GENETIC POLYMORPHISM ANALYSIS OF GENES INVOLVED IN LYSINE AND TRYPTOPHAN METABOLIC PATHWAYS AND HETEROTIC GROUPS OF QUALITY PROTEIN MAIZE INBRED LINES
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Date
2022
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Dr.RPCAU, Pusa
Abstract
A study was performed in order to establish a relationship between the genetic diversity of quality protein maize inbred lines and performance of hybrids thus obtained from these inbred lines, field experiment was conducted. The experimental hybrids were obtained from the 15-quality protein inbred lines when crossed with 3 tester inbred lines, which was observed in randomized block design having three replications. Parental lines and experimental hybrids were randomized separately in continuous plots at TCA, Dholi. Statistical analysis was performed based on 11 morphological characters was studied, including 50% tasseling in days, 50% silking in days, 75% brown husk in days, height of plant, cob girth, length of cob, ear height, kernels in a row, kernels in a cob, grain yield per plant and test weight during the Kharif and Rabi seasons. The tests were carried out in the Molecular Biology Laboratory of Dr. RPCAU, Pusa, Bihar, to assess the type and extent of divergence between eighteen QPM inbred lines based on the analysis of 20 SSR and 26 lysine and tryptophan metabolic pathways candidate gene-based marker targeted microsatellite sites-based polymorphism.
The ANOVA was done independently for both the season and across two environments. 18 QPM inbred parental lines and 45 single crosses, were subjected to statistical analysis for determination of significant differences among 11 quantitative features used in the experiment. ANOVA values revealed significant variations and degree of reliability in the performance of inbred lines and crosses in Kharif and Rabi seasons, as well as utilizing pooled data. The 63 entries in this study were analyzed for 11 quantitative characters, and ANOVA thus obtained revealed significant variations among them. To study the relevance of variation in the sources, the mean sum of squares for parents, hybrids, and parents vs. hybrids. For all of the characters studied, including 50% tasseling in days, 50% silking in days, 75% brown husk in days, height of plant, cob girth, length of cob, ear height, kernels in a row, kernels in a cob, grain yield per plant and test weight during the Kharif and Rabi seasons, categorization of variance showed substantial differences among the inbred parental lines. As deduced from the results obtained the mean sum of squares due to parents, hybrids, and parent vs. hybrids was significant for all traits during both Kharif and Rabi seasons, as well as across seasons.
For selecting economically important morphological traits, it involves taking into account the level of heterosis expression in grain yield, as well as hybrid performance per se, when evaluating the potential of hybrids. A careful examination of the related information for hybrid means performance and degree of heterosis expressed for grain yield per plant reveals that these 17 hybrids, namely: QPML-07×QPML-16, QPML-03×QPML-16, QPML-02×QPML-14, QPML-04×QPML-14, QPML-11×QPML-14, QPML-13×QPML-14, QPML-11×QPML-15, QPML-09×QPML-15, QPML-02×QPML-15, QPML-04×QPML-14, QPML-03×QPML-15, QPML-18×QPML-15, QPML-08×QPML-16, QPML-06×QPML-14, QPML-13×QPML-15, QPML-17×QPML-14, QPML-18×QPML-14, was observed to have higher mean performance as well as significantly positive heterosis for grain yield and appeared to be the most promising hybrids under consideration.
All the 18 parental QPM inbred lines were further classified into different clusters based on the dissimilarity units as observed in the dendrogram. The 18 QPM inbred lines were arranged into 3 clusters, out of these 3 clusters, 2 clusters, i.e. Cluster A and Cluster B were containing multiple genotype, however cluster C was containing only 2 genotypes. According to the dendrogram, QPML-13 and QPML-14 were showing maximum similarity when the 11 morphological characters were taken into consideration. However, QPML-11 and QPMLI-16 were showing minimum similarity when the 11 morphological characters were taken into consideration, and thus, was considered as the most divergent. Thus, from the analysis of genetic diversity performed in the current study, it was observed that 18 QPM inbred lines revealed abundant genetic diversity amongst each other.
An analysis of the genetic similarity, hybrid index value, and hybrid mean value of heterotic groups generated using microsatellite markers revealed that QPML-11, QPML-13, QPML-17, and QPML-18 all belonged to heterotic Group-1. In all cases, QPML-09 belonged to heterotic Group-2. In every case, QPML-01 belonged to heterotic Group-2. The overall coincidences were significantly high when inbred lines were classified on the basis of SSR markers in different heterotic groups according to hybrid index value and hybrid mean value. As a result, the values lead to the conclusion that microsatellite markers could be used for effective and efficient method for classification of inbred lines into diverse heterotic groups for minimizing the size of single crosses that is required for production and evaluation. Hence, enhancing the of hybrid maize breeding program.
The genomic DNA of maize was amplified utilizing following 26 candidate gene-based SSR primers. Different number of alleles were obtained for each marker and it ranged from 3 in ZmOPQ-2 to 13 in ZmAKH-1. A sum total of 193 alleles were obtained with 26 SSR marker, with an average value of 7.4 alleles per locus for the 18 inbred lines. Utilizing 26 SSR markers 78 unique alleles and 117 shared alleles were obtained. No unique allele was obtained for ZmHSDH-3. Only 1 unique allele was obtained for ZmOPQ-1, ZmOPQ-2, ZmAS-1, ZmAS-3, ZmOM-2, ZmOM-4, ZmAKH-4 and ZmOM-8. Maximum number of unique alleles were obtained for ZmAKH-1 (10) and ZmCS-1 (7). This showed varying degree of polymorphism amongst the 18 QPM inbred lines utilized under the present study.
Unique allele percentage is used for expressing the value Polymorphism Per cent (PP), thus maximum PP value was obtained for ZmAKH-1 (76.90%) and the minimum value was obtained for ZmHSDH-3 (0%), followed by ZmOPQ-01 and ZmAS-1 (14.20%). The mean value of PP thus obtained for all the 26 markers was 36.00%. Equally greater degree of PP was obtained for the following primers: ZmCS-1 (63.60%), ZmHSDH-5 (62.50%), ZmOM-3 (55.50%), ZmHSDH-1 (54.50%), ZmOPQ-4 (50.0%), ZmASK-1 (50.0%) and ZmHSDH-2 (50.0%). Maximum PIC value was obtained for ZmAKH-1 (0.902) and the minimum value was obtained for ZmOPQ-2 (0.500). The mean value of PIC thus obtained for all the 26 markers was 0.8. Thus, from the data obtained following are the primers pair having higher value of both PP and PIC as well as number of alleles: ZmOPQ-3, ZmOPQ-4, ZmASK-3, ZmCS-1, ZmAS-2, ZmOM-3, ZmAKH-1, ZmAKH-2, ZmHSDH-1, ZmHSDH-4, ZmHSDH-5 and ZmOM-6.
The genomic DNA of maize was amplified utilizing following 20 SSR primers. Different number of alleles were obtained for each marker and it ranged from 3 in Bnlg-1666 and UMC-1809 to 14 in UMC-1413. A sum total of 160 alleles were obtained with 20 SSR marker, with an average value of 8 alleles per locus for the 18 inbred lines. Utilizing 20 SSR markers 72 unique alleles and 88 shared alleles were obtained. No unique allele was obtained for Bnlg-1666 and Phi-002. Only 1 unique allele was obtained for Phi-064, Phi-046, Phi-011 and UMC-1370. Maximum number of unique alleles were obtained for UMC-1413 (11) and UMC-1159 (5). This showed varying degree of polymorphism amongst the 18 QPM inbred lines utilized under the present study.
Unique allele percentage is used for expressing the value Polymorphism Per cent (PP), thus maximum PP value was obtained for UMC-1413 (78.60%) and the minimum value was obtained for Phi-002, Bnlg-1666 and UMC-1809 (0%), followed by Phi-050 (16.60%), Phi-064 (20.0%) and Phi-046 (20.0%). The mean value of PP thus obtained for all the 20 markers was 32.42%. Equally greater degree of PP was obtained for the following primers: UMC-1159 (62.50%), UMC-1465 (50.0%), UMC-1587 (50.0%), UMC-1425 (40.0%), UMC-1117 (40.0%), UMC-1133 (40.0%), Phi-014 (40.0%) and UMC-1638 (40.0%). Maximum PIC value was obtained for UMC-1413 (0.908) and the minimum value was obtained for UMC-1809 (0.602). The mean value of PIC thus obtained for all the 20 markers was 0.77. Thus, from the data obtained following are the primers pair having higher value of both PP and PIC as well as number of alleles: UMC-1465, UMC-1117, UMC-1587, Bnlg-105, UMC-1413, Phi-014, UMC-1638, UMC-1370, Phi-050 and Phi-062, appeared to be more informative when the number of alleles generated by different primer combinations was compared to the level of polymorphism identified in the current study.
Genetic profile of the 18 inbred lines utilizing 26 candidate gene-based primers showed the inbred lines along the two main axes showed the accommodation of all the inbred lines into 3 well distinguished major genotypic groups. Thus, cluster analyses and principal coordinate analysis showed the existence of wide variation at genetic level that was present among the 18 QPM inbred lines. Thus, with the utilization of 26 candidate gene-based primer, significantly high degree of polymorphism was obtained among the 18 QPM inbred lines at genetic level.
Cluster analysis was performed for the evaluation of variability at genetic level amongst 18 QPM inbred lines utilizing 20 SSR primer pairs showed the accommodation of all the inbred lines into 3 well distinguished major genotypic groups. Thus, with the utilization of 20 SSR primer, significantly high degree of polymorphism was obtained among the 18 QPM inbred lines at genetic level. Thus, analysis showed the existence of wide variation at genetic level that was present among the 18 QPM inbred lines. Similarity coefficient was estimated based on the comparison of presence and absence of bands amplified utilizing 46 pairs of primers (genic and generic). All the 18 parental QPM inbred lines were further classified into different clusters based on the similarity units as observed in the dendrogram. Thus, with the utilization of 46 combined (genic and generic) primer pairs, significantly high degree of polymorphism was obtained among the 18 QPM inbred lines at genetic level.
Cluster analysis was performed for the evaluation of variability at genetic level amongst 8 QPM inbred lines and 4 non-QPM inbred utilizing candidate gene-based primer pairs. Similarity coefficient was estimated based on the comparison of presence and absence of bands amplified utilizing 26 pairs of primers. The 8 QPM inbred lines and 4 non-QPM were further classified into 2 different clusters. Likewise, when cluster analysis was done utilizing the 4 designed primers, it was also able to differentiate the 8 QPM inbred lines and 4 non-QPM taken as experimental material. The 8 QPM inbred lines and 4 non-QPM were further classified into 2 different clusters based on the similarity units.
Thus, cluster analysis and principal coordinate analysis showed the existence of wide variation at genetic level that was present among the 8 QPM inbred lines and 4 non QPM inbred lines. Cluster A comprised of the following 8 inbred lines: QPML-01, QPML-02, QPML-03, QPML-04, QPML-05, QPML-06, QPML-07 and QPML-08. Cluster B comprised of the following 4 non-QPM inbred lines: NQPML-01, NQPML-02, NQPML-03 and NQPML-04. The distribution pattern in 2-Dimension for inbred lines genetic profiles was in correspondence with the interrelationship exhibited by hierarchical classification on the basis of similarity coefficients (Fig. 4.). Genetic profile of the 8 QPM inbred lines and 4 non QPM inbred lines utilizing 26 candidate gene-based primers as well as the 4 designed primer pairs showed the inbred lines along the two main axes showed the accommodation of all the inbred lines into 2 well distinguished major genotypic groups.
The genetic structure illustrated by 46 combined SSR primers (26 candidate gene specific and 20 SSR primers loci showed that the genetic structure of 18 QPM inbred lines are composed of three major ancestral components. The results can infer that the computational analysis of the genetic structure of the QPM inbred lines unambiguously reflected that the genotypes subjected high tryptophan and lysine content are having related molecular characterization are the admixture of three ancestral components present in different combinations in different genotypes.