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.

No Correlation Between Pollen Fertility and Viability: Differential Measures of Male Gametophytic Fitness in Cannabis sativa L.

Pollen grains are male gametophytes, an ephemeral haploid generation of plants, commonly engaging in competition for a limited supply of ovules. Since differential male fertility may influence the direction and pace of population evolution, the relative fitness of pollen is regularly estimated as either pollen viability, the proportion of pollen containing intact cytoplasm's and regenerative nuclei, or pollen fertility, the frequency of pollen germinating under standardized conditions. Here, we estimated the relative fitness of pollen in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing pollen fertility and viability from multiple sires. Pollen fertility quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen viability declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both fertility and viability analysis displayed no significant correlation, implying researchers cannot predict pollen fertility from pollen viability, nor infer male gametophytic fitness from a single measure. Our work demonstrates two approaches to measure proxies of male fitness in C. sativa, and identifies new questions around what are valuable estimates of male fitness in plants.

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.

Diagnostic value of Rf1 gene molecular markers in sunflower

Background. Modern production of sunflower seeds is currently based on the cultivation of high-yielding heterotic F1 hybrids from cross-breeding of lines with cytoplasmic male sterility (CMS) of PET1-type and fertility restorer lines. The paternal parent serves as a donor of the nuclear Rf1 gene functional allele, which is responsible for pollen fertility restoration in F1 plants. The detection of carriers of the Rf1 locus recessive and dominant alleles using diagnostic molecular markers accelerates breeding of female and male parental lines for creating hybrids. Materials and methods. The material for the study included 75 lines of various origins from the VIR sunflower genetic collection as well as hybrids from crosses of VIR 116A sterile line with fertile lines differing in the type of cytoplasm (fertile or sterile) and the presence of molecular markers, most of which were linked to the Rf1 locus. For marker validation, two different approaches were used: either by analyzing associations between the ability of a line to restore pollen fertility and the presence of molecular markers in its genotype, or by estimating recombination frequency between the Rf1 locus and marker loci in four segregating hybrid populations. Results. According to the obtained results, no markers demonstrated 100% efficiency in the analysis of the sample of genotypes. The ORS511 marker was most frequently observed among the lines presumably carrying the dominant allele Rf1. Pollen fertility of F1 hybrids from interline crossings was 89-99%. The segregation for fertility/sterility in F2 fitted the theoretical ratio of 3:1 expected in case of the monogenic control of the trait. The markers HRG01, HRG02 and ORS511 were linked to the fertility restoration trait, with recombination rates between Rf1 locus and markers varying in different cross combinations. The analysis of VIR 116А × VIR 740 and VIR 116А × RIL 130 hybrids showed that among the marker loci studied, the ORS511 was closest to the Rf1 locus Rf1 (recombination frequency of 2.2 and 3.3%, respectively). The recombination rate between the Rf1 and ORS511 loci equaled 7.5% in the cross VIR 116А × VIR 210 and 8.9% in VIR 116 × VIR 195. Conclusion. The markers ORS511, HRG01 and HRG02 are the most efficient for the identification of alleles of the Rf1 gene and for the marker assisted selection in hybrid populations produced involving sunflower lines from the VIR collection.

Variation of morphological indicators of three-line hybrids sunflower and their parental forms

The article presents the results of three-year (2016–2018) studies of five three-line hybrids of the IMC selection - Agent, Agronomichny, Marshal, Kamenyar, Zaporozhskij 28 and their parent components - simple unreduced hybrids - ZL22A/102B, ZL42A/46B, ZL42A/58B and pollen fertility restorers – ZL512V, ZL678V and ZL7034V. It was found that individual indicators do not change synchronously. The Agent, Marshal and Kamenyar hybrids had the highest plant height in 2016, one each in 2017 – Agronomichny and in 2018 – Zaporozhskij 28. At the same time, three hybrids (Agent, Agronomical, Marshal) had the largest basket diameter in 2018 and two (Zaporozhskij 28, Kamenyar) in 2017. The shortest growing season hybrids Marshal, Zaporozhskij 28 and Kamenyar had in 2016 (90, 105 and 105 days), and two – Agent and Agronomichny in 2018 – 100 and 103 days, respectively. Among the simple unrecovered hybrids, two – ZL42A/46B and ZL42A/58B had high indicators of plant height, basket diameter and duration of the growing season in 2016. In the ZL22A/102B hybrid, the diameter of the basket was also the largest in 2016 (18.5 cm), the average plant height in 2016 – 124.5 cm was slightly lower than in 2018 (125.4 cm), also in these years in it almost coincided with the duration of the growing season – 97 days in 2016 and 96 in 2018 Fertility restorers had the highest indicators of plant height and basket diameter in 2017, also this year they had the shortest growing season, in 2016, on the contrary, they had the lowest height and the diameter of the basket, and the long growing season. The size of the baskets in the Marshal and Kamenyar hybrids correlates with the moisture supply of plants and the hydrothermal coefficient – the correlation coefficients are 0.997, 0.902 and 0.990, 0.973, respectively. The phases of organogenesis of plants of hybrids Kamenyar and Zaporozhskij 28 are greatly influenced by temperature, the correlation coefficient of this indicator with plant height and with the duration of the growing season is 0.996 for Kamenyar and 0.946 for Zaporozhskij 28, and with a hydrothermal coefficient – 0.939 and 0.753. In the Agent hybrid, the temperatures in June have the greatest influence on plant growth - the correlation coefficient is 0.997 and precipitation in May is 0.968, and the temperatures in May - 0.999 and June - 0.998 on the size of the basket. For the height of plants and the duration of the growing season at Agronomichny, moisture availability at the beginning of the growing season is very important, the correlation coefficients are 0.918 and 0.994, and in August during the filling of seeds 0.996 and 0.927, as well as July precipitation is 0.995. Of the simple unrecovered hybrids, the most demanding for heat is ZL22A/102B - the correlation coefficients are 0.941 with plant height and vegetation duration, 0.843 with the basket size. For ZL42A/58B, the most important are the May precipitation, their correlation with the vegetation duration of 1,000 and the basket diameter of 0.987, and the July temperatures - 0.999 and 0.993, respectively. Among the pollen fertility restorers, the line ZL512V turned out to be the most demanding to weather conditions. For plant growth, the temperatures of April (0.906), May (0.995) and June (1.000) are very important, for the duration of the growing season respectively – 0.958, 0.971 and 0.991, and for the size of the basket, precipitation in May (0.956) and July temperatures (0.943). The correlation coefficient with the sum of active temperatures is – 0.829 for plant height – 0.851 for basket diameter – 0.902 for the growing season.

A comprehensive approach to modern aspects in sunflower selection

According to scientists of the NAAS network, the genetic potential of sunflower hybrids of Ukrainian selection is used in production by no more than 30%. Due to the reduction of the rotation period in crop rotation, crop losses from diseases increased from 10-15% to 35%, and in some favorable years for the development of diseases, losses only from rot are 70% or more. Phytopathological studies were performed on a stationary artificial infectious background of the Institute of Oilseeds NAAS during 2017-2020. We studied the biological resistance to major fungal diseases of new promising lines and hybrids of sunflower breeding institutions of NAAS (Institute of Plant Breeding Yur`eva). Synthetic artificial infectious background was formed during 16 years with forced introduction of infection during sowing of selection genotypes of sunflower. To assess the resistance of sunflower varieties to dry rot and embelization, the infectious background began to be created in 2012. Infectious material was developed in the immunity laboratory of the Institute of Oilseeds of NAAS by increasing pure cultures by the method of V.Y. White Assessment of resistance of sunflower to a number of diseases in the field was carried out according to the method of VP Mistletoe. Sowing was carried out with a seeding rate of 55 thousand / ha. According to the results of field and laboratory studies, sunflower lines and hybrids were identified, which were characterized by the duration of the growing season of the phase "seedlings-physiological maturity" in hybrids 95-110 days, in lines 85-105 days. The selected linear samples had a yield higher than the linear standard (ZL42 with a yield of 0.97 t/ha), and the studied sterile lines had a yield from 1.76 (+0.79) to 1.96 (+0.99) t/ha. Pollen fertility restorer lines had a yield at the level of 0.36 (-0.05) - 0.55 (+0.14) t/ha, when forming the yield of the control line ZL260V - 0.41 t / ha. Created hybrids based on these lines (parental components) formed a yield: Firstborn 3.34 t / ha, Tour - 3.80 t / ha, which is higher than the control hybrid Darius from 0.4 to 0.86 t / ha, respectively. The weight of 1000 seeds of sterile analogues varied in the range of 55.2 g - 56.4 g. In the lines of pollen fertility restorers in the range of 29.4-35.0 g. In the presented hybrids, the average weight of 1000 seeds was in the range of 57.0-60,1 g, while the standard Darius formed a mass of 1000 seeds within 49.0 g. The selected samples had a high percentage of fat accumulation in the seeds. This figure ranged from 49.76 (ZL96A) to 51.12% (ZL72A) among sterile analogues, while the standard sample ZL42A had an oil content of 49.26%. New selected sunflower hybrids were marked by fat content in seeds at the level of 51.88 (Prize) and 52.12% (Firstborn), while the hybrid - standard Darius also had a fairly high level of oil content, namely 48.13%. The presented lines and their hybrid combinations for the study period (2017-2020) combined relatively high resistance to powdery mildew, dry rot, embelisation and high economic performance. According to the results of a comprehensive assessment, a number of hybrids were identified. Among them are Tour, Prize and Firstborn. With such indicators, the presented hybrids are able to compete with dignity in the sunflower seed market.

Characteristics of Fertility Transition Response to the Cumulative Effective Low Temperature in a Two-Line Male Sterile Rice Cultivar

Background Photo-thermo-sensitive genic male sterile (PTGMS) rice (Oryza sativa L.) is usually considered two-line male sterile rice because of its dual-purpose in two-line hybrid rice system: under short days and low temperatures, it is fertile and used for self-propagation, but under long days and high temperatures, it is sterile and used for hybrid seed production. Therefore, photoperiod and temperature conditions are extremely important for the fertility transition of two-line male sterile rice. In recent years, there have been frequent occurrences of abnormally low-temperature (ALT) resulting in failure of two-line hybrid rice seed production. The daily average temperature (DAT) during ALT events is sometimes higher than the critical sterility-inducing temperature (CSIT) of two-line male sterile rice, of which the night temperature is lower than the CSIT. DAT has been traditionally used as the single indicator of pollen fertility transition, but it is unknown why the fertility of two-line male sterile rice in seed production restored fertility under ALT conditions. Results For Hang93S (H93S), a newly released PTGMS line, we hypothesized fertility transition is determined mainly by the cumulative effective low temperature (ELT) and only a certain duration of low temperature is required every day during the fertility-sensitive period. This study simulated ALTs where the DAT was higher than the CSIT while some segments of night temperature were lower than the CSIT. The results showed H93S exhibited a fertility transition to varying degrees. Moreover, fertility was restored under simulated ALT conditions and pollen fertility increased with increasing cumulative ELT, indicating that the fertility transition was affected primarily by the cumulative ELT. Results also indicated that pollen fertility increased as the number of treatment days increased. Conclusions The fertility transition is caused mainly by the cumulative ELT. In two-line male sterile rice breeding, the effects of day length, ALT at night, and continuous response days should be considered together. The present study provides new insight into fertility transition so breeders can more effectively utilize the two-line male sterile rice, H93S, in breeding programs.

Jasmonic Acid Plays a Pivotal Role in Pollen Development and Fertility Regulation in Different Types of P(T)GMS Rice Lines

Two-line hybrid rice systems represent a new technical approach to utilizing the advantages of rice hybrids. However, the mechanism underlying the male sterile-line fertility transition in rice remains unclear. Peiai 64S (PA64S) is a photoperiod- and thermo-sensitive genic male sterile (PTGMS) line in which male sterility manifests at an average temperature above 23.5 °C under long-day (LD) conditions. Nongken 58S (NK58S) is a LD-sensitive genic male sterile (PGMS) rice that is sterile under LD conditions (above 13.75 h-day). In contrast, D52S is a short-day (SD)-PGMS line that manifests male sterility under SD conditions (below 13.5 h-day). In this study, we obtained fertile and sterile plants from all three lines and performed transcriptome analyses on the anthers of the plants. Gene ontology (GO) analysis suggested that the differentially expressed genes identified were significantly enriched in common terms involved in the response to jasmonic acid (JA) and in JA biosynthesis. On the basis of the biochemical and molecular validation of dynamic, tissue-specific changes in JA, indole-3-acetic acid (IAA) levels, gibberellin (GA) levels, and JA biosynthetic enzyme activities and expression, we proposed that JA could play a pivotal role in viable pollen production through its initial upregulation, constant fluctuation and leaf-spikelet signaling under certain fertility-inducing conditions. Furthermore, we also sprayed methyl jasmonate (MEJA) and salicylhydroxamic acid (SHAM) on the plants, thereby achieving fertility reversal in the PGMS lines NK58S and D52S, with 12.91–63.53% pollen fertility changes. Through qPCR and enzyme activity analyses, we identified two key enzymes—allene oxide synthase (AOS) and allene oxide cyclase (AOC)—that were produced and upregulated by 20–500-fold in PGMS in response to spraying; the activities of these enzymes reversed pollen fertility by influencing the JA biosynthetic pathway. These results provide a new understanding of hormone interactions and networks in male-sterile rice based on the role of JA that will help us to better understand the potential regulatory mechanisms of fertility development in rice in the future.