An AS-PCR assay for accurate genotyping of FAD 2 A / FAD 2 B genes in peanuts ( Arachis hypogaea L . )

395 (LDL) and beneficial high density (HDL) cholesterol levels. Oleate, however, is more stable; a high oleate peanut not only means prolonged shelf life (O’Byrne et al., 1997), but also helps to lower LDL cholesterol level, while maintaining HDL cholesterol level. The development of high oleate to linoleate ratio (O/L) varieties has therefore become a major breeding objective of the cultivated peanut. The cultivated peanut is an allotetraploid (2n=4x=40). FAD2A and FAD2B are homoeologous genes from A and B genomes coding for 12-fatty acid desaturases, which convert oleate (C18:1) to linoleate (C18:2). So far, several high oleate peanut mutants have been reported (Norden et al. 1987, Ashri 1988, Wang et al., 2010). In most cases, a 1-bp substitution (G:C→A:T) at position 448 after the start codon (FAD2A 448 G > A) in FAD2A, caused a missense amino acid substitution from aspartic acid to asparagine (D150N), and a 1-bp insertion (A:T) at position 442 after the start codon in FAD2B (FAD2B 441_442insA), resulted in a frame shift (Jung et al., 2000, López et al., 2000). The activity of both desaturases in these high oleate mutants was confirmed to be significantly lower that in normal oleate peanut genotypes by enzyme assays, mutagenesis and gene expression in yeast systems (Ray et al., 1993, Bruner et al., 2001). These high oleate mutants can be used in hybridization programs to incorporate this valuable trait into high yielding adapted peanut cultivars. For genetic studies and breeding applications, there is a need to identify peanut F1 and F2 hybrids with FAD2A/FAD2B mutant alleles or high oleate segregates in normal oleate × high oleate (FAD2A 448 G > A , FAD2B 441_442insA) crosses. Currently, some technologies have already been developed to partially meet the demand, which include, cleaved amplified polymorphisms (CAPS) (Chu et al., 2007), real-time PCR genotyping assays (Barkley et al., 2010, 2011), near infrared reflectance spectroscopy (NIRS) (Wang et al., 2010), direct sequencing of PCR products (Wang et al., 2010) and allele specific-PCR (AS-PCR) (Chen et al. 2010). The first three methods require complicated operations or special instruments. Although direct sequencing can be used to identify RESUMEN

(LDL) and beneficial high density (HDL) cholesterol levels.Oleate, however, is more stable; a high oleate peanut not only means prolonged shelf life (O'Byrne et al., 1997), but also helps to lower LDL cholesterol level, while maintaining HDL cholesterol level.The development of high oleate to linoleate ratio (O/L) varieties has therefore become a major breeding objective of the cultivated peanut.
The cultivated peanut is an allotetraploid (2n=4x=40).FAD2A and FAD2B are homoeologous genes from A and B genomes coding for 12-fatty acid desaturases, which convert oleate (C 18:1 ) to linoleate (C 18:2 ).So far, several high oleate peanut mutants have been reported (Norden et al. 1987, Ashri 1988, Wang et al., 2010).In most cases, a 1-bp substitution (G:C→A:T) at position 448 after the start codon (FAD2A 448 G > A) in FAD2A, caused a missense amino acid substitution from aspartic acid to asparagine (D150N), and a 1-bp insertion (A:T) at position 442 after the start codon in FAD2B (FAD2B 441_442insA), resulted in a frame shift (Jung et al., 2000, López et al., 2000).The activity of both desaturases in these high oleate mutants was confirmed to be significantly lower that in normal oleate peanut genotypes by enzyme assays, mutagenesis and gene expression in yeast systems (Ray et al., 1993, Bruner et al., 2001).These high oleate mutants can be used in hybridization programs to incorporate this valuable trait into high yielding adapted peanut cultivars.

SUMMARY
An AS-PCR assay for accurate genotyping of FAD2A/ FAD2B gene in peanut (Arachis hypogaea L.) FAD2A and FAD2B are homoeologous genes from A and B genomes in cultivated peanuts (Arachis hypogaea L.) encoding fatty acid desaturates which convert oleate to linoleate.To study the genetics of oleate and breed high oleate peanut cultivars, a simple allele specific-PCR (AS-PCR) protocol for the accurate genotyping of FAD2A/FAD2B was developed to discriminate the wild and mutant allele of both genes (FAD2A 448 G > A and FAD2B 441_442insA).The results may serve to develop a feasible procedure for producing highly desired high oleate peanut cultivars through hybridization.KEY-WORDS: Allele specific PCR (AS-PCR) -FAD2A/ FAD2B -Genotyping -Peanut.

INTRODUCTION
The cultivated peanut, Arachis hypogaea L., is an important cash crop all over the world.by the AS-PCR protocol described below were further tested for their real genotypes by direct sequencing.

AS-PCR primer design
Seven AS-PCR primers were designed according to the base difference between FAD2A/ FAD2B alleles.To ensure specificity in detection, a mismatched base was placed in the second or third base from the 3' end of 4 primers (Table 1).

PCR mixture components and thermal cycling profile
Four PCR reactions were established to detect the wild and mutant alleles of FAD2A/ FAD2B, respectively.As illustrated in Figure 1, Reaction I, Reaction II, Reaction III and Reaction IV were aimed to detect the FAD2A wild allele (Ol 1 ), FAD2A mutant allele (ol 1 ), FAD2B wild allele (Ol 2 ), and FAD2B mutant allele (ol 2 ), respectively.Each reaction (25 mL total volume) contained 12.5 mL of 2 × Taq PCR Mix (Tiangen, Beijing, China), 3 primers (volume listed in Table 2), and 1.5 mL of DNA template prepared from a slice of cotyledonary tissue (weight 3-5 mg) of a single seed following the method of Yu et al., (2010).
true F 1 hybrids, it is costly when handling large populations.NIRS cannot genotype the FAD2A/ FAD2B alleles.As mentioned above, Chen et al. (2010) has already developed an AS-PCR assay for genotyping the FAD2A/FAD2B alleles in peanuts, but failed to differentiate between the genotypes of Ol 1 ol 1 /Ol 2 Ol 2 and ol 1 ol 1 /Ol 2 Ol 2 , or Ol 1 Ol 1 /Ol 2 ol 2 and Ol 1 Ol 1 /ol 2 ol 2 .
The objective of the present study was to develop an AS-PCR assay for the accurate genotyping each of the FAD2A and FAD2B allele in the cultivated peanut, as long as the mutant type FAD2A and FAD2B were FAD2A 448 G > A and FAD2B 441_442insA, respectively.

Peanut material
Previously, FB4 and CTWE, 2 high-oleate peanut mutants developed by us, were identified as with FAD2A and FAD2B mutant alleles (FAD2A 448 G > A and FAD2B 441_442insA), and normal oleate Huayu 40 was verified to have wild type FAD2A and FAD2B (Wang et al., 2010).True hybrids had been selected by the direct sequencing of FAD2B PCR products of the resultant seeds (F 1 ) harvested from female parents in the normal oleate Huayu 40 × high oleate (FB4 or CTWE) crosses (Wang et al., 2010).
To test if the AS-PCR assay was accurate and reproducible, Huayu 40 was used to prepare an FAD2A/FAD2B wild allele homozygote DNA template (Ol 1 Ol 1 Ol 2 Ol 2 ), CTWE and FB4 were used to prepare FAD2A/FAD2B mutant allele homozygote DNA templates (ol 1 ol 1 ol 2 ol 2 ), and the true hybrid seeds (F 1 ) from Huayu 40 pollinated with FB4 or CTWE were used to prepare wild-mutant heterozygote DNA templates (Ol 1 ol 1 Ol 2 ol 2 ).
To test the accuracy of the AS-PCR protocol in a segregation population (derived from a normal oleate × high oleate cross) with various genotypes, randomly selected single F 1:2 seeds with known FAD2A/FAD2B "genotypes" identified

Primer
Sequence (5' to 3') The banding pattern of the AS-PCR products was satisfactory (Figure 2).As expected, Huayu 40 (Ol 1 Ol 1 Ol 2 Ol 2 ) produced a 557 bp target band in Reaction I and a 539 bp target band in Reaction III, CTWE and FB4 (ol 1 ol 1 ol 2 ol 2 ) produced target bands in Reaction II and IV (Figure 2a and Figure 2c), and the true F 1 hybrids (Ol 1 ol 1 Ol 2 ol 2 ) produced target bands in all 4 reactions (Figure 2b and Figure 2d), demonstrating that this method had potential to accurately detect each FAD2A/FAD2B allele from individual peanut seeds.

FAD2A/FAD2B genotyping in a normal oleate × high oleate F 1:2 population
A total of 86 randomly selected single F 1:2 seeds were genotyped by the AS-PCR assay.All 9 genotypes were detected in the population (Figure 3).Each of them appeared at least 2 times (Table 3).In all cases without exception, the trace files from the direct sequencing of FAD2A/FAD2B products amplified with these templates (Figure 4) were in full agreement with the results from AS-The 4 reactions used the same PCR program: 94 °C for 1min, followed by 30 cycles of 94 °C for 30 s, 53 °C for 30 s and 72 °C for 90 s, and a final extension of 72 °C for 5 min.

Agarose gel electrophoresis of PCR products and band separation
PCR products were separated on a 1% agarose gel (MDBio, Qingdao, China) in 1 × TAE buffer by electrophoresis at 120 V for 24 min.The agarose gels were stained with GelRed dye (Biotium, CA, USA).The resulting bands were visualized under UV light.
All of the 4 reactions were supposed to produce a ca 1.2kb internal reference band amplified by the forward primer (FAD2A-F or FAD2B-F) and the reverse primer (FAD2-R), which was an indication of PCR success.
The diluted PCR products (×100) were used as templates in the second amplification, with the primer pairs abf19F (AAGCCTCTTTCAAGGGTTCCA) and abf19R (GATGAGCCACATGCGTATCAGTT). The PCR mixture (50 mL) consisted of 25 mL of Tiangen 2 × Taq Platinum Master Mix (Tiangen, Beijing, China), 1ml of DNA template, and 2 mL of primers (10 mM) each.The thermal cycling profile was 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 sec, 54 °C for 30s, and 72 °C for 1 min, and a final extension of 72 °C for 4 min.
The PCR products were then recovered and purified, and were sent to Genscript Inc, Nanjing, China, for direct sequencing.Heterozygotes were identified by overlapped peaks (Wang et al., 2010).

DISCUSSION
The AS-PCR protocol for peanut FAD2A/FAD2B presented here was advantageous over the one developed by Chen et al. (2010).Genotyping with the current protocol was accurate and rapid.About 40 minutes were saved in electrophoresis, and less time was needed in thermal cycling.
Based on the present AS-PCR protocol and NIRS calibration models reported earlier by our research group (Wang et al., 2010), a feasible selection procedure for high oleate peanuts through hybridization can then be proposed, as long as the FAD2A/FAD2B genotypes involved are the same as in this study.Generally, normal oleate adapted   alleles can be identified by NIRS.Continuous selection for productivity and other agronomic traits should be carried out in F 3 and later generations.
Backcrossing is generally recommended to cope with the low yield of high oleate genotypes, a common problem in high oleate crop breeding.
Though proven useful, it is tedious and timeconsuming.The proposed selection procedure increases the possibility for obtaining desirable segregates with high oleate; consequently, it may hasten the process for developing peanut cultivars with both high yields and high oleate.
The AS-PCR method presented here can also be used to study the genetics of the high oleate trait in peanuts; earlier reports indicated that there were other genes responsible for this trait in addition to FAD2A and FAD2B (Lopez et al., 2000, Isleib et al., 2006).It is interesting to know how great the contribution of FAD2A/FAD2B to the high oleate trait is in different cross combinations of the cultivated peanut.

Figure 1
Figure 1Illustration of the 4 reactions used in AS-PCR for FAD2A/FAD2B genotyping in peanut.Wild and mutant alleles of FAD2A/FAD2B, primers in each reaction and their annealing sites on the genes were shown.