Understanding genetic diversity in drought-adaptive hybrid parental lines in pearl millet.

Information on genetic diversity and population structure is helpful to strategize enhancing the genetic base of hybrid parental lines in breeding programs. The present study determined the population structure and genetic diversity of 109 pearl millet hybrid parental lines, known for their better adaptation and performance in drought-prone environments, using 16,472 single nucleotide polymorphic (SNP) markers generated from GBS (genotyping-by-sequencing) platforms. The SNPs were distributed uniformly across the pearl millet genome and showed considerable genetic diversity (0.337), expected heterozygosity (0.334), and observed heterozygosity (0.031). Most of the pairs of lines (78.36%) had Identity-by-State (IBS) based genetic distances of more than 0.3, indicating a significant amount of genetic diversity among the parental lines. Bayesian model-based population stratification, neighbor-joining phylogenetic analysis, and principal coordinate analysis (PCoA) differentiated all hybrid parental lines into two clear-cut major groups, one each for seed parents (B-lines) and pollinators (R-lines). Majority of parental lines sharing common parentages were found grouped in the same cluster. Analysis of molecular variance (AMOVA) revealed 7% of the variation among subpopulations, and 93% of the variation was attributable to within sub-populations. Chromosome 3 had the highest number of LD regions. Genomic LD decay distance was 0.69 Mb and varied across the different chromosomes. Genetic diversity based on 11 agro-morphological and grain quality traits also suggested that the majority of the B- and R-lines were grouped into two major clusters with few overlaps. In addition, the combined analysis of phenotypic and genotypic data showed similarities in the population grouping patterns. The present study revealed the uniqueness of most of the inbred lines, which can be a valuable source of new alleles and help breeders to utilize these inbred lines for the development of hybrids in drought-prone environments.

Molecular characterization and SNP identification using genotyping-by-sequencing in high-yielding mutants of proso millet.

Proso millet (Panicummiliaceum L.) is a short-duration C4 crop that is drought tolerant and nutritionally rich and can grow well in marginal lands. Though the crop has many climate-resilient traits like tolerance to drought and heat, its yield is lower than that of common cereals like rice, wheat, and maize. Being an underutilized crop, the molecular resources in the crop are limited. The main aim of the present study was to develop and characterize contrasting mutants for yield and generate functional genomic information for the trait in proso millet. Gamma irradiation-induced mutant population was screened to identify high-yielding mutants, which were evaluated up to M4 generation. One mutant with a dense panicle and high yield (ATL_hy) and one with a lax panicle and low yield (ATL_ly) along with the wild type were sequenced using the genotyping-by-sequencing approach. The variants detected as single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) were annotated against the reference genome of proso millet. Bioinformatic analyses using the National Center for Biotechnology Information (NCBI) and UniProt databases were performed to elucidate genetic information related to the SNP variations. A total of 25,901, 30,335, and 31,488 SNPs, respectively, were detected in the wild type, ATL_hy mutants, and ATL_ly mutants. The total number of functional SNPs identified in high-yielding and low-yielding mutants was 84 and 171, respectively. Two functional SNPs in the high-yielding mutant (ATL_hy) and one in the low-yielding mutant (ATL_ly) corresponded to the gene coding for "E3 ubiquitin-protein ligase UPL7". Pathway mapping of the functional SNPs identified that two SNPs in ATL_ly were involved in the starch biosynthetic pathway coding for the starch synthase enzyme. This information can be further used in identifying genes responsible for various metabolic processes in proso millet and in designing useful genetic markers.

Development of ß-carotene, lysine, and tryptophan-rich maize (Zea mays) inbreds through marker-assisted gene pyramiding.

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased ß-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in ß-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1-BC3F1, and ICF1-ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3'TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45-88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the ß-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-ß1σ4-4-8-8, DBT-IC-ß1σ4-9-21-21, DBT-IC-ß1σ4-10-1-1, DBT-IC-ß2σ5-9-51-51, DBT-IC-ß2σ5-9-52-52 and DBT-IC-ß2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78-99.31% for DBT-IC-ß1σ4 population and 85.71-99.51% for DBT-IC-ß2σ5 population) like the recurrent parents and ß-carotene (14.21-14.35 µg/g for DBT-IC-ß1σ4 and 13.28-13.62 µg/g for DBT-IC-ß2σ5), lysine (0.31-0.33% for DBT-IC-ß1σ4 and 0.31-0.34% for DBT-IC-ß2σ5), and tryptophan (0.079-0.082% for DBT-IC-ß1σ4 and 0.078-0.083% for DBT-IC-ß2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs.

Gamma irradiation to induce beneficial mutants in proso millet (Panicum miliaceum L.): an underutilized food crop.

PURPOSE: Proso millet is a potential crop for food, nutritional security, and sustainable agriculture, particularly in the context of climate change. It is one of the traditional millet crops in cultivation in Tamil Nadu and India. Self-pollinated nature of this crop makes evolutionary process a terminal one and creating variability to broaden the genetic base is important. The objective of the study was to optimize mutagenic dose of gamma mutagen, document types of mutations and identify mutants for high grain yield (GY) and fodder yield (FY), hence to determine the feasibility of gamma mutagenesis in proso millet crop improvement. MATERIALS AND METHODS: A mutation breeding program with 10 doses of gamma irradiation, i.e. 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 Gy were imposed on seeds of variety ATL 1, a popular variety in India. Roll paper method, tray method and field evaluation were carried out to determine the LD50 and GR50 doses. Based on viable mutation frequency in M2 generation, mutagenic effectiveness and efficiency was estimated. Targeted selection for yield and yield contributing traits was carried out in M2, M3 and M4 generation to identify high yielding mutants. RESULTS: The LD50 and GR50 doses of gamma were estimated to be 418 Gy and 542 Gy, respectively. Based on results from probit analysis, mutagenic effectiveness, mutagenic efficiency and mutation spectrum, the optimum treatment dose of gamma was concluded to be 400 Gy. High frequency, i.e. 10.96% of phenotypic mutants was identified in the M2 generation. The broad range of mutants identified in M2 generation had mutations for plant height, plant habit, panicle shape, compactness, and length, days to 50% flowering (DFF), lodging resistance, tillering, leaf phenotype, apiculus color, culm branching, stem and leaf hairiness, sheath pigmentation, seed color and shape and seed coat attachment. Eight high yielding mutant families were isolated in M4 generation. The mean single plant GY and FY of these mutants ranged between 25 to 51 g and 40 to 68 g respectively while in control it was 15 g and 30 g, respectively. CONCLUSIONS: The wide spectrum and high frequency of mutations both for qualitative and quantitative traits suggest mutation induction as a promising method for creating novel variations in proso millet. The high yielding mutants identified can be utilized for varietal development both for grain and fodder purpose after further stability and quality evaluations in the advanced generations.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for ß-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in ß-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285ß+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the ß-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74-90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and ß-carotene content. The agronomic performance of the four lines was similar (78.83-99.44%) to that of the recurrent parent, and ß-carotene content (7.541-8.711 µg/g) was on par with the donor parent. Our study is the first to improve the ß-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.

Stability Analysis and Heterotic Studies in Maize (Zea mays L.) Inbreds to Develop Hybrids With Low Phytic Acid and High-Quality Protein.

Maize is a major staple crop with high value as food and feed in the poultry sector. Considering the overall nutritional value, maize-based diets comprise two major constraints, i.e., higher phytic acid (PA) and lack of tryptophan. To overcome these issues, a set of identified stable donors for low PA (lpa) and higher tryptophan were crossed in a line × tester fashion, and the hybrids obtained were evaluated at three locations with two replications. Among the inbreds for yield, UMI 1201 and UMI 1205 were the stable good combiners, and for PA, UMI 447 and LPA-2-285 were identified as efficient combiners across locations. Subsequently, 72 hybrids developed from these inbreds had a reduced phytate and higher tryptophan compared with checks having alterations in their yield levels. From Additive Main Effects and Multiplicative Interaction (AMMI) and Genotype main effect plus genotype-by-environment interaction (GGE) biplots, DMR-QPM-09-13-1 × UMI 1099 (PA:9.38 mg/g, trp:0.06%, and yield:184.35 g) and UMI 1205 × UMI 467 (PA:7.04 mg/g, trp:0.06%, and yield:166.39 g) were stable for their high yield with medium PA and tryptophan. Also, across environments, UMI 1200 × UMI 467 had a stable average yield of 129.91 g along with the lowest PA of 4.50 mg/g and higher tryptophan of 0.07%. Thus, these hybrids could be selected and evaluated in upcoming biofortification trials to benefit the poultry sector. Furthermore, the parental inbreds utilized were grouped into heterotic pools to serve as a source population for the development of lpa hybrids in future programs.

Enhancing ß-Carotene Concentration in Parental Lines of CO6 Maize Hybrid Through Marker-Assisted Backcross Breeding (MABB).

Vitamin A deficiency (VAD) is a global health problem; many people around the world, especially children and pregnant women, are VAD deficient or insufficient. Maize is known as an important source of provitamin A for humans. Hence, enhancement of provitamin A carotenoids (pVAC) in maize varieties through breeding or biofortification is a good option for alleviating VAD in developing countries, especially India. So far, numerous maize hybrids have been developed in India. Among them, CO6, derived from UMI1200 × UMI1230, is a popular maize hybrid and adapted to different agro-climatic zones of India, especially Tamil Nadu, a southern state of India. However, CO6 is deficient for pVAC carotenoid ß-carotene. Thus, the objectives of this study were to increase the ß-carotene concentration in UMI1200 and UMI1230 and generate the ß-carotene enriched hybrids through marker-assisted backcross breeding (MABB). For this purpose, the maize genotype HP467-15 was used as the donor for transferring the ß-carotene gene, crtRB1, into UMI1200 and UMI1230. In the MABB scheme, we used one gene-specific marker (crtRB1 3'TE) and 214 simples sequence repeat (SSR) markers for foreground and background selection, respectively. As a result, six improved lines with recurrent parent genome recovery (RPGR) ranging from 90.24 to 92.42%, along with good agronomic performance, were generated. The ß-carotene concentration of the improved lines ranged from 7.056 to 9.232 µg/g. Furthermore, five hybrid combinations were generated using improved lines and evaluated in a comparative yield trial (CYT) and multi-location trials (MLT) along with the original hybrid CO6 and commercial hybrids. It was revealed that ACM-M13-002 was a superior hybrid with a 7.3-fold increase in ß-carotene concentration and with a comparable yield to CO6. In summary, the improved maize inbreds can be used as possible donors for the development of ß-carotene-rich cultivars in maize breeding programs and the ß-carotene enriched hybrid developed in this study will hold great promise for food and nutritional security.

Marker aided introgression of opaque 2 (o2) allele improving lysine and tryptophan in maize (Zea mays L.).

Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311-0.331%) and tryptophan (0.040-0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize.

Marker-Assisted Selection to Pyramid the Opaque-2 (O2) and ß-Carotene (crtRB1) Genes in Maize.

Maize is an excellent nutritional source and is consumed as a staple food in different parts of the world, including India. Developing a maize genotype with a combination of higher lysine and tryptophan, along with ß-carotene, can help alleviate the problem of protein-energy malnutrition (PEM) and vitamin A deficiency (VAD). This study is aimed at improving lysine and tryptophan content by transferring opaque-2 (o2) gene from donor HKI163 to ß-carotene-rich inbred lines viz., UMI1200ß+ and UMI1230ß+. For this purpose, F1, BC1F1, BC2F1, BC2F2, and BC2F3 plants were developed using an o2 line HKI163 and two ß-carotene-rich inbred lines, UMI1200ß+ and UMI1230ß+, as the parents. Foreground selection using the associated marker umc1066 for the o2 gene and the marker crtRB1 3'TE for the crtRB1 gene was used to select the target genes. A total of 236 simple sequence repeat (SSR) markers distributed evenly across the maize genome were employed for the background selection. To fix the crtRB1 allele in the BC1F1 stage, individual plants homozygous at the crtRB1 locus and heterozygous at the o2 locus were selected and used for backcrossing to produce BC2F1 plants. Furthermore, the selected heterozygous BC2F1 plants from both crosses were selfed to obtain the BC2F2 plants, which were then selected for the target gene and selfed to generate the BC2F3 lines. From each cross, five improved lines with homozygous marker alleles for the crtRB1 and o2 genes with a recurrent parent genome (RPG) recovery ranging from 86.75 to 91.21% in UMI1200ß+×HKI163 and 80.00 to 90.08% in UMI1230ß+×HKI163 were identified. The improved lines had good agronomic performance and possessed high lysine (0.294-0.332%), tryptophan (0.073-0.081%), and ß-carotene (6.12-7.38 µg/g) content. These improved lines can be used as genetic resources for maize improvement.

Quantitative trait loci for thermal time to flowering and photoperiod responsiveness discovered in summer annual-type Brassica napus L.

Time of flowering is a key adaptive trait in plants and is conditioned by the interaction of genes and environmental cues including length of photoperiod, ambient temperature and vernalisation. Here we investigated the photoperiod responsiveness of summer annual-types of Brassica napus (rapeseed, canola). A population of 131 doubled haploid lines derived from a cross between European and Australian parents was evaluated for days to flowering, thermal time to flowering (measured in degree-days) and the number of leaf nodes at flowering in a compact and efficient glasshouse-based experiment with replicated short and long day treatments. All three traits were under strong genetic control with heritability estimates ranging from 0.85-0.93. There was a very strong photoperiod effect with flowering in the population accelerated by 765 degree-days in the long day versus short day treatments. However, there was a strong genetic correlation of line effects (0.91) between the long and short day treatments and relatively low genotype x treatment interaction indicating that photoperiod had a similar effect across the population. Bivariate analysis of thermal time to flowering in short and long days revealed three main effect quantitative trait loci (QTLs) that accounted for 57.7% of the variation in the population and no significant interaction QTLs. These results provided insight into the contrasting adaptations of Australian and European varieties. Both parents responded to photoperiod and their alleles shifted the population to earlier flowering under long days. In addition, segregation of QTLs in the population caused wide transgressive segregation in thermal time to flowering. Potential candidate flowering time homologues located near QTLs were identified with the aid of the Brassica rapa reference genome sequence. We discuss how these results will help to guide the breeding of summer annual types of B. napus adapted to new and changing environments.

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.).

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.