*4.6. Folates*

A wide exploration of available genetic resources is necessary to identify the rich source of folates for their potential use for the biofortification of pulse crops. Various methods have been employed to quantify folates from different food sources including pulse crops, and these methods include microbiological assays [209–211], liquid chromatography (LC) coupled with fluorescence detection (FD) [87,192], and mass spectrometry (MS) detection [15,30,191,193,194,212–214].

Previously, researchers have identified a wide range of variation in folates quantified from pulse crops (Table 1). For example, using a microbiological assay, Han and Tyler [190] reported 24.9–64.8 μg/ 100 g folates in green cotyledon peas and 23.7–55.6 μg/100 g in yellow cotyledon peas grown at multiple locations in Saskatchewan, Canada. Vahteristo et al. [189] and Hefni et al. [87] observed the folate concentration of 59 and 52 μg/100 g using LC-MS in vegetable peas consumed in Finland and dry green peas consumed in Egypt, respectively. Rychlik et al. [191] reported variable folate concentration in different pulse crops—275 μg/100 g (chickpeas), 106–164 μg/100 g (white beans), 110–154 μg/ 100 g (green lentils), and 10–20 μg/100 g (peas) using an LC-MS method, whereas Sen Gupta et al. [192] reported a higher total folate concentration in field peas (41–202 μg/100 g) grown in the USA compared to chickpeas (42–125 μg/100 g) using LC-FD.

Using ultra-performance LC (UPLC), six folate monoglutamates were quantified in four pulse crops, and the total folate concentration was the highest in chickpeas (351–589 μg/100 g), followed by common beans (165–232 μg/100 g), lentils (136–182 μg/100 g), and pea (23–30 μg/100 g) [15]. This method provided high accuracy in quantification of specific folates with the use of isotopically labeled internal standards. Zhang et al. [193] identified eight folate monoglutamates using an optimized one-step extraction approach in peas, chickpeas, beans, and lentils using UPLC-MS. They also observed the highest folate concentration in chickpeas and the lowest concentration in peas. Zhang et al. [194] quantified eight folate monoglutamates in six wild lentil species and one cultivated species using UPLC-MS. They observed that wild lentil species (195–497 μg/100 g) had generally higher folate concentration than cultivated genotypes (174–361 μg/100 g). Most recently, Jha et al. [30] quantified five folate monoglutamates in 85 diverse pea accessions originating from worldwide sources using UPLC-MS, and the results indicated a wide range of variation in the concentration of the sum of folates (14–55 μg/100 g dry seed weight).

Tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-MTHF), and 5-formyltetrahydrofolate (5-FTHF) were the most abundant in common beans, lentils, chickpeas, and peas [15,30,208]. Further, 5-MTHF represented 56% of the total folate concentration in peas [15,30], whereas 5-MTHF and 5-FTHF represented 35–39% and 33–51% of the total folates in common beans, lentils, and chickpeas, respectively [15]. Other studies also reported 5-MTHF as the predominant form of folate in common beans [87,215], lentils [87,191], and chickpeas [87]. 5-MTHF was also the major folate in cereals, vegetables, fruit, bread, milk, and meat products [87,216], and in humans [217]. Scaglione and Panzavolta [218] reported various advantages to using naturally occurring 5-MTHF over synthetic folic acid. For example, it helps in the prevention of the potential negative effects of unconverted folic acid in peripheral circulation. Thus, 5-MTHF may be the most important folate that could be targeted for improvement by breeders. Jha et al. [30] identified several pea accessions with greater folate concentration including MPG87 (42 μg/100 g), Kahuna-NIAB (40 μg/100 g), and OZP0902 (50 μg/100 g).

Using a GWAS, five SNP markers were associated with the sum of folates, and fifteen, eight, and three SNP markers were associated with major individual folates 5-MTHF, 5-FTHF, and THF, respectively [30] (Table 1). Further, SNP markers Sc\_6992\_86348 and Sc\_3060\_11265 were validated in an additional 24 accessions, and these markers have potential for marker-assisted selection in pea breeding.
