding restorer lines from remote hybrids, especially when they suffer from hybrid sterility.

Cytological and genetical studies of a male sterile celery

The inheritance and nature fo male sterility of a cerley (Apium graveolens L.) strain (MS1) is reported. Male sterility in MS1 is determined by a a recessive genotype for a single locus. Tests for linkage with the isozyme chromosome markers SDH-1 and PGM-1 were negative. MS1 male sterility was associated with a defective tapetum characterized by prominent vacuoles and premature degeneration. The stamens in the male sterile strain persisted in the flowers up to stigma receptivity, while in the male fertile plants they dropped before style expansion. The male sterile flowers produced normal amounts of nectar, resulting in cross pollination by various species of pollinators. It was estimated that the sterile strain produced 30% less seed than normal male fertile. Its possible use for hybrid celery seed production is discussed.

Sexual differentiation in Mercurialis annua L. (2n=16): Evidence for a control system of nucleo-cytoplasmic relationships by the study of tyrosyl-tRNAs from ‘developmental mutants’ of male sterility

In ‘developmental mutants’ of staminogenesis carrying sensitive cytoplasm (male sterile strains of the dioecious plant Mercurialis annua), the increasing levels of tRNAs tyr aminoacylation as a function of the degree of sterility confirm the classification of strains previously obtained by means of genetics and physiology: the series begins with fertile males with normal cytoplasm (0.61), restored fertiles (0.74), semisteriles (0.74) and total steriles (0.84), (all with S cytoplasm) and ends with normal females (1.06). The fractionation of tyrosyl-tRNAs shows that two chloroplast tyrosine tRNAs isoacceptors absent in normal fertile staminogenesis and present in female organogenesis are always expressed in strains with S cytoplasm, in the presence or absence of fertility nuclear restorer genes. In S cytoplasm the chloroplast escapes from its normal regulation during sexual differentiation. In restored fertile males (S cytoplasm), the nuclear restorer genes do not suppress but only compensate the chloroplast disfunction that remains of female type.

Studies on a male sterile strain having the Brassica campestris nucleus and the Diplotaxis muralis cytoplasm. I On the breeding procedure and some characteristics of the male sterile strain

1. D. muralis was used as a female parent in crosses with B. campestris (a commercial cultivar, Yukina). Successive backcrossing by the pollen of the latter species raised a male sterile strain having Yukina nucleus and D. muralis cytoplasm. 2. Morphological characters 0L this strain were quite alike to cv. Yukina, except flower organs; such as two nectaries, narrow petals and short filaments with stunted anthers in which was observed a small quantity of pollen. Pistils were normal in mor-phology as well as in function. 3. Two types 0L male sterility were recognized in the strain; one was completely male sterile (MS) without anther dehiscence and the other was partially male sterile (PMS), with a little emission of pollen. In order to obtain a family whose plants are all MS, maintainers were made by two methods, from a selfed progeny of the cultivar and from a cross Yukina><PMS. Completely male sterile families could be established by both the methods.