Assessment of genetic diversity among Chinese high-oleic peanut genotypes using miniature inverted-repeat transposable element markers

As compared with normal-oleic peanuts, high-oleic peanuts proved to be heart-healthier and had a prolonged shelf life and extended seed longevity. However, there have been concerns about the genetic diversity of present-day high-oleic peanut cultivars, which relied heavily on high-oleic donors with F435 type FAD2 mutations. In the study, a total of 104 high-oleic peanut cultivars/lines/mutants from main breeding teams in China were used to assess their genetic diversity with AhMITE markers. Of all the 31 cultivars tested, those from CTW (Chuan Tang Wang) team had the highest genetic variability. Again, of all the 73 lines studied, those from the same team ranked first in genetic diversity. As compared with cultivars from CTW and C&Y (Xiao Yuan Chi and Shan Lin Yu) teams, greater genetic diversity was detected in new lines of both teams, indicating that recent breeding efforts had resulted in improved genetic diversity in high-oleic peanuts.


Introduction
As a rich source of edible oil, dietary protein and phytochemicals, peanut is a main cash crop cultivated worldwide, which can be processed into a variety of food products (Bnoku and Yu, 2020).
In a report on the inheritance of high oleic acid in peanut, F 2 seeds with at least 70% oleic acid were classified as high-oleic (Moore and Knauft 1989). Likewise, in a similar study, Wang et al. (2013a) adopted the same standard based on the frequency distribution of oleic acid content in F 2 seeds. Notably, a qualified health claim was allowed by The US Food and Drug Administration (FDA) (2018) to be placed on the labels of oils with high levels of oleic acid, viz., no less than 70% oleic acid. The industry-accepted standard for high-oleic peanuts is, however, above 74% oleic acid (Davis et al. 2021). High-oleic peanuts are advantageous over their normal-oleic counterpart, having a prolonged shelf life, extended seed longevity and several health benefits (Wang and Zhu 2017;Nkuna et al. 2021). Up to now, much progress has been made in breeding high-oleic peanut cultivars. In China, related studies were mainly carried out at Shandong Peanut Research Institute (SPRI), Kaifeng Academy of Agriculture and Forestry (KFAAF) and Hebei Academy of Agriculture and Forestry Sciences (HBAAFS). Some high-oleic peanut cultivars were developed by breeders from Henan Academy of Agricultural Sciences (HNAAS) and Chinese Academy of Agricultural Sciences (CAAS) Oil Crops Research Institute (OCRS) (Wang and Zhu 2017).
As far as we know, only four research papers (in Chinese) on high-oleic peanut genetic diversity have been published, using high-oleic peanut genotypes with F435 type FAD2 mutations (G448A in FAD2A, 442insA in FAD2B). Hu et al. (2013) utilized 12 SSR (simple sequence repeat) primer pairs in the analysis of 5 high-oleic peanut genotypes along with a normaloleic cultivar (Huayu 22). Similarity coefficient was found to be 0.325-0.750, averaging 0.655. Yu et al. (2017) used 31 SSR primer pairs to assess the genetic diversity of 41 high-oleic peanut cultivars/lines. Similarity coefficient varied from 0.333 to 0.889, with an average of 0.626. In the study by Wang et al. (2020), genetic distance of 25 high-oleic peanut cultivars/lines, as revealed by 140 SSR primer pairs, ranged from 0.057 to 0.624, with a mean value of 0.451. Guo et al. (2020) used 217 SSR primer pairs to study the genetic diversity of 8 high-oleic peanut cultivars bred at KFAAF and found that similarity coefficient was 0.3438-0.9688, with a mean value of 0.6149.
As the output of continuous breeding efforts, more and more high-oleic peanut cultivars, lines and mutants are currently available. Under such conditions, there still have been concerns about the genetic diversity of present-day high-oleic peanut cultivars, which relied heavily on F435 type high-oleic donors.
Miniature inverted-repeat transposable elements (MITEs) are non-autonomous elements of less than 600 bp in length (Shirasawa et al. 2012a). In peanut, the number of AhMITE1 markers thus far developed has amounted to nearly 4000 (Shirasawa et al. 2012a, b;Gayathri et al. 2018). Being a new type of DNA molecular markers, through have not been widely used, AhMITE1 markers are most promising. Several reports regarding the identification of true hybrids, mapping, and evolutionary and genetic diversity studies using AhMITE1 markers all yielded satisfactory results Wang and Zhu 2017).
This study aimed to compare the degree of genetic diversity in high-oleic peanut cultivars/lines from different teams in China using AhMITE1 markers, and to find out if the gene base for high-oleic peanuts has been broadened by recent breeding efforts, through assessment of the levels of genetic diversity in representative high-oleic peanut genotypes.

Peanut material
Totally 104 high-oleic peanut cultivars/lines/mutants from main breeding teams in China were used in the study. These included 12 cultivars from Chuan Tang Wang (CTW) team, SPRI, 3 cultivars from Xiao Yuan Chi and Shan Lin Yu (C&Y) team, SPRI, 6 cultivars from Jian Zhong Gu (JZG) team, KFAAF, 3 cultivars from Yu Rong Li (YRL) team, HBAAFS, 7 cultivars from other teams, 32 lines from CTW team, 16 lines from C&Y team, and 10 mutants from CTW team (Table 1).

Primer pairs
38 AhMITE (Miniature Inverted-repeat Transposable Element) primer pairs producing discernable, reproducible and polymorphic bands were utilized to study the genetic diversity among the populations and groups (Tables 2, 3). DNA extraction, PCR and electrophoresis DNA was extracted from cotyledonary slices of peanut seeds according to a previously reported protocol (Yu et al. 2010). A 20 ll of PCR mixture consisted of 29Taq PCR Master Mix 10 ll (Tiangen Biotech, Beijing), forward and reverse primers 1 ll each (10 lM), template DNA 1 ll, and sterile double distilled water 7 ll. Thermal cycling profile was 5 min at 94°C for pre-denaturation, 35 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 40 s, followed by a final extension step of 5 min. PCR was performed on a thermocycler (Model Tc-s-B, Bioer, Inc.), and amplification prod-   (Table 3). Each polymorphic band was manually scored as present (1) or absent (0) for each peanut sample. Observed number of alleles, effective number of alleles, Nei's (1973) gene diversity, Shannon's information index, number of polymorphic loci, and percentage of polymorphic loci were calculated using the Popgene 1.31 software (Yeh et al. 1999). Analysis of molecular variance (AMOVA) was conducted using GenAlex (Peakall and Smouse 2012). Cluster analysis of populations and genotypes was done with Popgene 1.31 software using Nei's (1972) genetic distance and UPGMA (unweighted pair group method with arithmetic mean) method and DPS 14.50 package using Jaccard distance and flexible group average cluster method (Tang and Zhang 2013), respectively. Similarity coefficient was calculated with NTSYSpc 2.10e (Applied Biostatistics, Inc., 2000).

Results and analysis
AhMITE polymorphism 38 AhMITE primer pairs generated a total of 75 polymorphic loci (1.97 polymorphic loci per primer pair). AhMITE0017 banding pattern, as an example, was shown in Fig. 1. Among the 8 populations, the mean percentage of polymorphic loci (pp), Nei's (1973) gene diversity (h), and Shannon's Information index (i) were 100%, 0.2910 and 0.4518, respectively. Lines from CTW (L-CTW) had the highest level of variability, with pp, h and i being 94.67%, 0.2730 and 0.4211, followed by lines from C&Y (L-C&Y), with pp, h and i being 78.67%, 0.2468, 0.3724, respectively. Cultivars from CTW ranked third (pp, h and i were 68.00%, 0.2461 and 0.3663). CV-Other and Mu-CTW were in the fourth and fifth positions respectively. CV-JZG, CV-C&Y and CV-YRL had relatively low levels of variability (Table 3) The coefficient of genetic differentiation (G st ) between the cultivars of CTW and C&Y teams was 0.2640 (26.40% of total variation resided between, and 73.60% within the 2 populations) ( Table 3). Among  (Table 3).
Likewise, when the 8 populations were used in AMOVA, the results revealed that of the total genetic diversity, 9.2% was attributed to among-population diversity and the rest (90.8%) to within populations ( Table 4). Despite that among-population variation  Fig. 2, high-oleic peanut lines and mutants from CTW team were grouped together, and cultivars from YRL and JZG teams also had a close relationship. Cultivars from C&Y team were distantly related to other genotypes, however (Fig. 2). As compared with cultivars and lines from C&Y team, cultivars from CTW team were more closely related to the rest other genotypes (Fig. 2).

Genotype grouping
Based on AhMITE profiling, the 104 high-oleic peanut genotypes fell into two categories (I and II). Each category was further divided into two sub-categories (I-a, I-b, II-a, and II-b) (Fig. 3). Most of the lines from C&Y team were found in category I, whereas all the cultivars and most of the lines from CTW team were found in category II. All the cultivars from CTW team were in II-b, and 5 of the 6 cultivars from JZG team were in I-b. The mutants from CTW team could be   found in 3 of the 4 sub-categories (3 in I-b, 5 in II-a, and 2 in II-b). Cultivars from YRL team and lines from CTW team were in 3 of the 4 sub-categories (Fig. 3). Lines from CTW and C&Y teams were more genetically diversified than cultivars from both teams.

Conclusions and discussion
netic similarity coefficient of the 104 high-oleic peanut genotypes in the present study, as calculated by NTSYSpc 2.10e ranged from 0.4400 to 0.9333, with a mean value of 0.6953 (detailed data unshown), comparable to that in a previous study using AhMITE markers to analyze 115 peanut cultivars/lines (mean value = 0.6902) (Wang et al. 2013b). In earlier reports on high-oleic peanut, where only SSR markers were exploited, the minimum similarity coefficient was in the range of 0.3250-0.3438, and the average similarity coefficient ranged from 0.6149-0.6550 (Hu et al. 2013;Yu et al. 2017;Guo et al. 2020). However, in contrast to co-dominant markers, dominant markers tended to underestimate genetic diversity (Qian and Ge 2001), thus making a comparison between level of genetic diversity in the present study using AhMITE markers with that in the previous studies using SSR markers difficult. In the present study, with the 38 AhMITE markers from at least 10 of the 21 (C 47.62%) linkage groups of the cultivated peanut (Shirasawa et al. 2012b), it was still possible to assess the genetic diversity of high-oleic peanut genotypes from representative breeding teams at gene, population and group levels.
In this study, of all the 8 populations, lines from CTW ranked first in genetic variability, followed by lines from C&Y, and cultivars from CTW occupied the third position. CTW team and C&Y team have succeeded in broadening the gene base in high-oleic peanut, as new lines from both teams had higher levels of genetic diversity. Over 90% of the total genetic diversity could be ascribed to within population diversity. Like the situation in the 8 populations, as shown by G st of individual groups, for each group, within populations variation predominated (over 70% of total).
With the identification/creation of new high-oleic peanut mutants in CTW team (Nkuna et al., 2021;Wang and Zhu 2017), more and more natural and induced mutants were used in breeding. Also, more exotic germplasm lines were used in hybridization in the development of these lines. These were the reasons why the lines from CTW team had the highest genetic diversity and why the lines and mutants from the team had a close relationship. Since all of the 3 cultivars from YRL used the same high-oleic peanut donor (Kaixuan 01-6) from JZG team (Wang and Zhu 2017), cultivars from YRL and JZG were also closely related.
Compared to other types of molecular markers, handling of AhMITE markers in the laboratory is much easier and only requires fewer resources (Gayathri et al. 2018). AhMITE markers have shown considerably high polymorphisms . Thus, they have been considered a potent and convenient tool in genetic research. Since the genotypes used in the present report were of diverse origin, the generated information may provide a basis for Fig. 2 Dendrogram of 8 populations using Nei's (1972) genetic distance and UPGMA method further association studies on agronomically important traits. Using AhMITE markers, this study demonstrated the possibility of broadening the narrow gene base of high-oleic peanut, which is of global importance. In a world with erratic climates, it is imperative to explore novel germplasm resources of diversity and useful alleles to develop climate-resilient high-oleic peanut varieties. AhMITE markers may find wide use in the appraisal of parental lines, and marker-aided selection in peanut.
To summarize, much variability resided within populations in this study. Of the high-oleic peanut cultivars, those from CTW team had the highest genetic variability. Of all the high-oleic peanut lines tested, those from CTW team ranked first in genetic diversity. As compared with high-oleic cultivars from CTW and C&Y teams, greater genetic diversity was detected in new lines of both teams, indicating that recent breeding efforts were effective in improving the genetic diversity of high-oleic peanuts.