Verification of Soybean Seed Coat Colour Specific Markers Reveals I Loci Specific Markers Capable for Distinguishing Genotypes Differing in Seed Coated Colour

Soybean [Glycine max (L.) Merrill] is one of the important oilseed crops also known as “Golden bean”. It has an immense potential as a major source of edible oil (19.5%) and high quality protein (43.2%) food (Halvankar et al., 1992).
       
Being the richest, cheapest and easiest source of quality protein and fats and having a multiplicity of uses as food and industrial products, it is also called “Wonder crop.”
       
In soybean, seed coat colour trait shows a lot of variation. In India pigmented seed coat is not preferred in the market and fetches lower price. However, besides their role in protecting plants from various stresses, anthocyanins have diverse human health promoting capabilities (Lee et al., 2009; Tsoyi et al., 2008). Black soybean is long considered a medicinal food and consumed only occasionally in Japan and Korea Tsoyi et al., (2008). Black soybean plays a vital role in nutritional and livelihood security in foothills of Himalaya (Bhartiya et al., 2017; Joshi and Rahal, 2018). A small market also exists for black soybean, especially for Chinese foods. All modern high yielding cultivars feature yellow seed coats, with range of hila colours present-brown, black, imperfect black, buff and yellow. Cultivars with pale hila are highly prized for natto and tofu production.
       
The visual appearance of the soybean seed itself has been altered as a result of domestication. All the wild type Glycine soja germplasm possesses black soybean; whereas the majority of cultivated Glycine max germplasm possess yellow seed coat. Genetic studies conducted on soybean in the early 1900’s were concerned primarily with colour characteristics (Woodworth, 1921). Black soybean is similar in composition to cultivated yellow soybean, except that it accumulates high levels of anthocyanin and proanthocyanidin pigments in the seed coat. Interestingly, the black seed coat has a red color during mid-stages of seed development due to the presence of only moderate amounts of anthocyanins. By contrast, the seed coat of brown soybean accumulates only proanthocynidin pigments and browns during desiccation (Todd and Vodkin, 1993). Because hilum coloration is controlled by small number of genes, this trait is frequently used by breeders as a readily assayed visible morphological marker for presence of off-type in soybean seed lot.
       
Pigmentation of seed coat is induced via the deposition of large number of flavonoids in respective tissues ofsoybean. Synthesis of these compounds occurs mainly from anthocyanins by phenyl pathway of secondary metabolism. Thus far, alleles of at least 5 genetic loci (I, R, T, W₁ and O) are known to function epistatically to control seed coat pigmentation, six loci (W1, W2, W3, W4, Wm and Wp) control flower pigmentation and two loci (T and Td) control pubescence color in soybean (Palmer et al., 2004; Guo and Qiu, 2013) (7). There are 3 independent loci (I, R and T) that mainly control biosynthesis of pigments and determine the seed coat colours Palmer et al., (2004). I gene controls synthesis and spatial occurrence of pigments in the epidermal layer of seed coat, where as R and T gene are responsible for specific color. The dominant I allele exhibit colourless seed coat phenotype due to dominance inhibition, while the homozygous recessive i allele gives rise to self coloured seed coat. The R and T loci control specific seed coat colours, which include black (i, R, T), imperfect black (I, R, t), brown (i, r, T), buff (i, r, t) by controlling types of anthocyanin and proanthocyanin pigments.

The economic importance of problem is pigmented seed coat colour is not preferred in soybean and fetches lower price. However, it is also linked to some extent with the desired traits like seed longevity and nutritive quality. Morphological characters, including seed coat color, were found to be contributing to the high projections in the two principal components in phenotypic divergence study (DeChavez et al., 2017).
       
A major goal for molecular genetic studies in soybean involves the identification and characterization of genes associated with important agronomic traits. Microsatellite markers are ideal for varietal identification, genetic purity checking and to check specific soybean genotype in processed food (boiled/ steamed/roasted) for presence of mixture Kosaka et al., (2009). Thakare et al., (2017) tagged microsatellite markers that can be of help in the process of soybean breeding for characters such as shattering tolerance. In the present investigation molecular characterization of candidate genes specific markers controlling seed coat color in soybean was undertaken with the objective to identify markers controlling seed coat color in Indian soybean.

Materials
 
The materials for the study comprised of twelve soybean genotypes in which six yellow and six were black seed coat colour genotypes (Table 1; Fig 1). These materials were obtained from Agricultural Research Station, Kasbe Digraj, Dist-Sangli, Maharashtra. Seeds of each genotype were sown in plastic pots inside polyhouse and their seedlings were allowed to grow for a month for DNA isolation purpose.
 

 

 
Isolation of genomic DNA from leaves
 
The genomic DNA was extracted using cetyl trimethyl ammonium bromide (CTAB) protocol as described by Doyle and Doyle (1987). Pellets were air dried and dissolved in 100 µl of T₁₀E₁ (10 mM Tris, 1 mM EDTA) buffer. Concentration of purified DNA was measured using UV visible Spectrophotometer (Nanodrop, ND-1000 USA) at 260 and 280 nm. Two µl of all DNA extracts were electrophoresed (BioRad Sub Cell Model 96 USA) in 0.8% (w/v) agarose gel containing 0.5 µg/ml ethidium bromide at 6 v/cm in 1X TBE buffer. After electrophoresis the band intensity of genomic DNA was visualized on gel documentation unit (Flour Chem. ^TMAlpha Innotech, USA) and compared to that of known amount i.e. 100 ng Lambda phage DNA ladder loaded in the same gel. These gels also provided a visual measure of purity and integrity of DNA as sheared DNA indicate DNA degradation.
 
DNA amplification by SSR markers
 
For PCR amplification gene specific fourteen primers were investigated (Table 2). Amplification reaction mixture was prepared in 0.2 ml thin walled flat capped PCR tubes, containing the 1X Genei Taq DNA polymerase Buffer B, 2 mM  MgCl₂, mix with 250 µM of each dNTP, 10 picomoles of each primer, 1 unit of  Genei Taq DNA polymerase and 40 ng of genomic DNA. The total final volume of 20 µl in each reaction mixture was made up with sterile double distilled water, whichwas gently mixed and setup for DNA amplification in a Thermal Cycler (Eppendorf, Master Cycler Gradient, Germany). Gradient PCR amplification for different primers was carried out to determine the annealing temperature of each primer pair. The PCR conditions comprised of initial denaturation at 94°C for 5 minutes followed by 40 PCR amplification cycles of denaturation (94°C), annealing (as per primer) and extension (72°C) each for a minute, followed by final extension for 10 minutes. The samples were placed in thermal cycler until it reached to 50°C.
 

 
Agarose gel electrophoresis of amplified PCR products
 
2% Agarose gel in 1X TBE was used for electrophoresis of amplified PCR products (10 µl) at 80 volts. A 100 bp Low Range DNA Ruler was used as a molecular size reference for band yielded from PCR. The amplified PCR products were observed under UV transilluminator in gel documentation system (Flour Chem. ^TMAlpha Innotech, USA) and image was captured.

Conventionally the soybean genotypes are differentiated on the basis of plant morphological markers observed in the field. However, morphological characters such as seed colour are stage specific and the characterization is a laborious, time consuming process. Bhartiya et al., (2017) suggested the need to develop better performing black soybean varieties than the existing ones. Present study was conducted using SSR markers reported to be linked to five candidate genes (I, R, T, W₁ and W₂) known to epistatically control seed coat pigmentation in soybean Palmer et al., (2004). Results obtained are presented and discussed in the light of available literature.
       
The genomic DNAs isolated from 12 different soybean genotypes differing in seed coat color, were subjected to PCR amplification using 14 gene specific primers (Table 2). Annealing temperature of each primer was optimized by gradient PCR (Table 2). It was observed that all 14 primers amplified the genomic DNAs of 12 soybean genotypes in the present investigation. Out of 14 amplified primer pair, 10 primer pairs showed polymorphism and 4 primer pairs were monomorphic. Out of total of 50 bands amplified, 46 were polymorphic and 4 were monomorphic. The size of amplification product ranged from 130 to 600 bp with average number of bands found per primer pair being 4 and polymorphic bands per primer pair being 3.
 
Inhibitor locus (I) locus specific amplification
 
The I allele inhibits the production and accumulation of pigments over the entire seed coat, resulting in uniformly yellow-colored seeds, whereas the I allele leads to completely pigmented seeds by allowing the production and accumulation of pigments over the entire seed coat Senda et al., (2012). Classical analysis of I locus was performed in 1920s and 1930’s. Wang et al., (1994) reported that the reduction of CHS activity was found to be the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coat. Inhibitor locus (I) specific 4 primers (SM-303, SM-305, TM, TR) were used for present investigation Yang et al., (2010). These markers were scored in 12 diverse soybean genotypes with 6 each having yellow and black seed coat colors via 2% agarose gel separation. The homozygous recessive I allele gives rise to self coloured seed coat.
       
The PCR amplification pattern was highly polymorphic among soybean accessions. These primers amplified a total of fifteen bands with individual primers SM303, SM305, TR specific and TR specific primers amplifying 2, 7, 4 and 2 bands respectively. SM303 primer amplified 180 bp size band in genotypes having yellow seed coat color and 130 bp band in black seed coat color soybean genotypes (Fig 2). This marker showed polymorphism pattern matching with seed coat color. SM305 amplified four loci of size range in between 192 bp to 216 bp out of which a 200 bp band was monomorphic (Fig 3). Additional 208bp band was observed in four of six yellow seed coated genotypes, while unique locus of size 216 bp was present only in KDS-722; while another 192 bp band was unique to Phule Agrani (KDS-344) . This marker thus showed polymorphism pattern with common seed coat color specific band in 4 of six yellow seed coated genotypes.
 

 

       
The cold induced seed coat discoloration specific primer TM showed 100% polymorphism and amplified bands size in the range between 530-600 bp in present investigation and reported size was 532 bp. The polymorphism pattern produced by this primer did not matched with the seed coat color. This marker was reported to be specific to Toyomusume’ (TM) genotype which is CD sensitive with CD meaning Cold-induced seed coat discoloration (CD). Another cold induced seed coat discoloration specific primer TR distinguished two types of seed coat color genotypes by generating two polymorphic bands. TR primer amplified band within size range 336-344 bp in genotypes having yellow seed coat color and another of size range within 300-320 bp in black seed coat color soybean genotypes (Fig 4). A 340 bp was common in four of six yellow seed coated genotypes. This marker showed 100% polymorphism and polymorphism pattern is matching with the seed coat color. Ohinishi et al., (2011) observed that TR primer amplified a 329 bp marker specifically in cold-tolerant cultivar ‘Toyoharuka’ (thereby abbreviated as TR) and derived cold tolerant recombinant inbreed lines. They reported that the variation in GmIRCHS (Glycine max inverted-repeat CHS pseudogene) was linked to cold tolerance and can be a useful marker for selection of cold tolerance.
 

 
Senda et al., (2002) reported a correlation between the inhibition of pigmentation and I gene silencing in the seed coat. The ‘I’ locus controls synthesis of anthocyanin and proanthocyanin pigments. It is located at a region harboring a cluster of chalcone synthase (CHS) genes on MLG A₂ i.e. chromosome 8 (Todd and Vodkin 1996; Tuteja et al., 2004). The dominant I allele exhibits colourless seed coat phenotype due to dominance inhibition via a possible transcriptional mode of gene silencing Senda et al., (2004). Clough et al., (2004) identified a 103-kb gene-rich region in soybean with an inverted perfect repeat cluster of CHS genes comprising the I locus. A candidate for I allele has been identified in yellow soybean genome and designated as GmIRCHS (Glycine max inverted repeat of CHS pseudogene) Kurauchi et al., (2011).
 
R locus specific primers
 
The R loci along with T loci controls specific seed coat colour viz., black / imperfect black under dominant conditions (R) and brown / buff under recessive conditions (r) (Zabala and Vodkin, 2003). The locus controls the presence (R) or absence (r) of anthocyanin in black (iRT) or brown (irT) seed coats, respectively.
       
R locus specific three SSR primers (BARCSOYSSR-1492, BARCSOYSSR-1501, BARCSOYSSR-1504) were used for present investigation Gillman et al., (2011). The primer BARCSOYSSR-1492 amplified six loci which were polymorphic and show 100% polymorphism. BARCSO YSSR-1492 reported size was 183 bp and the size found in present investigation was 181 to 200 bp. BARCSOYSSR-1504 amplified monomorphic bands of size 230 bp. BARCSOYSSR-1501 amplified five bands of size 280-310 bp.
       
Gilman et al., (2011) fine mapped the r gene region to delimit genomic region containing the r gene to less than 200 kbp. Candidate gene analysis identified a loss of function mutation affecting a seed coat specific expressed R₂R₃ MYB transcription factor gene as a strong candidate for brown hilum phenotype. They observed a near perfect correlation between the mRNA expression levels of the functional R gene candidate and an UDP-glucose flavonoid 3-0-glycosyl transferase (UF3GT) gene, which is responsible for final step in anthocyanin biosynthesis.
 
Tawny (T) locus specific primer
 
The T locus of soybean controls pubescence and seed coat colour. Takahashi et al., (2005) reported that in addition to pubescence and seed coat colour, alleles at T locus are associated with chilling tolerance. T locus specific two SSR primers (SL-305 and SN-317) Yang et al., (2010) were used for the present investigation. The T loci control specific seed coat colours, which include black or brown under dominant condition (T) and imperfect black or buff under recessive conditions (t). Primer SL-305 amplified four polymorphic bands within size range 232-240 bp, while primer SN-317 amplified monomorphic band of size 300 bp in present investigation. Yang et al., (2010) generated molecular markers SL-305 and SN-317 by utilizing 262 insertion/deletion (indel) positions observed from alignment of the cDNA and genomic DNA sequences (Gen Bank accession No. AF501293-AF-501305) of F3’H genes. The plants genotypes scored by the two markers were identical. Considering the I allele containing plant lines, in which the genotypes of T locus could not be determined, the genotypes scored by 2 molecular markers correlated perfectly with genotypes scored by seed coat and hilum colors. Thus, they analyzed that all cultivars that could be predicted to contain dominant T allele on the basis of seed coat and hilum colour also harbour same allele as that of black seed coat and hilum at the T marker loci.
       
The T loci control specific seed coat colours along with R loci, which include black (i, R, T), imperfect black (I, R, t), brown (i, r, T), buff (i, r, t) by controlling types of anthocyanin and proanthocyanin pigments. The T locus and F3’H gene were located at the same position in the molecular linkage group C₂ Todaet_al(2002). They identified that a single base deletion in flavonoid 3’ hydroxylase gene is associated with gray pubescence colour. They further developed a PCR-RFLP marker that cosegregated with T locus in F₂ population segregating for T locus. The T loci encodes for F3’H gene (Zabala and Vodkin, 2003) responsible for synthesis of the anthocyanins and proanthocyanidins. The principal anthocyanin pigments in the seed coats of black soybeans are cyanidin-3-monoglucoside and delphinidin-3 monoglucoside. Pelargonidin-3-glucoside is not a major anthocyanin and is generated in special cases. The cloning and mapping of the soybean F3’-H genomic and cDNA sequences showed that the F3’-H gene cosegregates with the T locus on soybean linkage group C2 (Chromosome 6) (Zabala and Vodkin, 2003).
 
W₁ locus specific primer
 
W₁ gene influenced seed coat colour only under homozygous recessive (ir or it) genotypes i.e. when both R and T genes are not expressing. The W1 locus basically regulates flower colour i.e. purple (W1) or white (w1) flowers. Zabala and Vodkin (2005) identified F3’5’H gene being associated with the W1 locus on the basis of analysis of an F3’5’H mutant, a series of soybean cultivars and lines of F2 population. Chromatographic experiments and genetic analysis suggested that W1 gene encodes a flavonoid 3’5’ hydroxylase (F3-5-H) (Buzzell et al., 1987; Zabala and Vodkin 2007).
       
W₁ locus specific primer SN-019 amplified nine bands of which seven were polymorphic and two were unique. The polymorphic band size range was 280-300 bp. Two unique bands of size 180 bp and 170 bp were present in KDS-749 and VLS-65, respectively. Song et al., (2004) developed SN019 and SN020 markers which cosegregated and mapped between Satt-348 and Satt-160 on MLG F (Chromosome 13), which is the presumed location of W₁ locus in soybean map.
 
W2 locus specific primer
 
W₂ locus specific primer SL-017 produced five polymorphic bands of size range 180-200 bp. This polymorphic pattern was not matching with seed coat color pattern. Takahashi et al., (2013) reported that purple blue flower of soybean is controlled by W₂ locus and MYB transcription factor gene GmMYB G₂O-1 was located at a position similar to the W₂ gene.

Seed colour and durability markers
 
Seed colour and durability specific 3 primers, Satt-371, Satt-453 and Satt-618 (Hosamani et al., 2013) were used for present investigation. Together they amplified a total of 18 bands, however none of them was found to be associated with seed coat colour in the present investigation. Satt-371 primer amplified six polymorphic bands of range 180-220 bp. Satt-453 primer amplified four polymorphic bands in range of 210-220 bp. Satt-618 primer amplified eight bands (range 130-150 bp) out of which seven were polymorphic and one was unique. Unique band was present in Phule Sangam (KDS 726) was of 210 bp size.

Soybean seed coat colour is an important morphological trait on the basis of which its market price is calculated. Molecular analyses of soybean genotypes differing in seed coat color relation were assessed using SSR markers that have a higher discrimination capacity for seed coat colour. Out of the fourteen primers that amplified 52 alleles of which 48 showed the polymorphism. It can be concluded that I locus specific primers, SM303, SM305 and TR (cold induced seed coat discoloration specific) produced the amplification pattern that differentiated between yellow and black seed coat color genotypes. Primer SM303 amplified 180 bp allele in yellow seed coat color genotypes and a 130 bp allele in black seed coat color genotypes. Primer TR cold induced seed coat discoloration specific amplified larger sized allele within 336-344 bp range in yellow seed coat colored genotypes and a smaller sized allele within 300-320 bp range in black seed coat colored genotypes.  Primer SM305 amplified 208 bp band in four of six yellow seed coated genotypes, while unique alleles of 192/ 216 bp in other two yellow seeded genotypes. They need to be verified in larger natural as well as segregating population before used in marker assisted selection. This can of help in future breeding of yellow or coloured soybean cultivars as desired. Breeding for this pigmentation trait may help in improving anthocyanin content that plays an important role in defense against pathogens and protection from UV light exposure in addition to their nutritional value.

The authors are grateful to authorities of Mahatma Phule Krishi Vidyapeeth, Rahuri for providing necessary facilities to undertake this study. Help rendered by Officer Incharge, State Level Biotechnology Centre, M.P.K.V., Rahuri and Mr. B.D. Pawar during this study is also gratefully acknowledged.