Standardization of markers with rice
All 45 markers targeting 24 rice yield and quality trait governing genes were initially standardized using rice genomic DNA from NLR34449 at temperatures ranging from 50 to 65°C. Out of these, 41 primers successfully amplified with NLR34449, while four primers did not yield amplification. The primers that amplified with rice DNA were subsequently tested for their transferability to groundnut genotypes to assess their applicability in genotyping groundnut.
Standardization of selected markers with peanut genotypes
The 41 markers amplified with rice were tested on wild
(Arachis glabrata and
Arachis villosa) and cultivated groundnut varieties. 31 markers (76%) amplified with cultivated groundnuts, except sd1-h. 25 markers amplified with wild groundnuts (Table 1). 10 markers amplified exclusively with rice. Cross-transferability of markers to groundnuts requires further analysis.
Cross transferability of markers between rice and peanut
During the course of crop evolution, the genome content across plant kingdom is proved to be conserved and is evident from many synteny studies across genera/families. Further, conservation of functional genes and important motifs are reported by many earlier research groups
(Trivedi et al., 2013; Hussien et al., 2014). The transferability was analysed with similar allele sizes of groundnut genotypes to rice
i.e. which are observed across the genotypes under study.
Plant height
In this study, the sd1-h marker, linked to the sd1 gene responsible for plant dwarfism and encoding gibberellin 20-oxidase (GA20ox-2), was examined. While previous reports
(Monna et al., 2002) indicated the marker’s association with specific allele sizes (843 bp) in rice varieties, amplification in wild peanut yielded a 190 bp product size, whereas cultivated peanut showed no amplification. This discrepancy may imply significant changes at the primer locus or complete loss of relevant alleles/gene during evolution.
Plant architecture
OsSPL14 (Squamosa Promoter Binding Protein-Like 14), also known as wealthy farmer s panicle/ideal plant Architecture 1, regulated by microRNA OsmiR156, plays a crucial role in rice grain productivity and plant architecture. In 11 groundnut genotypes, the marker predominantly amplified a 500bp allele, akin to rice, with exceptions in certain genotypes showing a 70 bp allele size and Greeshma exhibiting a 390bp allele (Fig 1). Functionally, OsSPL14 promotes heavy panicle formation and increased secondary branches in rice
(Mohapatra et al., 2018). Exploring this gene in groundnuts offers potential for enhancing yield by modulating branching and flowering patterns, given the importance of secondary branch number in groundnut productivity.
The plant architecture and yield 1 (PAY1) gene, known for improving plant architecture and grain yield, was examined using the PAY1SP6 marker to assess transferability from rice to peanut. Notably, an allele of 200bp was consistently observed across rice and both wild and cultivated peanut genotypes (Fig 1).
Zhao et al., (2015) compared a wild rice introgression line, YIL55, with a mutant featuring modified plant architecture, PAY1, which exhibited superior traits such as increased plant height, reduced tiller number, thicker stems andlarger panicles. Analyses with NILs in Teqing or 9311 genetic backgrounds further confirmed PAY1's role in shaping superior plant architecture and enhancing grain yield in rice, suggesting its potential as a key regulator. Characterizing genes like PAY1 offers opportunities for enhancing groundnut plant architecture and reproductive unit production.
The grain number (GN2) gene, functioning as an OsWAK (Wall-Associated Kinase) receptor-like protein, is crucial for increasing grain number. In this study, the closely tagged marker RM3535 consistently amplified an 185bp fragment across all peanut genotypes, mirroring results observed in rice. Sequencing of this allele could further validate its utility for groundnut improvement breeding (Fig 1).
The
YLD (yield) gene linked SSR marker RM223 amplified 165bp in all genotypes of rice and peanut (Fig 1). This marker also has tagged to aromatic/non-aromatic trait of rice as reported in
Jewel et al., (2011).
The spikelet number (SPIKE) gene, linked to Narrow Leaf1 (NAL1), enhances spikelet number. The SPIKE-INDEL3 marker consistently amplified a 171 bp fragment in both rice and groundnut. However, in cultivated peanut genotypes, it showed non-specific amplification of a 250 bp fragment alongside the 171 bp fragment (Fig 1). Additionally, Erect Panicle 3 (EP3), associated with erect panicle formation, was noted. The S5-803 marker exclusively expressed a 243 bp allele size in rice
(Piao et al., 2009).
SCM2/Aberrant panicle organization 1 (APO1) gene, crucial for rachis branching and culm diameter regulation, features a beneficial allele known as SCM2 found in the Habataki rice variety
(Kim et al., 2016). The SCM2 INDEL1 marker amplified consistently at 117bp in both rice and peanut genotypes, although with lower intensity in peanut, possibly due to primer site variations. Further primer design targeting different gene regions may confirm the presence of SCM2 ortholog in groundnut.
The DEP1 gene which encodes phosphatidyl ethanolamine binding protien (PEBP), regulates dense and erect panicle traits in rice, impacting grain number per panicle
(Huang et al., 2009). Dep1s7 consistently amplified a 127 bp allele across rice and all peanut genotypes, indicating potential orthologous study from rice to peanut for identifying pod number increase per plant (Fig 1).
Seed quality genes (Seed Size and weight)
The GS2 gene plays a crucial role in regulating grain size and shape in rice, with the marker RM3212 associated with the medium-grain phenotype
(Zhang et al., 2013). This marker consistently expressed an 181bp allele size in both rice and peanut. GS3, linked to markers RGS1, SF28, RGS2 andSR17, was not detected in African rice cultivars due to the domestication process. However, the wild rice relative
O. meridionalis possesses unique alleles related to GS3, making it a candidate gene for genetic improvement in cultivated lines. Allelic variations at SF28, RGS1 andRGS2 loci within GS3 are strongly associated with grain length in Chinese rice germplasm. Meanwhile, SR17 marginally affects grain length
(Wang et al., 2010). RGS1 and SR17 markers amplified 180bp/200bp and 1400bp allele sizes, respectively, exclusively in rice, underscoring their roles in regulating grain size and length
.
The
GS5 gene, governing grain width
(Li et al., 2011) in rice, consistently amplified a 67 bp allele with GS5-INDEL1 in both rice and peanut genotypes, indicating potential transferability of the marker and suggesting wide grain size
(Kim et al., 2016). Additionally, RM574 expressed a 240bp allele associated with low grain width, which could be useful in rejecting undesirable traits in rice breeding
(Bidanchi et al., 2018). Exploring GS5 in groundnut could reveal key allelic variants influencing kernel size (Fig 2).
The
GW2 gene, integral to grain width and weight regulation, was analyzed utilizing the GW2SNP2 marker, which consistently revealed a 51 bp allele in both rice and peanut genotypes (Fig 2), mirroring previous findings of
Zhang et al., (2015). Furthermore, investigation into the
GW5 gene, associated with grain width, employed the RM3328 and RMw513 markers. RM3328 exhibited a 119 bp product in rice and a 95 bp allele in cultivated peanut genotypes, while no alleles were detected in wild groundnut. Conversely, RMw513 showcased a 600bp allele in both rice and cultivated peanut genotypes, with the wild peanut revealing a 700 bp allele (Fig 2). Further exploration of GW5 orthologs in peanut holds promise to unravel seed size variation.
The
SW5 (Seed Width) gene, targeted by the N1212 marker, is linked to increased grain width. Contrary to findings of
Zhang et al., (2015), our study revealed a 65bp product as the major allele in both rice and peanut genotypes (Fig 2).
GLW7 (Grain Length and Width), governed by the OsSPL13 transcription factor, regulates cell size in grain hulls, enhancing grain length and yield belongs to
Squamosa Promoter Binding Protein (SBP) family. This marker consistently expressed a 140bp size in both rice and peanut genotypes, with some cultivated peanut genotypes displaying a 160bp allele, indicating potential locus variations among groundnut genotypes. Further research on this gene could elucidate its function in peanut.
Another marker, RM505, linked to
qgrl7 responsible for grain length, exhibited varying (500 and 180bp) amplification patterns in different rice and peanut genotypes, with cultivated peanut genotypes displaying a 490 bp allele (Fig 3). Sequencing the major allele may clarify the gene’s orthologous nature in groundnut. RM21945, associated with grain length, grain weight (GW), length and width ratio and Gel Consistency
(Verma et al., 2015), consistently expressed a 292bp allele in all rice and peanut genotypes, but with lower intensity in peanut. These findings suggest potential transferability from rice to peanut, pending analysis with additional markers.
Seed filling
Shelling percentage, a vital trait in groundnut, prompted an examination of the rice GIF1 (GRAIN INCOMPLETE FILLING 1) gene, known for its role in grain filling. The GIF1 marker revealed a consistent 96bp allele across all rice and peanut genotypes, indicating potential transferability for seed filling/size regulation (Fig 2).
Flowering time
Groundnut farming in India relies on rainfed conditions, making early flowering crucial. Heading date genes like Hd3a and RFT1 (RICE FLOWERING LOCUS T) accelerate flowering under different day lengths
(Yano et al., 2000). Hd1AGC and Hd3a showed 140 bp and 90 bp alleles consistently across rice and peanut genotypes. This suggests the presence of similar genes in groundnut, aiding in the development of groundnut varieties with preferred flowering times.
Seed micronutrient content
Fe and Zn are important micronutrients towards human health, thus needs to be focused in research of food crops. For Fe and Zn content, primers using primer 3 software were designed for 3 reported genes
viz.,
YSL2,
YSL13 and
YSL15 which belongs to Yellow-Stripe Like gene family.
YSL2 gene reported for preferential expression in the leaf tissues which suggests that this
YSL2 functions as transporter which is responsible for the phloem transport of iron. The other gene
YSL15 proved for its significant expression in root and rhizome type of tissues indicating its role in the uptake/absorption of iron from the source
(Menna et al., 2011). Out of the five
indel markers used, only one marker (vf0226164188) tagged to
YSL2gene amplified alleles (137 bp) in both rice and peanut (Fig 2).
Assessment of cross transferability across genotypes
The assessment revealed that 76% of rice gene markers successfully amplified in groundnut, indicating high conservation between the two crops (Fig 3 and Fig 4). Genotype-level analysis (Table 2) showed common allele sharing ranging from 79.17% to 91.67% among groundnut genotypes (Fig 3). Seventeen markers from 14 genes showed consistent amplification across all groundnut genotypes (Fig 4), while two markers (Dep1 INDEL 1 and Hd1) exhibited limited amplification. Some markers showed lower intensity but retained rice allele size, suggesting the need for alternative marker regions to address amplification challenges.
