Genomic-regions associated with cold stress tolerance in Asia-adapted tropical maize germplasm.

Maize is gaining impetus in non-traditional and non-conventional seasons such as off-season, primarily due to higher demand and economic returns. Maize varieties directed for growing in the winter season of South Asia must have cold resilience as an important trait due to the low prevailing temperatures and frequent cold snaps observed during this season in most parts of the lowland tropics of Asia. The current study involved screening of a panel of advanced tropically adapted maize lines to cold stress during vegetative and flowering stage under field conditions. A suite of significant genomic loci (28) associated with grain yield along and agronomic traits such as flowering (15) and plant height (6) under cold stress environments. The haplotype regression revealed 6 significant haplotype blocks for grain yield under cold stress across the test environments. Haplotype blocks particularly on chromosomes 5 (bin5.07), 6 (bin6.02), and 9 (9.03) co-located to regions/bins that have been identified to contain candidate genes involved in membrane transport system that would provide essential tolerance to the plant. The regions on chromosome 1 (bin1.04), 2 (bin 2.07), 3 (bin 3.05-3.06), 5 (bin5.03), 8 (bin8.05-8.06) also harboured significant SNPs for the other agronomic traits. In addition, the study also looked at the plausibility of identifying tropically adapted maize lines from the working germplasm with cold resilience across growth stages and identified four lines that could be used as breeding starts in the tropical maize breeding pipelines.

Genetic trends in CIMMYT's tropical maize breeding pipelines.

Fostering a culture of continuous improvement through regular monitoring of genetic trends in breeding pipelines is essential to improve efficiency and increase accountability. This is the first global study to estimate genetic trends across the International Maize and Wheat Improvement Center (CIMMYT) tropical maize breeding pipelines in eastern and southern Africa (ESA), South Asia, and Latin America over the past decade. Data from a total of 4152 advanced breeding trials and 34,813 entries, conducted at 1331 locations in 28 countries globally, were used for this study. Genetic trends for grain yield reached up to 138 kg ha-1 yr-1 in ESA, 118 kg ha-1 yr-1 South Asia and 143 kg ha-1 yr-1 in Latin America. Genetic trend was, in part, related to the extent of deployment of new breeding tools in each pipeline, strength of an extensive phenotyping network, and funding stability. Over the past decade, CIMMYT's breeding pipelines have significantly evolved, incorporating new tools/technologies to increase selection accuracy and intensity, while reducing cycle time. The first pipeline, Eastern Africa Product Profile 1a (EA-PP1a), to implement marker-assisted forward-breeding for resistance to key diseases, coupled with rapid-cycle genomic selection for drought, recorded a genetic trend of 2.46% per year highlighting the potential for deploying new tools/technologies to increase genetic gain.

Genomic regions associated with heat stress tolerance in tropical maize (Zea mays L.).

With progressive climate change and the associated increase in mean temperature, heat stress tolerance has emerged as one of the key traits in the product profile of the maize breeding pipeline for lowland tropics. The present study aims to identify the genomic regions associated with heat stress tolerance in tropical maize. An association mapping panel, called the heat tolerant association mapping (HTAM) panel, was constituted by involving a total of 543 tropical maize inbred lines from diverse genetic backgrounds, test-crossed and phenotyped across nine locations in South Asia under natural heat stress. The panel was genotyped using a genotyping-by-sequencing (GBS) platform. Considering the large variations in vapor pressure deficit (VPD) at high temperature (Tmax) across different phenotyping locations, genome-wide association study (GWAS) was conducted separately for each location. The individual location GWAS identified a total of 269 novel significant single nucleotide polymorphisms (SNPs) for grain yield under heat stress at a p value of < 10-5. A total of 175 SNPs were found in 140 unique gene models implicated in various biological pathway responses to different abiotic stresses. Haplotype trend regression (HTR) analysis of the significant SNPs identified 26 haplotype blocks and 96 single SNP variants significant across one to five locations. The genomic regions identified based on GWAS and HTR analysis considering genomic region x environment interactions are useful for breeding efforts aimed at developing heat stress resilient maize cultivars for current and future climatic conditions through marker-assisted introgression into elite genetic backgrounds and/or genome-wide selection.

Genome-wide association mapping in maize: status and prospects.

Genome-wide association study (GWAS) provides a robust and potent tool to retrieve complex phenotypic traits back to their underlying genetics. Maize is an excellent crop for performing GWAS due to diverse genetic variability, rapid decay of linkage disequilibrium, availability of distinct sub-populations and abundant SNP information. The application of GWAS in maize has resulted in successful identification of thousands of genomic regions associated with many abiotic and biotic stresses. Many agronomic and quality traits of maize are severely affected by such stresses and, significantly affecting its growth and productivity. To improve productivity of maize crop in countries like India which contribute only 2% to the world's total production in 2019-2020, it is essential to understand genetic complexity of underlying traits. Various DNA markers and trait associations have been revealed using conventional linkage mapping methods. However, it has achieved limited success in improving polygenic complex traits due to lower resolution of trait mapping. The present review explores the prospects of GWAS in improving yield, quality and stress tolerance in maize besides, strengths and challenges of using GWAS for molecular breeding and genomic selection. The information gathered will facilitate elucidation of genetic mechanisms of complex traits and improve efficiency of marker-assisted selection in maize breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02799-4.

Beat the stress: breeding for climate resilience in maize for the tropical rainfed environments.

KEY MESSAGE: Intensive public sector breeding efforts and public-private partnerships have led to the increase in genetic gains, and deployment of elite climate-resilient maize cultivars for the stress-prone environments in the tropics. Maize (Zea mays L.) plays a critical role in ensuring food and nutritional security, and livelihoods of millions of resource-constrained smallholders. However, maize yields in the tropical rainfed environments are now increasingly vulnerable to various climate-induced stresses, especially drought, heat, waterlogging, salinity, cold, diseases, and insect pests, which often come in combinations to severely impact maize crops. The International Maize and Wheat Improvement Center (CIMMYT), in partnership with several public and private sector institutions, has been intensively engaged over the last four decades in breeding elite tropical maize germplasm with tolerance to key abiotic and biotic stresses, using an extensive managed stress screening network and on-farm testing system. This has led to the successful development and deployment of an array of elite stress-tolerant maize cultivars across sub-Saharan Africa, Asia, and Latin America. Further increasing genetic gains in the tropical maize breeding programs demands judicious integration of doubled haploidy, high-throughput and precise phenotyping, genomics-assisted breeding, breeding data management, and more effective decision support tools. Multi-institutional efforts, especially public-private alliances, are key to ensure that the improved maize varieties effectively reach the climate-vulnerable farming communities in the tropics, including accelerated replacement of old/obsolete varieties.

Genome wide association study and genomic prediction for stover quality traits in tropical maize (Zea mays L.).

Maize is rapidly replacing traditionally cultivated dual purpose crops of South Asia, primarily due to the better economic remuneration. This has created an impetus for improving maize for both grain productivity and stover traits. Molecular techniques can largely assist breeders in determining approaches for effectively integrating stover trait improvement in their existing breeding pipeline. In the current study we identified a suite of potential genomic regions associated to the two major stover quality traits-in-vitro organic matter digestibility (IVOMD) and metabolizable energy (ME) through genome wide association study. However, considering the fact that the loci identified for these complex traits all had smaller effects and accounted only a small portion of phenotypic variation, the effectiveness of following a genomic selection approach for these traits was evaluated. The testing set consists of breeding lines recently developed within the program and the training set consists of a panel of lines from the working germplasm comprising the founder lines of the newly developed breeding lines and also an unrelated diversity set. The prediction accuracy as determined by the Pearson's correlation coefficient between observed and predicted values of these breeding lines were high even at lower marker density (200 random SNPs), when the training and testing set were related. However, the accuracies were dismal, when there was no relationship between the training and the testing set.

Genetic gains with rapid-cycle genomic selection for combined drought and waterlogging tolerance in tropical maize (Zea mays L.).

Rapid cycle genomic selection (RC-GS) helps to shorten the breeding cycle and reduce the costs of phenotyping, thereby increasing genetic gains in terms of both cost and time. We implemented RC-GS on two multi-parent yellow synthetic (MYS) populations constituted by intermating ten elite lines involved in each population, including four each of drought and waterlogging tolerant donors and two commercial lines, with proven commercial value. Cycle 1 (C1 ) was constituted based on phenotypic selection and intermating of the top 5% of 500 S2 families derived from each MYS population, test-crossed and evaluated across moisture regimes. C1 was advanced to the next two cycles (C2 and C3 ) by intermating the top 5% selected individuals with high genomic estimated breeding values (GEBVs) for grain yield under drought and waterlogging stress. To estimate genetic gains, population bulks from each cycle were test-crossed and evaluated across locations under different moisture regimes. Results indicated that the realised genetic gain under drought stress was 0.110 t ha-1 yr-1 and 0.135 t ha-1 yr-1 , respectively, for MYS-1 and MYS-2. The gain was less under waterlogging stress, where MYS-1 showed 0.038 t ha-1 yr-1 and MYS-2 reached 0.113 t ha-1 yr-1 . Genomic selection for drought and waterlogging tolerance resulted in no yield penalty under optimal moisture conditions. The genetic diversity of the two populations did not change significantly after two cycles of GS, suggesting that RC-GS can be an effective breeding strategy to achieve high genetic gains without losing genetic diversity.

Use of Genomic Estimated Breeding Values Results in Rapid Genetic Gains for Drought Tolerance in Maize.

More than 80% of the 19 million ha of maize ( L.) in tropical Asia is rainfed and prone to drought. The breeding methods for improving drought tolerance (DT), including genomic selection (GS), are geared to increase the frequency of favorable alleles. Two biparental populations (CIMMYT-Asia Population 1 [CAP1] and CAP2) were generated by crossing elite Asian-adapted yellow inbreds (CML470 and VL1012767) with an African white drought-tolerant line, CML444. Marker effects of polymorphic single-nucleotide polymorphisms (SNPs) were determined from testcross (TC) performance of F families under drought and optimal conditions. Cycle 1 (C1) was formed by recombining the top 10% of the F families based on TC data. Subsequently, (i) C2[PerSe_PS] was derived by recombining those C1 plants that exhibited superior per se phenotypes (phenotype-only selection), and (ii) C2[TC-GS] was derived by recombining a second set of C1 plants with high genomic estimated breeding values (GEBVs) derived from TC phenotypes of F families (marker-only selection). All the generations and their top crosses to testers were evaluated under drought and optimal conditions. Per se grain yields (GYs) of C2[PerSe_PS] and that of C2[TC-GS] were 23 to 39 and 31 to 53% better, respectively, than that of the corresponding F population. The C2[TC-GS] populations showed superiority of 10 to 20% over C2[PerSe-PS] of respective populations. Top crosses of C2[TC-GS] showed 4 to 43% superiority of GY over that of C2[PerSe_PS] of respective populations. Thus, GEBV-enabled selection of superior phenotypes (without the target stress) resulted in rapid genetic gains for DT.

Genomic Regions Associated with Root Traits under Drought Stress in Tropical Maize (Zea mays L.).

An association mapping panel, named as CIMMYT Asia association mapping (CAAM) panel, involving 396 diverse tropical maize lines were phenotyped for various structural and functional traits of roots under drought and well-watered conditions. The experiment was conducted during Kharif (summer-rainy) season of 2012 and 2013 in root phenotyping facility at CIMMYT-Hyderabad, India. The CAAM panel was genotyped to generate 955, 690 SNPs through GBS v2.7 using Illumina Hi-seq 2000/2500 at Institute for Genomic Diversity, Cornell University, Ithaca, NY, USA. GWAS analysis was carried out using 331,390 SNPs filtered from the entire set of SNPs revealed a total of 50 and 67 SNPs significantly associated for root functional (transpiration efficiency, flowering period water use) and structural traits (rooting depth, root dry weight, root length, root volume, root surface area and root length density), respectively. In addition to this, 37 SNPs were identified for grain yield and shoot biomass under well-watered and drought stress. Though many SNPs were found to have significant association with the traits under study, SNPs that were common for more than one trait were discussed in detail. A total 18 SNPs were found to have common association with more than one trait, out of which 12 SNPs were found within or near the various gene functional regions. In this study we attempted to identify the trait specific maize lines based on the presence of favorable alleles for the SNPs associated with multiple traits. Two SNPs S3_128533512 and S7_151238865 were associated with transpiration efficiency, shoot biomass and grain yield under well-watered condition. Based on favorable allele for these SNPs seven inbred lines were identified. Similarly, four lines were identified for transpiration efficiency and shoot biomass under drought stress based on the presence of favorable allele for the common SNPs S1_211520521, S2_20017716, S3_57210184 and S7_130878458 and three lines were identified for flowering period water-use, transpiration efficiency, root dry weight and root volume based on the presence of favorable allele for the common SNPs S3_162065732 and S3_225760139.

Possible modulation of N-methyl-D,L-aspartic acid induced prolactin release by testicular steroids in the adult male rhesus monkey.

N-methyl-D,L-aspartic acid (NMA), an agonist of the neurotransmitter glutamate has been shown to acutely stimulate the release of prolactin (PRL) in intact rats and monkeys. To further investigate the role of neuroexcitatory amino acids in PRL secretion, the effects of NMA administration were examined on PRL release in long term orchidectomized adult rhesus monkeys, in both the absence and presence of testosterone. Intact and long term castrated adult male monkeys weighing between 8-13 kg, were implanted with a catheter via the saphenous vein for blood withdrawal and drug infusion. Blood samples were collected at 10 min intervals for 50 min before and 70 min after administration of the drug or vehicle. Plasma PRL concentrations were estimated using radioimmunoassay. Whereas a single iv injection of NMA (15 mg/kg BW) induced a prompt discharge of PRL in intact monkeys, an identical dose had surprisingly no effect on PRL secretion in orchidectomized animals. On the other hand, plasma PRL increases in response to a challenge dose of thyrotropin releasing hormone (TRH; 6 micrograms/kg BW, iv) were similar in magnitude in the two groups of monkeys. Testosterone replacement in orchidectomized animals by parenteral administration of testosterone enanthate (200 mg/wk) reinitiated the PRL responsiveness to acute NMA stimulation. These results indicate that N-methyl-D-aspartic acid (NMDA) dependent drive to PRL release in the adult male rhesus monkey may be overtly influenced by the sex steroid milieu.

Pituitary-testicular interrelationships during germinal involution in the vitamin A deficient rat.

Pituitary-testicular relationships in mature male rats were investigated during the period of germinal involution after the induction of vitamin A deficiency (VAD). Vitamin A deficiency caused a decrease in testicular weight, a gradual increase in the incidence of delayed spermiation, increased phagocytosis of spermatids and pyknosis of germ cell nuclei in rats aged 80 to 110 days. Both basal and gonadotrophin releasing hormone (GnRH)-stimulated serum FSH concentrations were increased by 100 days of age. During the same period, the per cent increment in GnRH-stimulated FSH secretion, pituitary FSH concentration and LH secretion remained unchanged. These results suggest that the increased serum FSH may mark specifically an alteration in the germinal epithelium. By 140 days of age, spermatogenic activity in the rats with VAD was limited to the spermatogonial proliferations so that only Sertoli cells, spermatogonia and preleptotene spermatocytes remained. At this time hypersecretion of FSH persisted while the per cent increment of GnRH-stimulated FSH secretion decreased. Concomitantly, basal and GnRH-stimulated LH concentrations were also increased in the presence of normal serum testosterone. These results indicate that a complete cessation of spermatogenesis beyond preleptotene spermatocytes is associated with a change in the secretion of both FSH and LH. The relationship between serum LH and testosterone was normal until at least 110 days of age. By 140 days the ratio between basal LH and basal testosterone, and between total LH and total testosterone, after GnRH administration, increased in the rats with VAD.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary and testicular functions in sexually mature rhesus monkeys under high-dose LRH-agonist treatment.

Treatment with high doses of LRH-agonists leads to a down-regulation of testicular LH/hCG receptors and is accompanied by a suppression of spermatogenesis in some laboratory animals. In order to test whether this may provide an approach to male fertility control, 4 adult rhesus monkeys were treated with very high doses of LRH-agonist, D-Ser(TBu)6-LHR-ethylamide (100 micrograms daily) for 10 weeks during the breeding season. There was a decrease in testicular volume after 3 weeks of treatment. Serum LH and testosterone levels were suppressed. Sperm counts were stimulated during the first 6 weeks of treatment. At the end of treatment sperm counts, although lower, were still in the normal range. The chronic treatment schedule resulted in a decrease in pituitary responsiveness to an acute challenge with 4 micrograms agonist iv, indicating a desensitization of the pituitary. However, the testosterone response remained unchanged. This study using extremely high doses of the agonist indicates that, compared to the rat, male primates are far more resistant to the suppressive effects of LRH-agonists.

Comparison of the antispermatogenic effects of a new D-homo-steroid and testosterone in rabbits.

In comparison to testosterone, 18 beta-hydroxy-18 alpha-methyl-16 alpha-methylene-D-homo-5 alpha-androstane-3-one (D-homo-S) shows more pronounced anti-gonadotrophic than androgenic properties in rats. The present study was initiated in rabbits to investigate the potential of D-homo-S to suppress spermatogenesis. D-homo-S in sesame oil was administered at the doses of 0.1 (DI), 3 (D II) or 10 mg (D III) per rabbit each day for 8 weeks. During treatment serum testosterone, sperm concentration and quality of sperm motility decreased, whereas sex drive, semen volume and seminal plasma concentrations of fructose and zinc were not changed in any of the groups. Testicular weight and intratesticular testosterone concentration decreased significantly in groups D II and D III, while weights of accessory sex glands increased in those groups. Testosterone in the same dose regimen did not suppress sperm count, motility or serum testosterone, however, seminal plasma zinc concentration in group T III and fructose in group TI increased. Testicular weight and intratesticular testosterone concentration decreased in group TIII only. On the other hand, the weight of the accessory sex glands increased in the same group. In conclusion, D-homo-S suppresses spermatogenesis and increases accessory sex gland weights at doses, when testosterone is still ineffective. Thus, in rabbits D-homo-S appears to be a more potent androgen than testosterone but a dissociation between antigonadotrophic and androgenic properties could not be observed.

The effects of ketamine HCl and barbiturate anaesthesia on the metabolic clearance and production rates of testosterone in the male rhesus monkey, Macaca mulatta.

The study compares metabolic clearance rates, production rates and serum levels of testosterone in conscious and ketamine and barbiturate anaesthetized adult male rhesus monkeys. Using the single injection technique for the determination of metabolic clearance rate, a significant increase in metabolic clearance rate from 430 +/- 71 l/day in conscious animals to 908 +/- 46 l/day for ketamine and 1224 +/- 174 1/day for barbiturate anesthetized animals was observed. Serum testosterone was increased from 106 +/- 9 ng/100 ml to 450 +/- 186 ng/100 ml in barbiturate anaesthetized animals, and this marked increase in serum testosterone was accompanied by a 13-fold increase in the production rate of testosterone from 0.44 +/- 0.04 mg/day to 6.0 +/0 3.3 mg/day. Serum testosterone levels and production rates were not significantly different in conscious and ketamine anaesthetized animals. Barbiturate anaesthesia caused continual changes in serum LH and testosterone levels over an observation period of 3.5 h. Serum LH gradually increased from 4.8 +/- 2.1 mIU/ml to 19.0 +/- 4.1 mIU/ml within 1 h, while serum testosterone rose from 214 +/- 63 ng/100 ml to 836 +/- 53 ng/100 ml within 2 h. In the light of these findings it may be concluded that barbiturate anaesthesia is not suitable for such prolonged studies of endocrine function. Effects exerted by ketamine anaesthesia are less pronounced.

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops).

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for 125 I-hCG, (0.9-2.5 × 109 M-1 , and 0.7-1.64 × 109 M-1 respectively, for green and rhesus monkeys) and low binding capacity (0.343-0.682 fmol/mg and 0.198-0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 109 M-1 ) and low binding capacity (1.04 fmol/mg tissue homogenate) for 125 I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.

Stimulation of pituitary and testicular functions with LH-RH agonist or pulsatile LH-RH treatment in the rhesus monkey during the non-breeding season.

Out-of-season male rhesus monkeys were used to compare the effectiveness of pulsatile treatment with LH-RH and administration of LH-RH agonist on testicular function. Treatment with LH-RH agonist (1.0 microgram Hoe 766/day) for 11 weeks resulted in partial stimulation of pituitary and testicular functions. The pituitary LH response to the agonist increased during treatment. Testosterone levels were stimulated to within the normal range and 2 of the 4 treated monkeys produced ejaculates, but sperm counts were below normal. Pulsatile treatment with LH-RH (100 ng every 96 min for 7 days alternating with LH-RH agonist treatment for 7 days) in 2 monkeys induced full testicular activity after 7 weeks. Ejaculations were induced at a time when the rhesus monkey is normally sexually inactive. Seminal characteristics at the end of treatment were similar to values in the normal breeding season. In samples collected from one monkey over a 24-h period before treatment there were no LH spikes and very low testosterone levels. During pulsatile LH-RH treatment distinct LH and testosterone spikes occurred, comparable to those in the breeding season.

Effects of gonadotrophin treatment in vivo on testicular function in immature rhesus monkeys (Macaca mulatta).

Changes in testicular histology and concentrations of testosterone and oestradiol 17 beta in testicular tissue and plasma have been studied following administration of gonadotrophins (oFSH, oLH, hCG and PMSG) to immature male monkeys. Treatment with FSH (1 mg/day) or PMSG (100 IU/day) for five days, induced a marked enlargement of the seminiferous tubules and increase in the Sertoli cell cytoplasm. Injections of LH (1 mg/daily) or hCG (100 IU/daily) administered similarly, failed to produce hypertrophy of the Sertoli cell. In LH, hCG and PMSG stimulated testes morphologically differentiated interstitial cells could be recognized. FSH did not produce any detectable effect on the intertubular tissue. A significant increase in testicular and plasma testosterone levels was observed with LH, hCG and PMSG. FSH was shown to be much less effective in stimulating androgenesis. An increase in testicular oestradiol production over that of controls, was observed in FSH and PMSG treated monkeys but not in animals treated with LH or hCG.

Seminal parameters in rhesus monkeys under physiological conditions and in studies for male fertility control using FSH antibodies or LH-RH-agonists.

To characterize the male rhesus monkey as a nonhuman primate model for human testicular functions, parameters of exocrine and endocrine testicular function were monitored in 16 adult male-rhesus monkeys for 1 and 5 years respectively. Testicular volumes in-season (October-January) were twice as great as in out-of-season animals (March-June). Ejaculations, both spontaneous and electrostimulated, ceased out-of-season. In 37 ejaculates obtained by electrostimulation in-season, sperm counts ranged from 110-1,100 million/ejaculate, 65% of sperm were motile and 60% were normally formed. Testicular histology showed regression of spermatogenesis out-of-season, with the diameter of the tubules being only one third of that in-season. Circannual changes in exocrine testicular function were accompanied by parallel fluctuations in pituitary and endocrine testicular functions, as evidenced by basal hormone levels and the production rate of testosterone, as well as the response to LH-RH throughout the year. As FSH is required for spermatogenesis in rhesus monkeys, we initiated a study on the long-term effects of active immunization against FSH as a possible means of fertility control. After the first 2 years of observation we can conclude that the production of specific antibodies to FSH results in suppression of spermatogenesis (oligospermia and occasional azoospermia) without affecting endocrine function. The lack of adverse side effects may encourage further investigations on this approach to fertility control. LH-RH-agonists exert degenerative effects on testicular function in rats via a down regulation of the pituitary and testis. A 12 week treatment of four adult monkeys in-season with Hoe 766 (Hoechst; 4µg/day for eight weeks, 20µg/day for 4 weeks sc) did not reveal any change in sperm counts or motility, although some pituitary desensitization was evident. It remains to be investigated whether even higher doses may result in a suppression of spermatogenesis.