Valuable Genetic Material Research Articles

Effect of PEG-induced drought stress on germination of ten chickpea (Cicer arietinum L.) genotypes

Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide, with its importance mainly relying on its high protein content. Chickpea productivity is strictly threatened by abiotic stresses, of which drought exerts the most crucial role in terms of growth inhibition and yield losses encountered. Given that germination is a critical stage that is negatively affected by drought, the aim of this study was to estimate the genotypic variability among ten chickpea genotypes and to determine the seed germination and seedling growth ability under drought stress conditions. Seeds were subjected to water stress by polyethylene glycol (PEG-6000) at five stress levels (0, 5, 10, 20, 30 and 50% PEG). Germination percentage, seed water content, seed water absorbance, root and shoot development and seedling vigour index were evaluated. The analyses revealed significant genetic variability in relation to genotypic performance under drought stress. Drought significantly affected germination as well as all other associated traits, with the effects of stress being analogous to the stress level applied. Findings point to the fact that seedling vigour index is a suitable selection criterion for drought tolerance as it allowed the classification of genotypes as tolerant, moderately tolerant, moderately susceptible and susceptible. Taken together, the commercial varieties ‘Thiva’, ‘Keryneia’ and ‘Gavdos’ as well as the landrace ‘Lemnos’ showed an increased drought tolerance at high stress level, indicating their possible exploitation as valuable genetic material for breeding programs or for commercial use.

Identification of rice blast (Pyricularia) resistance genes in the breeding samples of rice

The current paper has considered the challenge of breeding of rice lines adapted to the climatic conditions of the Southern Federal District of Russia by pyramiding a complex of Pi-1, Pi-2, Pi-33, Pi-ta and Pi-b rice blast resistance genes which is efficient in the southern part of Russia. The control of the resistance genes present in the breeding material was carried out using a marker-associated selection (MAS). The purpose of the current study was to identify the Pi-1, Pi-2, Pi-33, Pi-ta, and Pi-b rice blast resistance genes in the rice samples developed in the laboratory of rice breeding and seed production of the FSBSI “Agricultural Research Center “Donskoy” to identify valuable genotypes. The screening of 689 breeding samples of rice resulted in identification of the allelic state of the Pi-1, Pi-2, Pi-33, Pi-ta and Pi-b rice blast resistance genes, and the genotypes with functional alleles of these genes and their combinations. There were identified 19 breeding samples of rice possessing a combination of 4 functional alleles of rice blast resistance genes in various combinations. The identified rice samples are valuable genetic material and are recommended to use as the sources of rice blast resistance in the breeding process.

PSXIII-5 The age dynamics of the spermatogenic cells development in the roosters’ testes

Abstract The cells of the male gonads are considered as a valuable genetic material for the conservation of the gene pool of breeds and lines of agricultural birds, as well as the directed modification of the poultry genome. Mature germ cells – spermatozoa and their predecessors – spermatogonia, spermatocytes and spermatids can be used for these purposes. To obtain these types of cells, it is necessary to know the characteristics of their development (spermatogenesis). The dynamics of the development of certain spermatogenic cell types in the testicular tubules of different-aged roosters has been studied. Histological studies were performed on testes of roosters aged from 1 week to 6 months with an interval of 2 weeks. Samples of testis tissue were fixed in Bouin’s solution. Histological sections were stained with hematoxylin-eosin. Identification of different cell types (Sertoli, spermatogonia, spermatocytes, spermatids, sperm cells) was carried out according to their morphology. At the age of 1–6 weeks in the seminiferous tubule of roosters, the mainly presence of two cell types was noted: Sertoli cells and spermatogonia. From 7 weeks of age, spermatocytes were detected in the seminiferous tubules, in the 4 months - spermatids, in the 5.5 months - sperm cells. The number of Sertoli cells remained almost unchanged with age and was 21 ± 2. The percentage of these cells decreased with age from 71 ± 3 % to 5 ± 1 %. The percentage of spermatogonia also decreased with age from 75 ± 2 % to 7 ± 1 %. The number of spermatids and spermatozoa, on the contrary, increased to puberty (6 months) and reached 54 %. The study was supported by the RFBR within Project no.18-29-07079.

147 Dynamics of drake spermatogenesis

The examination of age dynamics for the development and differentiation of spermatogenic cells is of great importance to the study of spermatogenesis in poultry. Testicular stem cells, represented by spermatogonia, are a valuable genetic material for creating cryobanks of biomaterial. This is especially important when preserving and maintaining the gene pool of valuable breeds of poultry. In the process of differentiation, these cells give rise to a significant population of germ cells, so they can be used as donor cells for transplantation into the testes of male recipients. Thus, understanding species-specific characteristics of spermatogenesis in males is an important step for obtaining the spermatogenic cell population of interest. The aim of this research was to study the dynamics of spermatogenesis in drake. For the study, 10 groups of males were formed depending on age: 2 weeks and 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 7 months. There were eight males in each age group from 2 weeks to 6 months and six males in the 7-month age group. The testes were isolated postmortem, fixed in Bouin's fixative solution, and embedded in paraffin, and histological sections (5µm) were cut. The following indicators were evaluated: diameter of the seminiferous tubules, types of spermatogenic cells in the seminiferous tubules, and the amount of these cells within seminiferous tubules. Statistical analysis was performed using a t-test in SPSS ver. 15.0 (IBM Corp.). The types of spermatogenic cells were identified by morphology, and no fewer than 30 seminiferous tubules were examined from each individual. The diameter of the seminiferous tubules in the drake testes increased with age. At the ages of 2 weeks and 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 7 months, these indicators were 38±2, 55±2, 60±2, 61±3, 62±2, 62±4, 65±2, 76±3, 94±5, and 163±7µm, respectively. This was due to an increase in the number of spermatogenic cells within the seminiferous tubules to 23±1, 27±1, 38±1, 44±2, 47±3, 57±2, 68±2, 140±5, 187±7, and 466±13 at the ages of 2 weeks and 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 7 months, respectively. The presence, number, and ratio of the cell populations varied depending on age. At the ages of 1-12 weeks, the main cell types in the seminiferous tubules were Sertoli cells and spermatogonia. After the age of 4 months, primary spermatocytes began to appear in the seminiferous tubules. Secondary spermatocytes were visualised at 5 months of age, whereas spermatids could be detected at 6 months of age. Mature sperm cells were detected in the seminiferous tubules of drakes at the age of 7 months. Based on the data obtained, the following conclusion can be made: from 1-12 weeks of age, the generative cells of the seminiferous tubules in drakes are represented mainly by spermatogonia (P<0.05). Therefore, this period can be considered optimal for obtaining testicular stem cells and carrying out manipulations with them. This study was supported by the Russian Science Foundation within project no.16-16-04104.

Cryopreservation and transfer of pig embryos

Recent advances in cryopreservation and non-surgical transfer of pig embryos have made embryo transfer in pigs a commercially viable technology especially for the international transfer of valuable genetic material. Early research demonstrated that early stage pig embryos were highly sensitive to temperatures below 15°C and this sensitivity decreased with development to peri-hatching blastocysts which is accompanied by a reduction in lipid content. Removal of the lipid prior to freezing was found to improve the freezability and resulted in the birth of piglets. It was shown that by polarising the lipid material out of the blastomeres and using ultra rapid freezing (vitrification) the early stage zona pellucida intact embryo would also survive freezing and result in the birth of piglets. Other factors that have contributed to successful cryopreservation of the early stage pig embryo include the use of cytoskeletal stabilising agents, specific cryoprotectants, increasing cooling rates using open pulled straws or micro droplets and assisted hatching. The present review discusses the relative importance of each of these factors. The development of non-surgical transfer technology and its importance in the application of embryo transfer in the pig industry is also reviewed.

The development of a novel SNP genotyping assay to differentiate cacao clones

In this study, a double-mismatch allele-specific (DMAS) qPCR SNP genotyping method has been designed, tested and validated specifically for cacao, using 65 well annotated international cacao reference accessions retrieved from the Center for Forestry Research and Technology Transfer (CEFORTT) and the International Cocoa Quarantine Centre (ICQC). In total, 42 DMAS-qPCR SNP genotyping assays have been validated, with a 98.05% overall efficiency in calling the correct genotype. In addition, the test allowed for the identification of 15.38% off-types and two duplicates, highlighting the problem of mislabeling in cacao collections and the need for conclusive genotyping assays. The developed method showed on average a high genetic diversity (He = 0.416) and information index (I = 0.601), making it applicable to assess intra-population variation. Furthermore, only the 13 most informative markers were needed to achieve maximum differentiation. This simple, effective method provides robust and accurate genotypic data which allows for more efficient resource management (e.g. tackling mislabeling, conserving valuable genetic material, parentage analysis, genetic diversity studies), thus contributing to an increased knowledge on the genetic background of cacao worldwide. Notably, the described method can easily be integrated in other laboratories for a wide range of objectives and organisms.

25 Cryopreservation of horse testicular tissue as a model for rhinoceros

Cryopreservation of testicular tissue (TT) allows retention of valuable genetic material that can be used for conservation of endangered species, such as the northern white rhinoceros (NWR; Ceratotherium simum cottoni). Previously, we found that cryopreservation of NWR TT with a slow controlled cooling rate (CR) method induced morphological alterations in the seminiferous tubules (ST). However, the relative influence of CR, type of medium, and condition of TT from the aged NWR male on TT integrity was not clear. Due to the limited availability of rhinoceros TT, we used the horse as a model for optimization of TT cryopreservation. We evaluated the effect of (1) cryoprotectant solution [PBS (PBS +1.5M dimethyl sulfoxide) v. DMEM (DMEM/F12+10.0% fetal bovine serum+0.05M sucrose+1.5M dimethyl sulfoxide)] and (2) CR [CR1 (−2.0°C min−1 from 0°C to −4.0°C, −15°C min−1 to −12°C, and −0.3°C min−1 to −40°C in a programmable freezer) v. CR2 (same as CR1 but cooled to −8°C and held for 5min before cooling to −40°C) v. CR3 (−1.0°C min−1 from 0°C to −80°C in a CoolCell® freezing device; Corning, Corning, NY, USA)] on the structural integrity of ST from a 2-year-old horse (n=20 ST), cell viability, and expression of spermatogonial stem cells (SSC; GFRα1, and GRP125) and pluripotent markers (SSEA-4, SSEA-1, and OCT-4) in spermatogonial cells isolated from TT frozen with the above treatments (n=3). We found a positive interaction between CR and cryoprotectant solution on structural integrity of fixed and stained TT after freezing in PBS and CR2 that resulted in lower detachment of epithelium cells from the basement membrane (score±standard error of the mean; 0.50±0.1) than that of TT frozen in PBS and CR1 and CR3 (1.00±0.1 and 1.80±0.1, respectively; P<0.001) or in DMEM and CR1 (1.25±0.1), CR2 (1.35±0.1), and CR3 (1.40±0.1; P<0.01) and in lower incidence of basement membrane damage (0.75±0.1) than that of TT frozen in PBS and CR1 (1.17±0.07) and CR3 (1.16±0.07) or in DMEM and CR1 (1.10±0.1), CR2 (1.15±0.1), or CR3 (1.45±0.1; P<0.01). A lower rate of pyknosis was observed in TT frozen with PBS (1.15±0.06) than in TT frozen in DMEM (1.43±0.06; P<0.001). Overall, integrity of ST was improved when TT was frozen in PBS at CR2 having similar percentages of ST with intact epithelium (60%) and basement membrane (35%) as that of refrigerated TT (45 and 50%, respectively) but different from that of TT frozen with PBS at CR1 (10 and 15%, respectively; P<0.05). Flow cytometry analysis of spermatogonial cells revealed that the percentages of live cells from TT frozen in PBS (CR1: 61.5±7.4%; CR2: 59.7±4.8%; CR3: 51.5±4.1%) or DMEM (CR1: 66.2±6.0%; CR2: 59.8±6.0%; CR3: 58.9±6.9%), and expression of SSC and pluripotent markers was similar among all freezing treatments. However, the percentages of live cells from frozen-thawed TT were lower than those of cells isolated from refrigerated TT (80.6±2.2%; P<0.001). Overall, our results showed that (1) structural integrity of horse ST was better maintained when TT was frozen in PBS at CR2 and (2) SSC can be isolated from frozen-thawed TT with a similar relative frequency to that of refrigerated TT.

English

The bruchid, Callosobruchus maculatus (F.) is one of the most destructive pests and causes substantial losses to cowpea during storage in tropical and subtropical regions. The development of successful breeding strategy requires knowledge on gene action and trait inheritance in local and improved sources. In this study, the mode of inheritance, the types of gene action and maternal effects of cowpea resistance to bruchid was investigated. Nine parental lines and their 72 F2 segregating populations, created in a full diallel Griffing’s method 1 approach, were evaluated for resistance to bruchid attack in a randomized complete block design (RCBD) with three replications. Data were recorded on number of eggs laid by the bruchid (NE), adult bruchid emergence (NEI), median development period (MDP) and Dobie Susceptibility index (DSI) was computed. Genotype had highly significant effects on NE, NEI and MDP and DSI. General combining ability (GCA) effects of parents, specific combining ability (SCA) effects of crosses, and maternal and reciprocal effects were highly significant for all the traits. The ratios of GCA to SCA for all the traits were greater than 50% suggesting the preponderance of additive over non-additive gene action in the expression of the traits. Narrow sense heritability estimates were 64.12, 77.69 and 80.99% for NE, NEI and MDP, respectively. Parents 2419, TVu-2027 and IT84s-2246 were identified as promising general combiners for resistance to bruchid and the seven best selected crosses based on their SCA and DSI values were, IT84s-2246 × 2419, 2419 × MU9, TVu-2027 × SECOW2W and 2419 × IT90K-76, 2419 × WC69, 2419 × SECOW5T and 2419 × SECOW2W. The selected parents and/or crosses could be valuable genetic materials for breeding cowpea resistance to bruchid in Uganda or similar environments. Key words: Additive gene action, heritability, median development period, reciprocal effects.

Genetic diversity loss and homogenization in urban trees: the case of Tilia × europaea in Belgium and the Netherlands

Urban trees form a vital component of sustainable cities but the use of a restricted range of species and genotypes may pose a risk to global biodiversity. Despite several studies investigating tree species diversity, intraspecific genetic diversity of urban trees remains largely unexplored. Here, we characterized the genetic diversity of Tilia × europaea, one of the most widely planted urban tree species in Northwest Europe. We compared the genotypic diversity of historical plantings of Tilia spp. from the 17th century with the genotypic diversity of currently available planting stock in Belgium and the Netherlands. In total, 129 trees were sampled and genotyped with 14 microsatellite loci and 150 polymorphic Amplified Fragment Length Polymorphism markers. In Northwest Europe, homogenization of urban T. × europaea plantings already started at the 17th century. Genetic diversity within contemporary commercial planting stocks was extremely narrow and consisted mainly of two clones, sold under the name ‘Pallida’ and ‘Zwarte linde’. The genetic diversity found within the historical plantings was about four times higher than in the current commercial planting stocks. We recommend that tree nurseries should enlarge the genetic diversity of T. × europaea commercial planting stocks. The old clones have shown long-term disease resistance and could provide tree breeders with the valuable new genetic material. The range of available Tilia species and genotypes needs to be explored in future urban tree planning to optimize desired ecosystem services.

PLANTS GENETIC RESOURCES IN THE REPUBLIC OF BELARUS – THE FUNDAMENTAL PRINCIPLE OF FOOD, ENVIRONMENTAL AND BIOLOGICAL SECURITY OF THE COUNTRY

Breeding of new domestic varieties of economically useful plants for different purposes (food, fodder, technical, medicinal, decorative, etc.) is the basis for food, environmental and social security of the republic population. Therefore, preservation, study, mobilization and rational use of genetic resources in the context of global climate change and steady increase of anthropogenic pressure on nature is a planetary problem. The welfare and life of the present and future generations of mankind depends on successful solution of this problem. The paper presents history in chronicles of genetic resources preservation in the Republic of Belarus – from conception of this trend to the present day. Particular attention is paid to the presentation of the results obtained by the implementing organizations of the State Program “Plant Gene Pool” over a 17-year period (2000-2017), including 11 academic institutions and 2 higher educational institutions of the republic. The contribution of each organization to the national genetic bank formation is reflected. Basic, active, working, field, double, target, indicative, core and training collections of the main resource crops, economically significant crops and forest-forming species were formed by their efforts. The largest and most valuable genetic collections of 7 implementing organizations are included in the State Register of Scientific Objects, which constitute the national treasure of the Republic. These collections are a source of valuable genetic material and, at the same time, they are successfully used in scientific, environmental, educational and other programs. Currently, genetic collections in general include over 78,300.00 specimens belonging to 1,680.00 species of crop plants and their wild relatives. 1,016.00 varieties of crop plants were created based on them. The paper also reflects the main approaches and perspectives of preservation and rational use of the plant gene pool. Sophisticated and time-consuming processes of genetic collections formation in modern hard economic and regional natural conditions, taking into account the results of similar studies of other states, will serve as a scientific and information basis for increasing efficiency and improving work in this direction. In practical terms, the high level of species, variety and form biological diversity of genetic collections serves as a guarantee for their efficient use in crop production and selection as sources and donors of valuable genetic material.

Identification of stem rust resistance genes in wheat cultivars in China using molecular markers.

Wheat stem rust caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn. (Pgt), is a major disease that has been effectively controlled using resistance genes. The appearance and spread of Pgt races such as Ug99, TKTTF, and TTTTF, which are virulent to most stem rust-resistant genes currently deployed in wheat breeding programs, renewed the interest in breeding cultivars resistant to wheat stem rust. It is therefore important to investigate the levels of resistance or vulnerability of wheat cultivars to Pgt races. Resistance to Pgt races 21C3CTHQM, 34MKGQM, and 34C3RTGQM was evaluated in 136 Chinese wheat cultivars at the seedling stage. A total of 124 cultivars (91.2%) were resistant to the three races. Resistance genes Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were analyzed using molecular markers closely linked to them, and 63 of the 136 wheat cultivars carried at least one of these genes: 21, 25, and 28 wheat cultivars likely carried Sr2, Sr31, and Sr38, respectively. Cultivars “Kehan 3” and “Jimai 22” likely carried Sr25. None of the cultivars carried Sr24 or Sr26. These cultivars with known stem rust resistance genes provide valuable genetic material for breeding resistant wheat cultivars.

Biomass productivity of snow algae and model production algae under low temperature and low light conditions

This study was designed to determine biomass productivities of specific algal species under low temperature and low light conditions. The algal species examined in this study included two psychrophilic algal species (Chlamydomonas yellowstonensis and Chlamydomonas augustae). These species are commonly known as “snow algae” due to their ability to grow in low temperature water bodies including ice and snow deposits. Additionally, two model production algal species used in high biomass productivity pilot studies (Scenedesmus bijuga and Chlorella sorokiniana) were evaluated. Currently, temperature dependent growth data within known optimal limits exists for these model production species but there is no detailed information about their biomass productivity under low temperatures. In addition, little information can be found about the potential for productivity of these species under limited light exposure. This study examined biomass productivity of these four species at four relatively low temperatures (5, 10, 15, and 20 °C) with three relatively low light exposures (50, 100, and 300 μmol/m2 s). It was hypothesized that the two psychrophilic algae species would produce more biomass per day than model production algal species under these limiting conditions. This study found that both snow algae species performed better than model production species at the lowest temperature (5 °C) and two lower light intensities (50 and100 μmol/m2/s). C. augustae growth rate was shown to have a positive correlation with temperature and a negative correlation with light intensity for the values observed in this study. This finding has significant implications for the use of C. augustae as a cool-season algal crop and a source of valuable genetic material for future engineering of algae. This could lead to the development of cool-season algal crops for sustainable, year-round, industrial production of algae in temperate climates. Furthermore, both of the snow algae species studied here showed inhibited growth at the highest light intensity studied here.

Метод вирощування апоміктичного насіння селекційно-цінних генотипів огірка посівного (Cucumis Sativus L.)

Метод вирощування апоміктичного насіння селекційно-цінних генотипів огірка посівного (Cucumis Sativus L.)

Cryopreservation effects on sperm function and fertility in two threatened crane species

The capacity to cryopreserve semen from captive cranes facilitates production of offspring from behaviorally incompatible or geographically separated pairs, and allows for long-term preservation of valuable genetic materials. The present study sought to develop effective cryopreservation protocols for whooping (Grus americana) and white-naped (Grus vipio) cranes, through examining the influences of two permeating (DMA and Me2SO) and one non-permeating (sucrose) cryoprotectants, as well as vitamin E on post-thaw sperm survival. In Study 1, ejaculates (whooping: n = 10, white-naped: n = 8) were collected and cryopreserved in one of six cryo-diluents (crane extender with: DMA; DMA+0.1M sucrose; Me2SO; Me2SO+0.1M sucrose; 0.1M sucrose; 0.2M sucrose) using a two-step cooling method. Frozen samples were thawed and assessed for overall motility, motion characteristics, membrane integrity, morphology, and ability to bind to the inner perivitelline membrane (IPVM). In Study 2, whooping crane ejaculates (n = 17) were frozen in crane extender containing Me2SO alone or with vitamin E (5 μg/mL or 10 μg/mL). Frozen samples were thawed and assessed as in Study 1, except the binding assay. White-naped crane sperm were more tolerant to cryopreservation than whooping crane (15% vs 6% post-thawed motility). In both species, sperm cryopreserved in medium containing Me2SO alone displayed higher post thaw survival and ability to bind to IPVM than the other cryodiluent treatments. Vitamin E supplementation exerted no benefits to post thaw motility or membrane integrity. The findings demonstrated that there was species specificity in the susceptibility to cryopreservation. Nevertheless, Me2SO was a preferred cryoprotectant for sperm from both whooping and white-naped cranes.

Strong Heterogeneity in Advances in Cryopreservation Techniques in the Mammalian Orders.

Between 1970 and 2012, vertebrate abundance has declined by 58% with an average annual decline of 2%, calling for serious action to prevent a mass extinction and an irreversible loss of biodiversity. Cryobanks and cryopreservation have the potential to assist and improve ex situ and in situ conservation strategies by storing valuable genetic material. A great deal of studies concerning cryopreservation have been performed within the class Mammalia, although no systematic overview has previously been presented. The objective of this study is therefore to evaluate the status, pattern and future of cryopreservation within Mammalia. A strong disproportional distribution of studies in examined orders is displayed. For the majority of examined orders less than 10% of species has been examined. However, the cryopreservation of germplasm has in several cases been successful and resulted in successful applications of assisted reproductive techniques (ARTs). Various obstacles are associated with the development of cryopreservation protocols, and among them the most prominent is interspecific differences in cryotolerance. Extrapolation of protocols in closely related species is considered the most applicable procedure, and a future supplement to overcome this problem is the examination and comparison of cryobiological traits. Successful protocols have been developed for the vast majority of domesticated mammals, which gives incentive for the further extrapolation of protocols in threatened species.

The quality of leguminous vegetables as influenced by preharvest factors

Abstract The cultivation of most legumes, aims to the production of either dry seeds consumed by humans, also known as pulses, or animal fodder. However, some legumes are cultivated for fresh consumption either as pods or as immature seeds. The economically most important legumes consumed as vegetables are green pods of common bean, cowpea, faba bean, snow pea (mangetout) and green pea seeds. As a rule, the legume vegetables are consumed after cooking and in many countries, they may be used to cover primary nutritional needs, because their protein content is high in comparison with most other vegetables. Furthermore, the legume vegetables, which have distinct organoleptic properties when compared to pulses, are also considered important sources of carbohydrates, essential minerals, vitamins, several other antioxidants and health promoting compounds, and dietary fiber. On the other hand, legume vegetables constitute a low-fat foodstuff. Legume vegetables may contain some antinutritional factors, particularly lectins, phytic acid, saponins, oligosaccharides belonging to the raffinose-family, and vicin and convicin in faba bean, but the levels of most of them are lower than in dry pulses and generally do not constitute a constraint to their consumption. Breeding is one of the most efficient tools to reduce the concentrations of antinutritional factors and increase the levels of health promotic compounds and the taste in legume vegetables. Among the legume species frequently consumed as vegetables, a relatively large number of local accessions and landraces are available, which constitute a valuable genetic material for breeding programs aiming at improving their quality. Breeding of more resilient cultivars to combined stress conditions characterized by a higher nutritional value entails also a better understanding of the physiological mechanisms underlying the biosynthesis of health promoting and antinutritional compounds, as well as the plant adaptation to adverse environmental conditions. Last but not least, agronomical practices, such as crop establishment and plant density, fertilization, irrigation, weed control, and harvesting time and practices play a crucial role for the quality of legume vegetables and deserve special attention.

БИОХИМИЧЕСКИЙ СКРИНИНГ ОТЕЧЕСТВЕННОЙ И МИРОВОЙ КОЛЛЕКЦИИ ПРОСА НА СОДЕРЖАНИЕ АМИЛОЗЫ В ЗЕРНЕ

Amylose is the most important biochemical indicator of grain quality in cereals. In this research are presented the results of biochemical screening for the quantitative amylose content in grain of 112 proso millet samples domestic and world collections (Afghanistan, Belgium, Hungary, China, Canada, India, Iran, Mexico, Pakistan, Russia, USA, Turkey, Ukraine, France). On the basis of the obtained data was made conditional classification of proso millet collection on amylose content. The results of biochemical screening of proso millet genotypes showed that the studied samples have different by the amylose content and their number varied from 5.5 to 34.90%. In domestic samples the amylose content ranged from 14.6% to 34.8%. A low amylose content were shown samples K-3742, PI 436626 (Lung Shu 18), PI 436625 (Lung Shu 16) and Ma zha Yan, averaging from 5.5 to 5.9%. Selected samples represent valuable genetic material for the creation of glutinous proso millet varieties and will be involved in the breeding process. On the basis of the conditional classification, it was found that the amylose content was higher than 25% (high amylose) in 68% of the studied samples , 23% were medium-amylose (15-25%) groups, and only 5% and 4% were low-amylose (6-14%) and glutinous (to 6%) groups, respectively. Key words: millet, collection, amylose, screening, classification.

5 Live Offspring Produced from Reproductive Material Recovered During the Annual Cull of Bison from Yellowstone National Park

Bison from Yellowstone National Park (YNP) have a unique genetic history that makes them a conservation priority for the species. Unfortunately, there is a high prevalence of the zoonotic disease brucellosis in the herd. Part of the management strategy for controlling the disease and herd size in YNP is an annual culling process. This culling may result in loss of valuable genetic material and, until now, has been a missed opportunity to recover and preserve those genetics. The goal of this project was to demonstrate the feasibility of producing healthy offspring from reproductive material collected during the nonbreeding season from bison postmortem and to determine effects of donor parameters, including sexual maturity and pregnancy status on IVF outcomes. Oocytes were collected from ovaries harvested from bison within 2 h of slaughter during winters of 2014 to 2017 for use in in vitro embryo production (IVP). Frozen-thawed semen from 7 YNP bulls collected via electroejaculation in vivo or from epididymal flushes postmortem was used for IVF. Embryos were produced using standard cattle IVP procedures. On Days 7, 7.5, and 8 of in vitro culture, embryos were assessed for developmental stage and quality, and Grade 1 and 2 embryos were vitrified on Cryotops® (Kitazato, Tokyo, Japan) in a two-step equilibration process. Embryos were then stored until the breeding season when they were warmed, cultured for 6 h, evaluated for survival, and transferred to healthy bison recipients. Data were analysed using a Student’s t-test. Age did not affect the mean number of oocytes collected from a mature female (n = 254) v. juvenile (n = 27; 24 ± 16 v. 24 ± 13), embryo cleavage rates (54 ± 24% v. 45 ± 24%), or the rate of blastocyst development (8 ± 12% v. 7 ± 8%, respectively). Pregnancy status did have a significant effect on the number of oocytes collected per female (pregnant 22 ± 15%, n = 258; nonpregnant 26 ± 20%, n = 82; P = 0.04) and the percent of oocytes that cleaved (pregnant 56 ± 23%, n = 189; nonpregnant 47 ± 24, n = 61; P = 0.005). No difference was detected in the percent of embryos that reached the blastocyst stage of development (pregnant 8 ± 10%, nonpregnant 8 ± 15%). In August 2016, 12 embryos were transferred singly or in pairs to 10 recipient bison synchronized using a standard synch protocol. Ten days following removal of the CIDR, embryos were transferred to recipient bison with a palpable corpus luteum. In spring of 2017, a healthy female calf was born to a recipient who had received 2 Grade-2 blastocysts as graded at the end of the 6-h incubation period. The genetic dam of the calf was a pregnant adult bison cow. This calf demonstrates that live offspring can be generated from oocytes collected from bison postmortem in the nonbreeding season. Although cleavage rates were higher with oocytes from pregnant bison, viable embryos were generated from nonpregnant adults and juvenile females, warranting continued collection of their oocytes and banking of resulting embryos. To our knowledge, this is the first bison calf produced by in vitro production using postmortem reproductive material.

201 Spermatogonia Transplantation in the Chicken

Spermatogonia are the precursors of male germ cells. They are a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the spermatogonia from male donor’s testes, transformation, and transplantation of donor cells into the sterilized recipient’s testes. The transplanted spermatogonia subsequently differentiate into male sex cells (sperm). The aim of this study was to optimize the individual stages of donor spermatogonia transplantation into the recipient’s testes to increase the effectiveness of spermatogenesis recovery. In the first stage, the spermatogenesis in male chicken was examined to determine the optimal age for isolation of spermatogonia from testes. Histological examinations of male chicken testes (n = 80 birds) were done for 8 age categories, from 1 week to 3 months. It was found that under the age of 4 weeks, the cell population in the seminiferous tubules of male chickens was represented mainly by Sertoli cells and spermatogonia. Maximum percentage of spermatogonia was 69 ± 3% at 4 weeks. At the next stage, a culture of spermatogonia was obtained. Testes of 3-week-old male chickens were used. Separation of the spermatogonia from other types of cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population was established by transfer (3 times) of the supernatant containing unattached cells after 24 h of cultivation into a new culture dish for further cultivation. The cell population is represented mainly by the spermatogonia (89 ± 3%). The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the spermatogonia. The efficiency of spermatogonia infection with lentiviral particles (TU/mL = 2.5 × 108) was 65 ± 2%. After transformation, spermatogonia were introduced into the testes of busulfan-sterilized recipients. The optimal concentration of busulfan treatment after series of experiments from 40 to 100 mg/kg was determined. The effective dose for the removal of own spermatogenic cells was revealed at a concentration of 80 mg/kg of live weight. With complete elimination of other types of spermatogenic cells, the number of Sertoli cells and spermatogonia in the testicle tubules decreased by 39 ± 2% and 98 ± 1%, respectively, compared with the control group. The efficiency of spermatogenesis recovery was assessed based on sperm analysis that was obtained from male recipients (n = 5 birds) 4 months after the introduction of donor cells using PCR. The presence of recombinant DNA (ZsGreen) in recipients’ sperm was shown. Thus, our results indicate the prospect of using spermatogonia as a genetic material for the production of transgenic poultry. Study was supported by the Russian Science Foundation (Project no.16-16-10059).

143 The Dynamics of Spermatogenesis in Guinea Fowls

Male gonads are valuable genetic material for creation of biomaterial cryobanks to preserve the genes of various animals, including poultry. Spermatogonia, which are stem cells of the testes, are of greatest interest. For effective selection of spermatogenic cells, including spermatogonia, it is necessary to know the specific features of spermatogenesis of the species of interest. In this regard, the aim of this study was to investigate the dynamics of spermatogenesis in guinea fowl. Histological examinations of guinea fowl testes (n = 90 birds) were done for 9 age categories, from 2 wk to 6 months. For each individual, at least 30 seminiferous tubules were examined. Seminiferous tubule diameters and numbers and types of spermatogenic cells (based on morphology) were determined. Overall, the histologic structure of guinea fowl testes was similar to that of mammals. Cell populations of the seminiferous tubules included Sertoli cells and generative cells, including spermatogonia, spermatocytes, spermatids, and sperm, at various stages of differentiation. Diameter of seminiferous tubules was (mean ± SEM) 36 ± 1, 58 ± 1, 64 ± 1, 65 ± 1, 110 ± 3, 178 ± 4, 233 ± 4, 274 ± 6, and 295 ± 5 µm at 2 wk, 1, 1.5, 2, 2.5, 3, 4, 5, and 6 months, respectively. Furthermore, at those ages, the number of spermatogenic cells per tubule was 18 ± 1, 20 ± 1, 29 ± 2, 30 ± 2, 68 ± 5, 114 ± 8, 186 ± 10, 400 ± 20, and 447 ± 24. Maximum percentage of spermatogonia was 72 ± 2% at 6 wk. Primary and secondary spermatocytes were first observed at 10 and 12 wk of age, respectively, whereas spermatids were first apparent at 4 months. Sperm were first identified at 5 months, with more present at 6 months. We concluded that the optimal age for retrieving testicular germ cells in guinea fowl was no later than 8 wk, as that represented the age when seminiferous tubules were dominated by spermatogonia. The study was supported by the Russian Science Foundation (Project no.16-16-04104).