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Physiological and Molecular Analysis of Soybean Seed Longevity and Validation of Candidate Markers

P. Sirisha1, S.N.C.V.L. Pushpavalli1,*, P. Sujatha2, S. Vanisri1, M. Rajendar Reddy3
1Institute of Biotechnology, Professor Jayashankar Telangana State Agricultural University, Rajendranagar, Hyderabad-500 030, Telangana, India.
2Department of Seed Science and Technology, Seed Research and Technology Centre, Professor Jayashankar Telangana State Agricultural University, Rajendranagar, Hyderabad-500 030, Telangana, India.
3Agricultural Research Station, Adilabad, Professor Jayashankar Telangana State Agricultural University, Rajendranagar, Hyderabad-500 030, Telangana, India.

  • Submitted26-07-2023|

  • Accepted09-03-2024|

  • First Online 24-05-2024|

  • doi 10.18805/LR-5213

ABSTRACT

Background: Seed longevity is a major constraint in soybean seed production. The major focus of this study is to analyze the physiological and molecular changes associated with seed longevity and identify promising germplasm which are good storers for soybean breeding program. 

Methods: Nineteen genotypes were studied for seed longevity using accelerated ageing test and genetic integrity based on SSR marker data. Genotypes were clustered into distinct groups based on seed morphological and physiological parameters (Mahalanobis D2 analysis). SSR markers for seed longevity were validated in the germplasm.

Result: Per cent reduction in germination after accelerated ageing was significantly and positively correlated with traits associated with seed storability such as seed length, seed width, seed thickness and 100 seed weight and negatively correlated with seedling vigour indices. Hence, it would be worthwhile to rely upon these parameters for enhancing the seed storability in soybean. Genetic integrity of the germplasm was evaluated based on SSR markers in accelerated ageing seeds. SSR markers (Satt 285, Satt 534, Satt 538, Satt 281, Satt 162, Satt 631 and Satt 371) revealed significant association for the seed longevity characters such as seed length, seed width, seed thickness and seed weight. Candidate markers (Satt 371, Satt 281, Satt162, Satt 285, Satt 534) which can differentiate the soybean genotypes for storability have been identified in this study. The genotypes were grouped into seven clusters with monogenotypic cluster III (PSPB23) having minimum reduction in germination after accelerated ageing. 

INTRODUCTION

Seed ageing is commonly described as the loss of seed quality or viability under long storage conditions (Priestley, 1986, Coolbear, 1995). It is a complex biological activity and involves a network of physiological, biochemical, metabolic and molecular processes (McDonald, 1999; Zaheer et al., 2016). Seed physical and physiological traits like seed coat, seed thickness, germination percentage, vigour and seed leachate conductivity are the common considerations for seed longevity. Seed ageing affects germination (Rehman et al., 1999), emergence (Verma et al., 2003; Basra 2003), shoot and root dry weight (Verma et al., 2003), seedling length (Verma et al., 2003), normal seedling percentage and seed size (Dell’Aquila and Di Turi, 1996). Accelerated ageing is a technique (AA) widely used to evaluate seed storability and is based on increased seed deterioration after exposure to high temperature and relative humidity (Mali et al., 2014). The decrease in germination, increase in abnormal seedlings and dead seeds after AA is the result of progressive loss of seed viability and vigor (Jain et al., 2006; Radha et al., 2016). Soybean [Glycine max (L.) Merrill] is an important legume crop of Fabaceae family, which meets both protein and oil requirements across the globe. Soybean seed deterioration is a major problem in agricultural production systems as seed rapidly deteriorates than most of the other crops (Shelar et al., 2008; Finch-Savage and Bassel, 2016) due to high temperature and high relative humidity during storage. Soybean is classified as a “poor storer” when compared to other grain crops (Jagadish et al., 2013). Loss of germination potential or viability is more severe in tropical and subtropical areas than in temperate habitats. Soybean germplasm identified with good storability traits can be utilized as parental lines in the breeding program.

MATERIALS AND METHODS

Nineteen soybean genotypes with variable seed coat colour were used for this study (Table 1). The experiments were conducted at PJTSAU, Hyderabad. Soybean seed was categorized as black, brown, yellow, yellow green and green based on seed coat colour and on the basis of size as bold, medium and small seeded types. Three replications of 10 seeds each were randomly selected from each genotype to measure seed length, width and thickness by using digital vernier calipers. Weight of 100 seeds was measured for test weight/100 seed weight.

Table 1: Classification of soybean genotypes based on colour of seed coat.



Freshly harvested soybean seeds (42 g) of each genotype were placed in ageing boxes and subjected to accelerated ageing for 72 hr at 42°C and 90% relative humidity (RH) in accelerated ageing chamber. Samples were drawn after 72 hr and immediately kept for germination test (ISTA, 2016). Seed physiological parameters such as germination (%), seedling length (cm), seedling dry weight (mg), seedling vigour index I and II, per cent reduction in germination, percent reduction in field emergence were recorded for both freshly harvested seeds and seeds subjected to AA.
 
 
 
 
  
Seedling vigour index I and II were calculated as suggested by Abdul-Baki and Anderson (1973).
                             
Seedling vigour index I = Germination (%) x Seedling length (cm)  
 
Seedling vigour index II = Germination (%) x Seedling dry weight (mg) 
 
Genomic DNA was isolated from young leaves following the CTAB procedure as described by (Saghai-Maroof et al., 1984). A total of 21 SSR markers associated with seed longevity traits were used for PCR (Table S4 and S4a). Completely randomized design (CRD) was followed for the physiological experiments. Correlation analysis and genetic divergence was carried out using INDOSTAT software. A measure of group distance based on multiple characters was given by Mahalanobis (1936) using D² statistic by which, genetic divergence between genotypes was estimated (Windostat software). Correlations were computed according to Al-Jibouri et al. (1958). Single marker analysis was done with t-test and regression analysis to identify association between SSR markers and seed traits.

RESULTS AND DISCUSSION

Seed ageing can be defined as progressive deterioration of the structures and functions of the seed over time which ultimately leads to death of the organism. The quality of soybean seed during storage has been reviewed and seed deterioration has been identified as one of the basic reasons for low productivity in soybean (Shelar et al., 2008). Accelerated ageing is the most commonly used stress test to assess the storability of crop seeds in a short period of time (Mali et al., 2014). The genotypes were grouped on the basis of seed coat colour (Table 1).
 
Analysis of variance (ANOVA) and variability for seed physical and physiological traits
 
Seed physical traits of soybean genotypes were recorded in fresh seed lot, while seed physiological parameters were recorded in both freshly harvested seeds and seeds subjected to AA. ANOVA for seed physical parameters of fresh seed exhibited significant variation among all the genotypes for seed length, seed width, seed thickness and 100 seed weight. Significant variation was observed for the seed physiological traits viz percent reduction in germination, seedling length, seedling dry weight, seedling vigour index-I, seedling vigour index-II (Table S1, S2 and S3). Genotypic coefficient of variability (GCV) of soybean genotypes for seed, seedling traits and vigour ranged from 7.65% to 41.12%. The seed physical and physiological traits expressed high heritability estimates ranging from 82.3 to 99.5% (Table 2).

Table 2: Estimates of GCV, PCV, heritability in broad sense (h²) and genetic advance over mean (GAM) for different characters in soybean.



Table S1: Mean values of soybean genotypes for seed physical parameters.



Table S2: Mean values of soybean genotypes for physiological parameters before and after accelerated ageing.



Table S3: Analysis of variance for seed physical and physiological parameters.


 
Correlation between seed physical/physiological traits and seed longevity
 
Physical parameters associated with seed storability like seed coat colour, seed size, seed weight can be helpful in making a decision on duration of soybean seed storage (Mosavi et al., 2011; Tubic et al., 2011). Seed physical and physiological traits were recorded before and after AA to study longevity of soybean germplasm and phenotypic correlation coefficients were computed (Table 3). Per cent reduction in germination after AA exhibited significant positive correlation with seed traits associated with storability such as seed length (0.40*), seed width (0.58***), seed thickness (0.51***) and 100 seed weight (0.52***). Significant negative correlation was observed with SV-I (-0.55***) and SV-II after ageing (-0.50**). SV-I displayed significant positive correlation with seedling length (0.81 ***), while SV-II displayed significant positive correlation with seedling dry weight (0.55 ***) after AA (data not shown). Similar positive and significant correlations were reported by Kuchlan et al. (2010b) for seed size and 100 seed weight in soybean. Black-seeded genotypes were better than yellow-seeded genotypes. Traits such as hard seed coated seeds; small seed size and black seed coat were identified to have better storability in soybean (Subash et al., 2017). Negative correlation is observed in the present study for germination (Naik et al., 2019) and seedling vigour indices after AA (Pallavi et al., 2018). 

Table 3: Correlation between seed longevity parameters and per cent reduction in germination.


 
Evaluation of genetic integrity using SSR markers
 
Genetic integrity is also of equal concern as much as the physiological integrity during seed storage. Decline in germination percent after accelerated ageing was observed in all genotypes and was drastic in NRC 130 and Karune (Table S2). Variation in the SSR profiles after ageing was observed and four markers viz., Satt 434, Satt 281, Satt 286, Satt 390 were able to detect polymorphisms among AA seeds of soybean. A drastic decrease in the number of alleles has been observed after AA indicating loss of DNA integrity (Table 4). Only one allele was detected for SSR markers Satt 523, Satt 371 and SOYGPATR after AA, while 4-5 alleles were observed in freshly harvested seeds. Loss of alleles due to failure of PCR amplification may be attributed to loss in genetic integrity during AA. It is interesting to note that most of the genotypes with black/brown seed coat have amplified few alleles after AA, indicating loss of genetic integrity was more prominent in green and yellow green seeded genotypes. Reduction in the number of alleles after AA has also been reported in several crops such as rice (Adeboye et al., 2015), barley (Parzies et al., 2000), rye (Chebotar et al., 2003), bread wheat (Fu et al., 2015), Siberian wild rye (Huang et al., 2019), soybean and safflower (Vijay et al., 2009). Assessment of DNA integrity combined with a germination test efficiently characterizes the vigour of seeds after AA and may be a promising tool for long term conservation in seed banks.

Table 4: SSR markers showing variation in number of alleles before and after accelerated ageing.


 
Validation of SSR markers for seed longevity
 
SSR markers associated with seed longevity such as seed length, seed width, seed thickness and 100 seed weight (single marker analysis) were validated in our study. Seven markers (Satt 285, Satt 534, Satt 538, Satt 281, Satt 162, Satt 631, Satt 371) having significant association with seed storability traits have been identified (Table 5). SSR marker Satt 285 was significantly associated with seed physical parameters such as seed length, seed width, seed thickness and seed weight, while Satt 534 was significantly associated with seed thickness, seed width and 100-seed weight. Despite the small sample size and minimum number of markers used in our study we observed significant association of seven SSR markers with seed longevity. This is corresponds with previous findings (Singh et al., 2008a, b; Mian et al., 1996). We also identified a few candidate SSR markers that discriminated the good storer soybean genotypes from the rest of the genotypes based on allele size. These markers (Satt 371, Satt 162, Satt 463, Satt 281, Satt 285) were associated with seed specific traits such as seed coat colour, seed size and storability. Our results are in conformity with earlier reports where SSR markers Satt 371, Satt 453 and Satt 618 produced specific allelic bands making them candidate markers for linkage with seed storability and testa colour (Jagadish et al., 2013a). SSR marker Satt 281 exhibited distinct banding pattern that could clearly differentiate good and poor seed longevity in soybean genotypes (Naik et al., 2019).  Markers Satt 162, Satt 523 and Satt 453 which are either linked with seed coat colour or seed permeability exhibited a specific size allelic fragments in soybean genotypes and crosses revealing better seed longevity (Pawar et al., 2018). On the other hand, marker Satt 285 showed allelic variation based on seed size.

Table 5: SSR markers associated with seed longevity in soybean.


 
Candidate markers identified for seed storability
 
SSR marker Satt 371 amplified in black seed genotypes exclusively at 268-278 bp. In our study, 10 out of 11 good storer genotypes amplified within this size range. Thus, preliminary studies indicate that Satt 371 may have close linkage with good storability that may be attributed to black seed coat. Few other markers such as Satt 162 amplified at 300 bp in 10 out of 11 good storer genotypes and Satt 463 amplified five out of seven black seed genotypes at 110 bp. Eight out of 11 good storer genotypes were amplified at 190-225 bp using Satt 281 marker. Marker Satt 619 amplified at 125 bp in 8 out of 11 good storers, which were black seeded suggesting that Satt 619 is specific to seed coat colour. Marker Satt 534 amplified at 175 bp in extremely poor storer genotypes, while Satt 656 amplified in 8 out of 11 good storer genotypes at around 150 bp indicating its possible association with storability.
 
Genetic divergence (D2 statistics) of soybean germplasm
 
The genotypes were grouped into seven clusters on the basis of seed physical and physiological attributes with cluster I being the largest comprising of ten genotypes. Five out of six brown seeded genotypes were grouped in cluster I (Table 6). The average inter-cluster distances varied from 378.08 to 56,445.64 while intra cluster distance ranged from 0.00 to 1300.32. The highest inter-cluster distance was recorded between clusters IV and VII indicating greater diversity (Table 7). Cluster means were computed for six seed traits and four seedling traits (before and after AA) (Table 8). Monogenotypic cluster V (NRC 2755) recorded maximum cluster mean for seedling length, seedling vigour-I & II, seedling dry weight both before and after AA indicating better storability of the germplasm. The monogenotypic cluster III (PSPB-23 [green]) recorded lowest per cent reduction in germination and field emergence after accelerated ageing followed by cluster IV. Crosses may be attempted between genotypes of cluster III and IV with cluster V for enhanced seedling vigour and storability traits. Monogenotypic cluster (VII) consisted of bold seeded germplasm DS91-3 (black seeded) that recorded highest value for seed traits studied such as seed length, width, thickness and 100-seed weight. The genotype NRC 2755 with diverse genetic base is the most prominent contributor for seedling vigour- I and II after AA. The robust vigour after AA is attributed to seedling length and dry weight.

Table 6: Distribution pattern of soybean genotypes based on seed longevity traits.



Table 7: Average inter and intra cluster distances (D2).



Table 8: The mean values of 14 parameters in seven clusters of soybean genotypes.



NRC 2755 can be used in crop improvement program for seedling vigour trait. Percent reduction in germination showed maximum contribution (34.5%) towards genetic divergence (Table S5). 

Table S4: List of reported SSR markers associated with seed longevity in soybean.



Table S4a: Primer sequences and annealing temperature of SSR markers associated with seed longevity in soybean.



Table S5: Per cent contribution of characters towards genetic divergence in soybean genotypes.

CONCLUSION

Significant variation in seed physical and physiological traits was observed in fresh and AA seed among soybean genotypes with varying seed coat colour. D2 analysis grouped the genotypes into seven clusters and monogenotypic cluster V (NRC 2755) recorded maximum cluster mean for seedling length, seedling vigour-I and II, seedling dry weight both before and after AA indicating better storability of the germplasm. Significant positive correlation was observed between seed storability traits (seed length, seed width, seed thickness and 100 seed weight) and per cent reduction in germination, while negative correlation with seed vigour-I and II. Loss of genetic integrity after AA (decreased number of alleles) has been identified using SSR markers. Single marker analysis confirmed potential SSR markers associated with seed longevity parameters (seed size and seed weight). Candidate markers for seed coat colour and storability differentiated the soybean genotypes.

CONFLICT OF INTEREST

All authors declared that there is no conflict of interest.

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