Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.

Inter-varietal sterility and fertility restoration in Oryza sativa Linn.

This study explored the underlining reproductive factors that cause sterility in rice, using hybrids obtained from crosses among landrace selections and improved varieties. The study was carried out between 2016 and 2018. Nine landrace cultivars and two improved varieties were involved in the hybridization experiment. Among the 17 putative hybrids that were obtained, only 5 were confirmed as true hybrids. Meiotic chromosome studies and pollen studies in the F1, and Mendelian segregation studies for fertility in the F2 were carried out. Some F2 lines were monitored to F3 to ascertain the level of fixation of gene combinations for fertility. The results obtained from the chromosomal studies showed that phenomena such as laggards, precocious movements, formation of multivalents, and unequal segregation to the poles are associated with pollen sterility in all - 5 hybrids, at the F1 and F2 generations. The indehiscence of anthers contributed to infertility due to pollen shortage than the fertility of the pollens themselves. Even though in males, there is a preponderance of male sterility, female sterility is also a phenomenon that is possibly contributing to inter-varietal sterility. The segregational pattern of 13:3 was observed for fertility in the F2 which suggests the inhibition of fertility by a gene in the dominant state. There was the restoration of fertility in many of the lines advanced to the F3 to up to 93 % fertility. It was, therefore, concluded from the study that landraces of rice still hold the key for the rice crop improvement and should therefore be conserved.

The Unstable Restorer-of-Fertility Locus in Pepper (Capsicum Annuum. L) is Delimited to a Genomic Region Containing PPR Genes

The use of cytoplasmic-genic male sterility (CGMS) systems greatly increases the efficiency of hybrid seed production. Although marker development and candidate gene isolation have been performed for the Restorer-of-fertility (Rf) gene in pepper (Capsicum annuum L.), the broad use of CGMS systems has been hampered by the instability of fertility restoration among pepper accessions, especially sweet peppers, due to the widespread presence of the Unstable Restorer-of-fertility (Rfu) locus. Therefore, to investigate the genetic factors controlling unstable fertility restoration in sweet peppers, we developed a segregation population from a cross between a male-sterile line and an Rfu-containing line to examine the inheritance of Rfu. Individuals with unstable restoration vs. sterility segregated at a 3:1 ratio, indicating that a single dominant gene controls unstable fertility restoration. Genetic mapping delimited the Rfu locus to a 479 kb genomic region on chromosome 6 flanked by two markers, which is close to but different from the previously identified Rf-containing region. The Rfu-containing region harbors a pentatricopeptide repeat (PPR) gene, along with 13 other candidate genes. In addition, this region is syntenic to the genomic region containing the largest number of Rf-like PPR genes in tomato. Therefore, the dynamic evolution of PPR genes might be responsible for both the restoration and instability of fertility in pepper. During genetic mapping, we developed various molecular markers, including one that co-segregated with Rfu. These markers showed higher accuracy for genotyping than previously developed markers, pointing to their possible use in marker-assisted breeding of sweet peppers.

Development and validation of CAPS-marker associated with the Rf2 gene in sorghum (Sorghum bicolor (L.) Moench)

Background. The development of heterotic hybrids based on cytoplasmic male sterility (CMS) is the leading strategy in breeding sorghum (Sorghum bicolor (L.) Moench). The trait of pollen fertility restoration in forms with CMS A1 (milo), predominantly used in sorghum breeding, is determined by at least two dominant complementary genes Rf1 and Rf2, and also gene Rf5. The development of accessible molecular markers of sorghum Rf genes is highly relevant for hybrid breeding, since they can significantly accelerate the process of creating female sterile forms (A lines), sterility maintainers (B lines) and pollen fertility restorers (R lines).Material and methods. The studied material included 36 sorghum accessions from the VIR collection, which differed by the ability to restore pollen fertility in forms with A1-type CMS. The nucleotide polymorphism of 935 bp fragments of the PPR genes Sobic.002G057050, Sobic.002G054100, and Sobic.002G054200 located at the chromosome 2 was studied.Results. The fragments obtained with the use of a pair of 2459403fw and 2459403 primers were 935 bp long and included parts of three genes: Sobic.002G057050, Sobic.002G054100, Sobic.002G054200. For identifying the sequence variant Sobic.002G057050-1090 associated with the Rf2 gene, Tru9 I restrictase was chosen, which allows obtaining a 572 bp fragment unique for all the studied R lines. Such a marker was found in 10 sorghum lines from West China and Kyrgyzstan, which are widely used in breeding as fertility restorers. The fragment was found neither in three lines with sterile cytoplasm and their fertile analogues, nor in 7 accessions of kafir sorghum, which lacked functional alleles of Rf genes.Conclusions. It has been demonstrated that the marker can be used for selection and checking purity of R and B/A lines. It is also applicable for verifying hybridity of F1 seeds and analyzing hybrid populations from crosses of R lines 924-4, 928-1, 929-3, 931-1, 933-1/6, 1237-3, 1243-2, 1251, 1150-1, F10BC2 with A lines Nizkorosloe 81s, А-83 and А-10598. It may be suggested that the ability to restore pollen fertility in R lines, which lack the marker CAPS- 572, is determined by another Rf gene. The studied 935 bp fragment of Sobic.002G057050 harbours 22 SNP, therefore the development of CAPS-markers for their identification and differentiation can be promising.

Restoration of Fertility in Patients with Spontaneous Premature Ovarian Insufficiency: New Techniques under the Microscope

Premature ovarian insufficiency (POI), a condition affecting up to 1% of women by the age of 40 years, is characterized by an extremely low chance of spontaneous pregnancy. Currently, fertility restoration options are virtually nonexistent for this population. To become pregnant, the only solution is egg donation. Interestingly, animal studies have provided encouraging results in terms of fertility restoration, and consequently, research has begun into the most promising approaches for women suffering from POI. The PubMed database was searched for studies in which techniques aiming at restoring fertility in women with spontaneous POI were tested. Although robust studies are lacking, the literature suggests a positive effect of certain techniques on fertility restoration in women with POI. The most promising approaches seem to be intraovarian injection of autologous platelet-rich plasma or of mesenchymal stem cells. In addition to these, in vitro and mechanical activation of dormant follicles and etiology-driven therapies have also been studied with mixed results. No safety concerns were raised in these studies. The absence of robust studies does not allow us to draw meaningful conclusions on the efficacy or superiority of any single technique at this stage, and so research in this area should continue using robust study designs, i.e., multicenter randomized controlled trials including sufficient subjects to achieve statistical power.

Molecular Analysis Uncovers the Mechanism of Fertility Restoration in Temperature-Sensitive Polima Cytoplasmic Male-Sterile Brassica napus

Temperature-sensitive male sterility is a heritable agronomic trait affected by genotype-environment interactions. In rapeseed (Brassica napus), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is commonly used for two-line breeding, as the fertility of pol TCMS lines can be partially restored at certain temperatures. However, little is known about the underlying molecular mechanism that controls fertility restoration. Therefore, we aimed to investigate the fertility conversion mechanism of the pol TCMS line at two different ambient temperatures (16 °C and 25 °C). Our results showed that the anthers developed and produced vigorous pollen at 16 °C but not at 25 °C. In addition, we identified a novel co-transcript of orf224-atp6 in the mitochondria that might lead to fertility conversion of the pol TCMS line. RNA-seq analysis showed that 1637 genes were significantly differentially expressed in the fertile flowers of 596-L when compared to the sterile flower of 1318 and 596-H. Detailed analysis revealed that differentially expressed genes were involved in temperature response, ROS accumulation, anther development, and mitochondrial function. Single-molecule long-read isoform sequencing combined with RNA sequencing revealed numerous genes produce alternative splicing transcripts at high temperatures. Here, we also found that alternative oxidase, type II NAD(P)H dehydrogenases, and transcription factor Hsfs might play a crucial role in male fertility under the low-temperature condition. RNA sequencing and bulked segregant analysis coupled with whole-genome sequencing identified the candidate genes involved in the post-transcriptional modification of orf224. Overall, our study described a putative mechanism of fertility restoration in a pol TCMS line controlled by ambient temperature that might help utilise TCMS in the two-line breeding of Brassica crops.

Exploitation of Heterosis for Seed Yield and its Contributing Traits in CMS Based Hybrids of Indian Mustard [Brassica juncea (L.) Czern and Coss]

In the present study, line× tester analysis was carried out at Castor-Mustard Research Station, S. D. Agricultural University, Sardarkrushinagar, Gujarat, India during rabi 2018–19 (October 2018 to February 2019) in order to estimate all three types of heterosis (relative heterosis, heterobeltiosis and economic heterosis) for identification of superior cross combinations of Indian mustard [Brassica juncea (L.) Czern&Coss]. Thirty-five hybrids along with five CMS lines, seven testers and check GDM 4 were evaluated for ten different traits. The F1 generation of all the crosses exhibited fertility restoration with pollen fertility except F1 crosses with Vardan, Rohini and SKM 319 fertile line (0%). The remaining crosses exhibited pollen fertility ranging from 68.26% (Kranti×SKM 303) to 85.17% (Kranti×Mori ‘R’ 1-18). The analysis of variance for parents, hybrids and parents vs. hybrids revealed that mean sum of squares of parents were highly significant for majority of the characters except days to maturity. Whereas, hybrids differed highly significant for all the characters. Comparison of mean squares due to parents vs. hybrids was found significant for almost all the characters except number of seeds siliqua-1 and oil content. This indicates that considerable amount of genetic variability present among the parents and hybrids for all the characters studied. On the basis of per se performance, three hybrids viz., Kranti×Mori ‘R’ 1-18, SKM 9928×PusaAgrani and SKM 9928×Mori ‘R’ 1-18 were foundpromising for seed yield plant-1 over the standard check GDM 4.With respect to heterosis, one of the hybrid Kranti×Mori ‘R’ 1-18 (17.85 %)manifested significant and positive standard heterosis for seed yield plant-1.

Development of Brassica Napus L. Ogu-INRA CMS Restorers Using Recurrent Full-Sib Selection.

The Ogu-INRA CMS system in canola and rapeseed (Brassica napus L.) uses a cytological variant of the radish- (Raphanus sativus L.) derived Ogu CMS pollination control system introduced through interspecific introgression. The restorers (R-lines) contain an introgression that is associated with poor agronomic performance due to a large undesired segment of the radish chromosome that was introgressed along with the Rfo gene. The introgression contains pentatricopeptide (PPR) motif repeats that confer fertility restoration abilities to the R-lines. The objective of this research was to test the hypothesis that multiple cycles of intermating will result in R-lines with improved agronomic performance. A base population was developed by designing five R-line by R-line crosses. Twelve plants from each initial cross were grown and chain-crossed at random, without selection, other than the presence of the Rfo gene. Twelve flowers from each plant were crossed and the remainder of the plant was selfed. Three intermating crossing cycles (C0, C1 and C2) were completed and each was selfed three times for evaluation. Total pod number, seeds per pod, a visual pod rating, thousand seed weight and yield were evaluated. The visual pod rating showed a positive correlation with seeds per pod. Improvements for all traits were found at C0 and C1 when compared to the best parent. Individual families from two of the crosses showed a yield increase of over 78 % from the best parent. This suggests that improvements in yield components can be obtained from intermating R-lines.