*Article* **Ultra-Orthodox Nursing Students' Cultural Challenges Inside and Outside Their Community during the COVID-19 Pandemic**

**Sara Genut 1,\*, Yaacov G. Bachner 2, Zvika Orr 3 and Adi Finkelstein 3**

**Abstract:** In line with findings that nurses from minority groups have an important role in making health services accessible to their community, our study aimed to identify the challenges ultra-Orthodox Jewish nurses faced during COVID-19 in their encounters with patients and health staff from other communities, as well as their own community. The ultra-Orthodox community is a highly religious group that maintains isolation from general society, a phenomenon that affected its member experiences during COVID-19. Our research followed sequential explanatory mixed methods. The quantitative phase included a questionnaire completed by 235 female students (111 ultra-Orthodox and 124 non-ultra-Orthodox), followed by a qualitative phase, which included six focus-groups (*n* = 15). The quantitative analysis showed that the ultra-Orthodox students felt a higher sense of responsibility toward their community. They used their authority and knowledge to guide their community during the pandemic. The qualitative analysis identified two themes expressed as challenges ultra-Orthodox nursing students encountered within their community and with other sections of Israeli society. Our research shows the important role that transcultural nurses play in mediating updated health information otherwise inaccessible to their community, especially in times of crises. It is important to address dilemmas this group faces inside and outside their respective communities.

**Keywords:** ultra-Orthodox; transcultural; minority; cultural competence; COVID-19; mixed methods; nursing students

#### **1. Introduction**

Nurses must recognize patients' needs based on their cultural background [1]. The recommendations of culturally competent baccalaureate nurses to be achieved during nursing education include awareness of personal culture, values, beliefs, attributes, and behaviors; skills in communicating with individuals from other cultures; and cultural knowledge and opportunities for students to interact with different cultures [2–4].

Providing culturally competent healthcare to diverse populations is important for reducing health inequalities and loss of critical information [5,6] and increasing patient satisfaction, which is a key factor in quality of care and correlated with patient compliance [7]. Studies have shown that language barriers [8] and lack of trust in the healthcare system [9] were reasons why minority populations were dissatisfied with the healthcare services they received [10]. Instead, they were likely to be satisfied with the healthcare services received from providers with the same race or ethnicity [11,12].

Considerable research has been performed on transcultural nurses as essential frontline workers providing culturally appropriate services to minority groups [13–16]. Transcultural nursing and ethnic minority nurses are attracting attention because of their central role

**Citation:** Genut, S.; Bachner, Y.G.; Orr, Z.; Finkelstein, A. Ultra-Orthodox Nursing Students' Cultural Challenges Inside and Outside Their Community during the COVID-19 Pandemic. *Int. J. Environ. Res. Public Health* **2022**, *19*, 9215. https:// doi.org/10.3390/ijerph19159215

Academic Editors: Richard Gray and Sonia Udod

Received: 17 June 2022 Accepted: 25 July 2022 Published: 28 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in making medical care accessible to patients from diverse cultural backgrounds, and especially to patients in their community [14,15,17–19].

One of the goals of the US National Coalition of Ethnic Minority Nurse Associations, specifically highlighted during the COVID-19 pandemic, includes increasing the number of ethnic minority nurses because of their special contribution to society [20]. Dawson (2021) [17] described AA/Black nurses as frontline healthcare providers to patients in hospitals and nursing homes during this pandemic. Given their large network, they were among the first to inform and educate the AA/Black communities regarding COVID-19, working with politicians, social service agencies, healthcare systems, public health departments, etc.

#### **2. The Aim of the Study**

Our study aim was to examine the challenges of female Jewish ultra-Orthodox (UO) nursing students in encounters with other communities and their roles within their own community, especially during the COVID-19 pandemic.

Unlike other minorities, the ultra-Orthodox community stays isolated from the general society as part of religious ideology to preserve its traditional beliefs and behavior, a phenomenon that has both social and economic implications [21,22]. These implications influenced the experiences of the community during the COVID-19 pandemic. Ultra-Orthodox people made COVID-19-related decisions based on both health and medical rationalizations and religious considerations, and among them, obedience to their community leaders [23]. This community possesses characteristics typical of minority groups, which make it difficult to cope with the COVID-19 pandemic, such as large families, low socioeconomic status, and crowded living conditions [24]. This was part of the reason why a disproportionately large number of UO people contracted this disease and died [25].

#### **3. Background**

#### *3.1. The Characteristic of the Ultra-Orthodox Community in Israel*

According to the [26] (p. 40), in the general Jewish Israeli population of 2016, 14% defined themselves as UO, 16% as religious or very religious, 25% as traditional, and 45% as secular.

Important factors to consider in relation to religion and health are perceptions, behaviors, and access to health-related information [27]. First, the UO community lives an intense social and community life, built around prayers in synagogues, studying in groups, and multi-participant social events [24,28–30]. To keep themselves isolated from the influence of the so-called secular culture, they object to the use of virtual media and do not use TV or smartphones [31,32]. This factor was crucial in coping with COVID-19 as they lacked access to updated information about the virus and about measures taken by authorities, such as physical distancing. Due to their ideological non-use of the Internet, they were unable to use services remotely, such as food shopping, work, healthcare, and distance learning [33].

Second, there is a science literacy gap between UO and secular education, as sciences are either not taught or taught at a minimal level in UO schools. Moreover, relevant scientific knowledge is accessible nowadays in the media, yet, due to their ideological desire to remain isolated from the secular society, their members forsake the Internet, thereby lacking access to updated scientific knowledge and information [23,34].

Finally, the UO community mistrusts state authorities, which are perceived as interfering with their social isolation [31]. Thus, official instructions regarding COVID-19 were taken by this community with suspicion. Ultra-Orthodox leaders significantly shape believers' perceptions in ways that affect health promotion and use of health services [23]. They influence the health behaviors as well as health-related decisions of individuals, often using "health mediators" between the UO community and professionals and service providers outside the UO world [35].

#### *3.2. Ultra-Orthodox Jewish Nurses in Israel*

Ultra-Orthodox Jewish nursing students represent a new population in the nursing profession in Israel. This phenomenon derives from the recent changes and needs of the UO society [36,37]. Women are the main providers in UO households because men devote their lives to religious study [38,39]. These women usually get married young and lack professional qualifications, which results in low-paying jobs. Therefore, for them, nursing studies offer an opportunity to acquire guaranteed employment and a stable source of income [40].

Additionally, there is growing awareness that community clinics and hospital wards must properly meet UO unique needs to maximize cultural competence and gender access [41–43]. This need was also recognized by the government, especially during the COVID-19 pandemic [44], and, therefore, there is a need for both male and female ultra-Orthodox nurses. As part of the national plan for integrating the UO population in academic nursing studies, several schools in Israel provide dedicated, gender-segregated programs to UO men [36] and women [37].

Nevertheless, nursing is not yet an acceptable profession in this community because it challenges a few of their religious norms, and the entry of UO men and women into the nursing profession entails multiple challenges [36,37]: (1) This community generally opposes integrating in academic studies; (2) Nursing is a science-based discipline, and potential students need a strong foundation of science. The elementary and high school programs of the UO education system lack knowledge and skills in many domains; and (3) The *Halakhah* (Jewish law) specifies strict sex-based roles to avoid physical or other connection between men and women [45].

Our study advances the understanding of the experiences of nurses from ethnic minority populations who serve the majority population, and, at the same time, could take advantage of their profession to help their minority community [20]. Research questions include:

#### **4. Materials and Methods**

#### *4.1. Study Settings*

All participants were female nursing students at the Jerusalem College of Technology (JCT) [46]. JCT specializes in providing science and technology education to the religious Jewish community and about 45% of JCT's 4700 students are UO Jews; among them, 55% are women. Nursing is the largest department of the institute, and out of the nursing students, 88% were women. Men and women study in separate campuses according to their religious norms. All research participants studied a unit in cultural competence as part of a mandatory first-year academic course named "Sociology of Health and Illness". The course was monitored by the last author; classes were supplemented by theoretical reading and class discussions.

#### *4.2. Study Design*

Our main research tools included a questionnaire and focus-group interviews according to sequential explanatory mixed methods [47]. According to this strategy, the analysis of quantitative findings was followed by the analysis of focus-group interviews to explain the quantitative findings.

#### *4.3. Data Collection*

#### 4.3.1. Quantitative Phase

For the quantitative phase, we composed a questionnaire comprising 14 items ranked on a 1–10 Likert-scale, (ranging from "strongly disagree" to "strongly agree"). We adopted six items from the Hebrew version of the RCTSH Cultural Competence Assessment Tool (CCATool), student nurse specific, which consists of four sections measuring cultural awareness, cultural knowledge, cultural sensitivity, and cultural practice [48]. The questionnaire

focused on the importance accorded by the students to the need of supplying medical information and guidelines with sensitivity to the beliefs, values, and norms of patients of different cultures, specifically in light of the experience during the COVID-19 pandemic. The questionnaire included items to assess the insights of the UO students, who constitute a large proportion of students at the college. Of the 235 female respondents, 111 (47%) were UO.

The questionnaire was administered to nursing students at JCT in June 2020, and 285 of them completed the questionnaire. We statistically analyzed the responses of 235 female students who completed the questionnaire. Fifty questionnaires were omitted from the study because they had a high percentage of missing data.

#### 4.3.2. Qualitative Phase

Six focus group were conducted between January and February 2021, with two to three students in each group (*n* = 15). The participants were all UO female nursing students from all four years of the nursing program at JCT. Each group contained students from the same school year. Purposive sampling was performed and participants were recruited by the first author who knew the participants well and thought that they would contribute to the research. Six students refused to participate in the study.

Each focus group interview lasted 60–90 min, conducted using a script designed under the guidance of the last author, a qualitative research expert. Following our quantitative phase results, the questions were specifically related to participants' dilemmas around the decision to study nursing and their community's response to that decision, as well as students' experiences of encountering different cultures during their clinical rotations, most of which occurred during the COVID-19 pandemic. The first group was a pilot with three students from the second, third, and fourth years; their insights helped finalize the script questions.

All focus group were conducted online (via Zoom) and recorded with the participants' consent. Audio recordings were later fully transcribed by a professional transcriptionist. Transcripts were compared to audiotapes to ensure accuracy by the last author.

#### *4.4. Data Analysis*

#### 4.4.1. Quantitative Phase

We conducted factor analysis using a varimax technique to define the categories within the questionnaire items. Twelve of the original fourteen items were categorized into four factors that were significant to the research questions.

Confidence in coping with the effects of racism at work as a nurse; sense of mission as a nursing student in using knowledge and authority in their community; cultural identity and community belonging; and cultural sensitivity. Cronbach's α values for each category showed good internal consistency. Table 1 presents the items of each category, the number of items for each factor, and Cronbach's α.

#### 4.4.2. Qualitative Phase

We chose an inductive approach, which means that the themes identified were closely linked to the data without trying to fit them into an earlier analytic theory [49]. We utilized thematic analysis—a recommended technique for analyzing focus group transcripts [50]. The six phases of thematic analysis as described by Braun and Clarke (2006) [51] were followed: (1) familiarizing with the data; (2) generating initial codes; (3) searching for themes; (4) reviewing themes; (5) defining and naming themes; and (6) producing the report. Phases one and two were conducted individually by the first and the last authors. The initial codes were compared at joint meetings. After comparing the two lists and reaching an agreement, a joint list was prepared. The third and fourth phases were jointly conducted by the two authors and shared with team members for feedback. The fifth and sixth phases were performed by the last author. The analysis was performed manually without the use of any software [52].

**Table 1.** Categories, Items, and Cronbach's α.

#### *4.5. Trustworthiness*

To establish the trustworthiness of the present study, we implemented various strategies. *Consistency* was achieved with all focus group sessions being conducted by a single researcher with a single set of questions during a short period [53]. *Credibility* [54] was achieved through practices, including: (1) *a thick description*: we provided in-depth illustrations and abundant details about the culture and social context of our study; (2) *triangulation*: we used multiple coding. Two researchers analyzed the data independently and compared their findings [55]; and (3) *multivocality*: we provided empathic understanding and space for a variety of opinions. To facilitate *transferability* [53], we reported in the article clear descriptions of the research context, selection procedure, characteristics of participants, data collection, and the process of analysis.

#### **5. Results**

#### *5.1. Quantitative Phase*

A series of independent *t*-tests were conducted to examine any statistically significant differences between the UO students and the general students in the factors identified in the questionnaire. Table 2 presents the results of independent *t*-tests.

**Table 2.** Independent Samples *t*-Test (Factors).

\* *p* < 0.05.

As presented in Table 2, there is a statistically significant difference between the two groups in terms of the *sense of mission as a nursing student in using knowledge and authority in their community* (*p* = 0.01; t (233) = 2.46). The UO nursing students revealed a higher sense of mission toward their community (M = 6.67, SD = 2.46), compared to other nursing students. Differences related to other factors were not statistically significant between the groups.

Additionally, we conducted the Spearman's correlation coefficient test to examine correlations between the various factors regarding the school year of the female students in the nursing study program. The results are shown in Table 3.

\* *p* < 0.05, \*\* *p* < 0.01.

As presented in Table 3, there is a significant positive correlation of the school year with the *sense of mission as a nursing student in using knowledge and authority in their community* factor (*p* < 0.01; r = 0.28). This correlation signifies that as the student advances with nursing studies, the sense of mission toward her community increases. Additionally, there were significant positive correlations between the *sense of mission as a nursing student in using knowledge and authority in their community* factor and the *confidence to cope with the effects of racism at work as a nurse* factor (*p* < 0.05; r = 0.13); the *cultural identity and community belonging* factor (*p* < 0.05; r = 0.27); and the *cultural sensitivity* factor (*p* < 0.05; r = 0.14). Moreover, there was a significant positive correlation between the *confidence in coping with the effects of racism at work as a nurse* factor and the *cultural sensitivity* factor (*p* < 0.05; r = 0.40).

#### Additional Results

We also conducted Levene's test for equality of variances of each item of the questionnaire to identify any significant statistical differences in specific items between the UO and other students and the non-UO students. The results are presented in Table 4.

**Table 4.** Independent Samples Test (Items).

\* *p*-value < 0.05; \*\* *p*-value < 0.01.

As presented in Table 4, statistically significant differences exist between the UO and the non-UO students in four items: *The pandemic has made me critical of other people's attitudes towards my community* (*p* < 0.001); *I used my authority as a nursing student to encourage others in my community to obey the instructions of the Ministry of Health during the pandemic* (*p* < 0.01); *I used my knowledge as a nursing student to guide others in my community on how to behave properly during the pandemic* (*p* < 0.01); and *I am very confident to challenge racism and discrimination toward clients* (*p* < 0.05).

#### *5.2. Qualitative Phase*

Two themes were identified in the analysis as follows (see Table 5 for quotations):

**Table 5.** *Cont.*

**Table 5.** *Cont.*

5.2.1. Challenges in the Encounters Ultra-Orthodox Nursing Students Experience within Their Community

*Students had to overcome objections within their community in relation to exercising the students' wish to enter nursing school.*

Most of the participants shared that they faced objections to academic studies from within their close community (high school teachers, friends, cousins, etc.). Some of them specifically faced objections against entering the nursing field and had to keep their plans secret until they began their studies. They all asked for permission from their parents and their rabbi prior to enrolling in nursing school. When asked, participants strongly recommended that other girls from their community study nursing. However, they did not see themselves as activists for social change for girls or women in their community. Rather they emphasized their personal interest in nursing and the mission they see in the nursing profession.

*Students expressed ambivalence about their role as health information providers to their community.*

Although they encountered objection, participants said that they are highly valued in their community for having studied nursing. Participants noted that people considered them as health authorities and sought their advice regarding health, even though they were still students. Participants explained they were flattered as it raised their confidence and self-pride. They felt obliged to share the health and medical information they acquired in the nursing school with their community. Nevertheless, participants expressed ambivalence since they hesitated to answer medical questions because, as they said, they are still students and preferred to direct such questions to a professional, such as a nurse or physician.

*Students described having mixed feelings in relation to providing care to patients from their community.*

Participants emphasized their mission as UO nurses familiar with the cultural sensibilities of their community. Participants mention their responsibility to help patients from their community feel comfortable when they arrive at general hospitals and clinics. However, a few of the participants noted that the cultural background they share with patients from their community was not always beneficial. Under certain circumstances, clinical procedures may create situations that conflict with cultural norms and embarrass both parties. For example, in cases where they needed to touch or even talk to young UO men, where both parties were aware of the restrictions about relations between men and women in UO communities. The participants explain how they overcome these difficulties and emphasized that in moments of embarrassment, they focus on their task and remind

themselves that everything they are doing is professional with the purpose of helping the patient.

#### *Students mediated reliable health and health behavior information to their community at the time of the COVID-19 pandemic.*

The pandemic increased the participants' sense of responsibility for conveying medical information to their community. They described their role during the COVID-19 pandemic amid massive confusion among their community members who have less exposure to smartphones, the Internet, television, and radio. Community members therefore sought and relied on COVID-19-related information from participants. Participants explained that they were surprised to see that the guidelines of the Ministry of Health were often misleading and sometimes contradictory of UO community customs. For example, during the holidays that occurred at the beginning of the epidemic, the directives of the Ministry of Health were to maintain social isolation and the UO people wanted to observe their custom of offering prayers in public. Participants described mediating for their close community between the formal guidelines of the Ministry of Health and those given by community leaders.

5.2.2. Challenges in the Encounters of Ultra-Orthodox Nursing Students with Other Sections of Israeli Society

*Students appreciated the opportunity to challenge their perceptions about others outside of their community but also mentioned the difficulties involved.*

The experiences of nursing students during their clinical training provided them with the opportunity, sometimes the first opportunity they had experienced, to meet people from other sections of Israeli society, such as nonreligious Jews and non-Jews (i.e., patients from the Arab sector). Participants discussed how this experience was sometimes surprising and made them more open-minded although the encounters were challenging because of exposure to different—sometimes contradictory—values, norms, and customs. Few participants described feeling more comfortable with people from their own community who they treated more warmly—than other patients, who they treated "by the book".

*Students encountered racism during the COVID-19 pandemic, although they raised criticism towards the behavior of the members of their community.*

Interactions with patients and staff from various communities of the Israeli society exposed the participants to prejudices toward their community. Participants said that they learned for the first time that the public blames the UO community for the spread of the virus because they did not maintain social distance in the first period of the of the COVID-19 pandemic. The participants said that they are not always comfortable when others see them as representatives of the ultra-Orthodox society. For some participants, the exposure to stigmas about their community increased their sense of identification with their community. These participants were angry with the critical attitude of outsiders, since, as they said, the guidelines did not always reach the ultra-Orthodox community. Some saw their role as nurses as an opportunity to change these prejudices against their community. However, a few criticized the behavior of their community members during the pandemic.

#### **6. Discussion**

Previous studies have addressed the factors related to multicultural nurses' experiences in encounters with the general population, as well as factors related to their work with patients from minority groups, especially from their community [14,15,17–19]. This study focused on female UO nursing students, their challenges, and dilemmas in their encounters with the general Israeli population, as well as their own community members, especially during the COVID-19 pandemic.

The nursing profession is noble, as nurses save human lives—a value highly regarded in the Jewish tradition and especially in the UO community [56]. However, the UO female nursing students are exceptional in their community that rejects academic pursuits. Their vocational nursing training in school, and later their profession, require extensive encounters with different worldviews, which can lead to a clash with their lifestyles [36].

*Ultra-Orthodox nursing students' sense of mission*

The participants sense of mission was the main finding in both the quantitative and qualitative analyses in our study. Arieli and Hirschfeld (2010) [57] found that, compared to Palestinian–Israeli nursing students, most Jewish students' reason for choosing nursing was an inner desire to serve. Pragmatic considerations for choosing nursing were mentioned but were usually presented by Jewish students as a relatively minor motivation. The quantitative phase of our study showed how the UO nursing students revealed a significantly higher sense of mission toward their community compared to the other nursing students in our study [t (233) = 2.46, 95% confident interval of the difference 0.15–1.39]. In the focus groups, participants emphasized nursing as a mission to help and serve others. They described their mission to provide reliable health-related information to their community. This was especially noticeable during the COVID-19 pandemic, which exposed the trust deficit between their community and health authorities [27].

Entering nursing school gives UO students an opportunity for social mobility [58]. They acquired higher education, and it will help them find a stable and well-paying job in the future, which will improve their social status [59,60]. Nursing education also gives participants in our study a chance for social mobility within their community because they hold valuable social capital: their professional—and potentially lifesaving—knowledge about health issues [14,15,19]. However, although these students are pioneers of their community in terms of entering nursing school, the participants in the focus groups emphasized not regarding themselves as "flag bearers" of social change for other women in their community. They stressed that they entered nursing school after receiving approval from the rabbinical authority and their parents. They reported remaining committed to the values of their community and stated that their choice of the nursing profession was not driven by a desire for social mobility. They were happy to recommend this direction to other UO girls.

#### *Ultra-Orthodox nursing students' culture competence challenges*

Our study shows the dilemmas and challenges that the UO nursing students faced in their intercultural encounters with the general society, coming—as they did—from a closed, distinct group that faithfully upholds religious and traditional values and practices [61]. At the same time, they discussed their struggle when facing patients from their own community. Their deep acquaintance with the cultural norms of the UO community brings the UO participants to tread cautiously so as not to offend these patients.

The findings of studies on changes in cultural competence over the years in nursing schools are contradictory. A few studies indicate that there has been no change over the years [62,63], whereas others show that there has been a change [64]. The cultural competence factors examined in our study sample, especially the sense of mission, increased with advances in nursing studies. This was true for the UO participants, as well as the non-UO participants. It is in line with the significant positive correlations between the examined factors, among themselves, and in relation to the school year of the program studies, as it is well confirmed by Spearman's correlation coefficient test.

Utilizing the CCATool [48], Repo et al. (2017) [4] found cultural competence to be positively associated with Finnish nursing students' minority backgrounds and frequency of interacting with different cultures. Following the above finding, we assumed that in our study, as students' progress academically, they perform more practical training in hospitals and in the community and interact with a diverse population both among patients and staff, which enhances their sensitivity toward other cultures. The academic progress of our UO participants also enhances their sense of responsibility toward their community.

Repo et al. (2017) [4] also reported that students with a minority background had higher cultural competence than general Finnish students. This has also been reported by reported by Mesler (2014) [65]. We agree with Repo et al.'s ([4], p. 102) explanation that "this may be due to the fact that minority background students already are culturally aware since they have had to reflect on their own ethnic and cultural identity while living as foreigners in Finland." In our focus groups, the UO participants declared that they believe in equal medical care for all people and that they follow this tenet at work. Compared to other students in our sample, the UO participants were not only more sensitive to the issue of racism toward patients, but they felt more confident in dealing with it. It was manifested in a statistically significant difference between the UO and the non-UO students in the quantitative phase in the item: *I am very confident to challenge racism and discrimination toward clients*. However, they did not state that they were trying to develop awareness or acquire knowledge about patients from other communities.

*Ultra-Orthodox nursing students in the COVID-19 pandemic*

Their clinical experience, especially when the COVID-19 pandemic first appeared, exposed our study participants to the ignorance of parts of their community members about health issues, their lack of access to the latest and most reliable information due to cultural–religious restrictions, and the serious consequences that all of these had on the community members' health.

Previous studies [14,15,17,18] have noted language and communication problems as barriers, especially for minorities, in gaining access to health services. The UO Jews constitute a minority group in Israeli society, although most of them speak Hebrew, which is the common language of Israel [66]. However, UO communities lack health literacy [27], which refers to "the degree to which individuals have the ability to find, understand, and use information and services to inform health-related decisions and actions for themselves and others" [67].

The participants in the focus groups shared that their community members recognized them as a source of reliable medical information, and they often made the guidelines of the Ministry of Health accessible to their community. It is in line with the findings of the quantitative phase, which showed significant differences between the UO and the non-UO students in the items: *I used my authority as a nursing student to encourage others in my community to obey the instructions of the Ministry of Health during the pandemic; I used my knowledge as a nursing student to guide others in my community on how to behave properly during the pandemic*.

The COVID-19 period accentuated for the participants not only their roles within their community, but also what the public thought about their community. The UO community faced instances of racism during the COVID-19 pandemic. There were cases where people blamed them for spreading the disease by disobeying the official instructions for limiting the spread of the virus [27].

Some of the UO participants in our research were exposed, due to their work as nurses, to the prejudices about their community for the first time, largely because the UO community stays isolated from the general society as part of their religious ideology. The UO students described how patients regarded them as representatives of the UO society, which was not always appropriate, especially when their community was blamed for the spread of the disease. It is in line with the statistically significant difference between the UO and the non-UO students in the quantitative phase in the item: *The pandemic has made me critical of other people's attitudes towards my community.* Moreover, a few participants in the focus groups recounted the comments of the medical staff, especially nurses who worked with them. Iheduru-Anderson et al. (2021) [68] (pp. 122–123) found that "racism in nursing remains under-investigated. There is a collective denial and a culture of silence around racism, which perpetuate systemic racism and its consequences on the nursing profession . . . ". Following their experience, the UO participants in our focus groups acknowledged the importance of treating members of their own community with greater warmth because of the difficulties and barriers they face in the society.

#### *Limitations*

This study has several limitations. First, the small number of items in the questionnaire limits the number of identified factors in the factor analysis. Hence, significant differences between the UO and the non-UO students in specific items, which are relevant to the research questions, are found in Levene's test for equality of variances of each item but are not revealed in the comparison of the factors between the two groups. Using a larger number of items might define factors, which would include the "missed" items within the categorized groups of items. However, the factors are highly supported by the themes identified in the analysis of the qualitative phase of this study. Second, in the quantitative phase, we compared the UO students—this study's focus—with the non-UO students in the same college. However, the latter are also religious Jews and do not represent the secular section of Israeli society. This might limit the generalization of the study. Our assumption is that because the non-UO participants in the study resemble general Israeli women in terms of their previous education and other social variables, such as participating in the military service (or national service instead) [69], they are integrated into Israeli society, and, therefore, can represent a control group for the UO participants.

#### **7. Conclusions**

Our research shows that transcultural nursing students play a decisive role in mediating updated and accurate health information to their community, especially at times of crises, from the initial stages of their training. However, they face dilemmas both inside and outside their respective communities. We believe that insights derived from this study can be utilized to build programs to address the challenges of nurses from minority communities and transcultural nurses. At the policy level, we join the call of Drach-Zahavy et al. (2021) [70] to prepare in advance a crisis plan for nursing schools that could be rapidly implemented, and to incorporate in the nursing curricula crisis-related topics, such as developing flexible coping strategies. We propose that these policies should leverage the substantial potential of transcultural nursing students in working with their communities and consider these students' distinct needs and dilemmas. This will help channelize the social role of transcultural nurses for conveying otherwise inaccessible information to isolated communities.

**Author Contributions:** Conceptualization, S.G. and A.F.; Methodology, S.G. and A.F.; Validation, S.G., A.F., Y.G.B. and Z.O.; Formal Analysis, S.G., A.F., Y.G.B. and Z.O.; Investigation, S.G. and A.F.; Resources, S.G. and A.F.; Writing—Original Draft Preparation, S.G. and A.F.; Writing—Review & Editing, S.G., A.F., Y.G.B. and Z.O.; Supervision, S.G. and A.F.; Project Administration, S.G. and A.F.; Funding Acquisition, S.G. and A.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financed by the Jerusalem College of Technology.

**Institutional Review Board Statement:** The study was conducted with the approval of the Jerusalem College of Technology Ethics Committee No. 011\_20. All participants signed consent forms and were assured of complete confidentiality and anonymity. Participation in the study was voluntary.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The study did not report any data (all data is reported in the article).

**Acknowledgments:** The authors would like to acknowledge the nursing students who took part in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article* **Gluten-Free Breadsticks Fortified with Phenolic-Rich Extracts from Olive Leaves and Olive Mill Wastewater**

**Paola Conte 1,\*, Simone Pulina 1, Alessandra Del Caro 1, Costantino Fadda 1, Pietro Paolo Urgeghe 1, Alessandra De Bruno 2, Graziana Difonzo 3, Francesco Caponio 3, Rosa Romeo 2 and Antonio Piga 1**

**Abstract:** Nowadays, food processing by-products, which have long raised serious environmental concerns, are recognized to be a cheap source of valuable compounds. In the present study, incorporation of phenolic-rich extracts (500 and 1000 mg kg−1) from olive leaves (OL) and olive mill wastewater (OMW) into conventional gluten-free formulations has been exploited as a potential strategy for developing nutritious and healthy breadsticks with extended shelf-life. To this end, moisture, water activity (aw), visual and textural properties, the composition of biologically active compounds (soluble, insoluble, and bio-accessible polyphenols), antioxidant activity, oxidation stability, and consumer preference of the resulting breadsticks were investigated. Fortified breadsticks had higher moisture and aw, lower hardness, and similar color in comparison to the control, especially in the case of OL extract supplementation. All enriched formulations significantly affected the phenolic composition, as evidenced by the decrease in insoluble/soluble polyphenols ratio (from 7 in the control up to 3.1 and 4.5 in OL and OMW, respectively), and a concomitant increase in polyphenol bio-accessibility (OL: 14.5–23% and OMW: 10.4–15% rise) and antioxidant activity (OL: 20–36% and OMW: 11–16% rise). Moreover, a significant shelf-life extension was observed in all fortified breadsticks (especially in case of OMW supplementation). Sensory evaluation evidenced that 61% of the assessors showed a marked, but not significant, tendency to consider the sample supplemented with high levels of OL as a more palatable choice.

**Keywords:** breadsticks; gluten-free; olive oil by-products; antioxidants; oxidation stability


*δt* = (306.50, 348.60, 420.01) mm for three hazard levels

The building passes Life Safety (LS) performance under MCE level earthquake in case of 0.16 g and 0.18 g seismic hazard in Ahmedabad city for IS code-design case (refer **Figure 4**). Also, the building designed with IS code had performance of Collapse Prevention (CP) level while the building design with ACI-318 and EC-8 will collapse in 0.22 g hazard level. However, the location of failure hinge shown in **Figure 5**, describes the better behavior of structure designed using US and EN code provisions for buildings as only the base columns are failing. The columns at 4th and 7th level are failing in IS design case, hence the energy dissipation in the graph is poor. Important to note the strength of building designed as per IS code is highest and ductility achieved is more than the other design codes though the location of failure is not acceptable.

**Figure 4.** *Comparison of capacity curve of building as per design codes.*

**Figure 5.** *Location of failure hinge at the end of POA. (a) IS design. (b) ACI design. (c) EC8 design.*

coefficient method (DCM) is a better means to employ performance-based design procedure (PBD) as displacement gives better control. The target

level (refer Eq.(3)).

failure is not acceptable.

*Comparison of capacity curve of building as per design codes.*

*Location of failure hinge at the end of POA. (a) IS design. (b) ACI design. (c) EC8 design.*

**Figure 4.**

**Figure 5.**

**216**

displacement ð Þ *δt* is estimated to obtain nonlinear displacement limit at structure

*T*2 *e*

4*π*2 *g* (2)

*δt* ¼ *C*0*C*1*C*2*Sa*

The building passes Life Safety (LS) performance under MCE level earthquake in case of 0.16 g and 0.18 g seismic hazard in Ahmedabad city for IS code-design case (refer **Figure 4**). Also, the building designed with IS code had performance of Collapse Prevention (CP) level while the building design with ACI-318 and EC-8 will collapse in 0.22 g hazard level. However, the location of failure hinge shown in **Figure 5**, describes the better behavior of structure designed using US and EN code provisions for buildings as only the base columns are failing. The columns at 4th and 7th level are failing in IS design case, hence the energy dissipation in the graph is poor. Important to note the strength of building designed as per IS code is highest and ductility achieved is more than the other design codes though the location of

*δt* = (306.50, 348.60, 420.01) mm for three hazard levels

*Natural Hazards - Impacts, Adjustments and Resilience*


Constraints in DPF at Present

Following [15] and analogous to [14], we use in DPF until now, instead of sketch constraints in the sense of Definition 29, only plain sketch arrows *ϕ* : L → R and call them (positive) universal constraints or negative universal constraints, respectively [18,21]. We define the satisfaction of universal constraints in DPF by means of the closedness property in Definition 23. That is, a sketch G satisfies the "universal constraint" *ϕ* : L → R if, and only if, for any strict sketch arrow *τ* : L → G there is a strict sketch arrow : R → G such that *ϕ*; = *τ*.

By Proposition 7, we can transfer many findings in Sections 5.2.3 and 5.2.4 into the sketch constraints setting. Corollary 6 and Definition 28 ensure that the satisfaction of a universal constraint *ϕ* : L → R in a sketch G can be equivalently expressed by the assertion that G satisfies the general constraint (*uc*, !*G*) with (compare Corollary 3):

$$\mathcal{g}\mathcal{c} := \mathbf{0} \blacktriangleright \forall (L: (\bigwedge \mathcal{S}t^{\mathbb{L}} \to \exists (\varphi, \mathcal{R}: \bigwedge \mathcal{S}t^{\mathbb{R}}))).$$

Be aware that the identifier *ϕ* in *gc* does not refer to the sketch arrow *ϕ* : L → R but to the underlying context morphism *ϕ* : *L* → *R*. Note further that we can replace *St*R by (*St*R \ *ϕ*(*St*L)) without losing the equivalence!

Furthermore, we say that a sketch G satisfies the "negative universal constraint" *ϕ* : L → R if, and only if, for any strict sketch arrow *τ* : L → G, there does not exist a strict sketch arrow : R → G such that *ϕ*; = *τ*. This requirement is equivalent to the statement that G satisfies the general constraint (*ngc*, !*G*) with:

$$\mathfrak{mgc} := \mathbf{0} \blacktriangleright \forall (L : (\bigwedge \mathcal{S}t^{\mathbb{L}} \to \neg \exists (\mathfrak{q}, \mathcal{R} : \bigwedge \mathcal{S}t^{\mathbb{R}})))$$

What can we do if a sketch G does not satisfy a general constraint (*c*, !*G*) for a simple condition of the form *c* = **0** ∀(*L* : ( *St*1 → *∃*(*ϕ*, *R* : *St*2))) where *St*1 is a set of statements in *L* and *St*2 a set of statements in *R*, respectively?

We can repair this flaw by applying the sketch arrow *ϕ* : (*L*, *St*1) → (*R*, *St*2 ∪ *ϕ*(*St*1)) as a transformation rule for all sketch morphisms *τ* : (*L*, *St*1) → G not satisfying the conclusion in condition *c*. In other words, a match of the transformation rule is given by a context morphism *τ* : *L* → *G* such that G |= ( *St*1, *τ*) and G |= (¬∃(*ϕ*, *R* : *St*2), *τ*). Note that the negative application condition G |= (¬∃(*ϕ*, *R* : *St*2), *τ*) ensures that we do not apply the rule twice for the same match *τ* : (*L*, *St*1) → G. Applying the rule *ϕ* via the match *τ* means nothing but to construct a pushout in the category Sk*a s* of sketches and strict sketch arrows (compare Remark 32).

(*L*, *St*1) *ϕ τ PO* (*R*, *St*2 ∪ *ϕ*(*St*1)) *τ*∗ G *ϕ*∗ H

Depending on the properties of the context morphism *ϕ* : *L* → *R* the pushout construction may have different effects. The context *G* can be extended and/or factorized and, if *St*2 = ∅, we will add new statements to the statements originating from G.

In terms of sketch constraints, we can describe the crucial effect of the rule application as follows: H satisfies the constraint ( *St*2, *τ*∗) in addition to the constraint ( *St*1, *τ*; *ϕ*∗) inherited from G.

**Example 52** (Repairing Stm*CT*-sketches)**.** *As discussed in Example 51, there is one violation of the general constraints* (*guc*, !*G*) uniqueness of composition *by the* Stm*CT-sketch* G = (*G*, *St*G) *in Example 49 and one violation of* (*ct*3, !*G*) decomposition of monomorphisms*. Repairing these two violations by pushout constructions, as described above, will result in a* Stm*CT-sketch* H *like the one visualized below.*

G *also does not satisfy the general constraint* (*gec*, !*G*) definedness of composition *and the general constraint* existence of identities *that has not been formalized in Example 51. We do not want to require that any* Stm*CT-sketch satisfies these two general constraints since we do not intend to use* Stm*CT-sketches just as encodings of categories but rather as (hopefully finite) representations of (possibly infinite) categories. This is the original purpose of sketches in category theory. See also the later discussion in Remark 40.*


*4.1. Calcium*

In human health, calcium (Ca) is required in several systems, like musculoskeletal, nervous and cardiac. It is essential to maintain good bones, teeth, and mineral homeostasis. It also acts as a cofactor in many enzyme reactions and contributes to the function of the parathyroid gland [42]. The RDA of Ca ranges between 1000 and 1300 mg day−1. The UL for adults is 2500 mg day−1 [43]. Calcium is an important nutrient for plant metabolism, involved in structural functions of cell, acting as a counter-cation for organic and inorganic anions trafficking across the tonoplast and as an intracellular, cytosolic messenger [44]. It is one of most abundant nutrients in the earth's crust, with an average concentration of about 36.4 g kg−1[45]. Ca2+ concentration in the soil solution is usually enough (0.1–20 mM) to meet the plants' demands or, in neutral and calcareous soils, to exceed their requirement, thus leading to Ca accumulation in the vicinity or inside the roots [44]. However, some Cadeficient conditions can sometimes be encountered, especially in highly weathered tropical soils or in saline/sodic soils. Calcium is absorbed as divalent cation by the root apex and/or regions of lateral shoot initiation [46], where Casparian band between endodermal cells is absent or disrupted, and/or the endodermal cells surrounding the stele are not suberized [47]. Once inside the plant, Ca moves primarily through the xylem [46] with the water flow driven by transpiration [48,49], either as Ca2+ or complexed with organic acids [50]. However, Ca2+ movement inside the xylem vessels cannot be explained simply in terms of mass flow, as Ca2+ ions are also absorbed by adjacent cells and are complexed to nondiffusible anions in the xylem walls [48]. Due to its slow phloematic mobility, this element is present at lower concentrations in mostly phloem-fed organs (e.g., young leaves, fruits, and tubers) than in the older leaves ( ≈10-times less). Considering the mineral partitioning inside the plant, leafy vegetables can play a primary role in the dietary intake of Ca, so being possible targets for Ca biofortification [51]. This last point should be addressed at increasing the Ca content of the edible portions, without adversely impacting both plant growth and production costs [27]. Most plant species can accumulate high Ca contents in leaf laminae (up to 100 g kg−1 DW) without any symptoms of toxicity, because Ca exceeding plant's needs is detoxified by sequestering as insoluble Ca oxalate and deposited either in the cell wall or stored inside the vacuole [44,47]. Depending on the plant species, tissues, and growing conditions, Ca concentration in plants varies between 1 and >50 mg kg−1. However, some species may have insufficient detoxification mechanisms, so their growth can be severely depressed at high Ca tissue content [44]. Excessive Ca can cause toxicity symptoms such as the presence of yellow flecks formed by crystals of calcium oxalate and growth inhibition, the latter can be observed even in calcicole species (plants occurring in calcareous soils) when submitted to a soil solution

with a concentration higher than 10 mM Ca [46]. Strategies for Ca biofortification should include (i) increasing Ca supply to cells; (ii) increasing Ca uptake into cells; (iii) removing compounds making Ca unavailable and/or (iv) increasing Ca storage at the cellular and/or tissue level [27,52,53]. The application of Ca fertilizers can increase its concentration mostly in leafy vegetables (Table 1), whereas for grain, seeds, and fruits, sound indications are still to be reached. In 21-day old *Brassica rapa* plants grown on soil, the increased Ca supply to roots (compost mix supplemented with 0.4 vs. 3.5 g CaCl2 L−1) significantly enhanced its concentration in leaves (0.75 and 25 g kg−1 DW, respectively). The result was not influenced by the different supply of Mg fertilizer [54]. To reduce the effects of different soil characteristics (e.g., minerals concentration, pH) on Ca availability, soilless cultivation on inert substrates or water (e.g., floating system) allows a better control of the ion concentration in the root environment. In some leafy vegetables, D'Imperio et al. [24] increased the Ca concentration by adding calcium phosphate and calcium chloride in the nutrient solution (from 100 to 200 mg L−1), determining an increase of Ca concentration in leaves of basil ( ≈15%) and mizuna ( ≈12%), but not in tatsoi or endive (Table 2). Moreover, the biofortification process did not influence their oxalate content nor Ca bioaccessibility. A higher Ca content (up to 5-fold higher than control) in lettuce (*Lactuca sativa* L.) grown in a floating system was obtained by Borghesi et al. [55] with a nutrient solution containing 800 mg Ca L−1 (as CaCl2), compared to the control with no Ca addition. However, the high salt content increased both the Cl concentration and electrical conductivity of the nutrient solution, so reducing the marketable quality and yield ( −32%). Foliar applications of soluble Ca fertilizers are commonly made for several horticultural crops, to prevent Cadeficiency disorders. However, only few experiments refer to Ca biofortification through foliar applications. Moreover, these applications are expected to have limited effects on Ca content of roots, tubers, and seeds, because of the typical translocation patterns of the element. In one of few experiments, Yuan et al. [56] observed a significant increase of Ca concentration in lettuce sprayed three times every 20 days with 120 mg L−1 of CaCl2 compared to 60 and 180 mg L−1 (21.4% and 5.2%, respectively), although this effect was genotype-dependent. Overall, Ca biofortification of vegetables using Ca chloride proved to be effective in the majority of the studied leafy crops even if negative effects on yield cannot be excluded; besides, one of the main challenges is related to the presence of oxalate, which can partially limit Ca bioavailability.

**Table 1.** Response of some vegetable crops to biofortification (1).

**Table 1.** *Cont*.

(1) The list reports the most representative horticultural crops. In this and in the following tables, data refer to research on Scopus**®** using "biofortification" and "vegetables" as keywords performed in November 2020. Papers which tested more than one species were counted more than one time. (2) Calculated in the edible portion.

*Foods* **2021**, *10*,

**Table 2.** Chemical forms of each mineral used in the biofortification of some vegetable crops.

 223

initial and final concentration of mineral element in the edible part of vegetables.


**6. Medicinal plants with demonstrated anti-asthmatic activity**

Asthma is a complex inflammatory disease and congestive respiratory disorder brought about by airway narrowing. It symptoms may include episodic wheezing, cough and chest tightness resulting in airflow block. It leads to changes in the levels of eosinophils, mast cells, lymphocytes, cytokines and other inflammatory cell products. There is increased prevalence worldwide especially in industrialized countries and among children with increased morbidity and mortality rate [73, 74]. Medicinal plants have been screened for properties that enhance their activity as anti-asthmatic agents, since current medications have adverse side effects. Few of such plants with demonstrated activity are reviewed below.


**Abstract**

The high conservation of miRNA sequences provided an opportunity to develop an effective type of markers that is useful not only for genetic diversity study but also as potential biomarkers in plant stress responses. The fundamental potential of miRNA-based markers relies on the primer design based on the sequences of mature miRNAs, which are part of the step-loop structures. The advantages of this marker system include high polymorphism, reproducibility, and transferability across species. The abundance of mature miRNAs, which is linked to the expression of *MIRNA* genes, varies greatly among miRNAs, tissue types, or developmental stages, indicating the spatially and temporally regulated expression patterns of plant miRNAs. The results confirm the significance, reliability, and the position of miRNA-based markers as stress-sensitive biomarkers, indicating their potential in a wide range of applications of agricultural research.

**Keywords:** molecular markers, miRNAs, genotyping, stress-biomarkers

#### **1. Introduction**

Systematic documentation and evaluation of plant genome based on molecular markers that capture variability at the level of DNA or its protein products is essential for plant genetic resources management. These markers complement the morphological, agronomic, and other characteristics necessary to classify and identify plant genotypes within the species. Their importance and benefits are significant both for research and breeding, and for practice [1, 2]. The study and comparison of molecular information of individual organisms involves the search for DNA polymorphisms [3]. Polymorphisms are differences in DNA sequence caused by mutations. Molecular techniques in which polymorphisms can be visualized without sequencing are called molecular marker techniques [4]. A molecular marker is a specific fragment of DNA that can be identified within the whole genome and that is transmitted to the next generation following the standard rules of inheritance (Mendel's Laws). Marker may be located in or close to a gene or in noncoding regions.

The presence of polymorphisms between individuals will lead to a different pattern of markers after electrophoresis. These patterns are comparable with a "fingerprint"; therefore, these techniques are referred to as fingerprinting techniques.

These patterns reveal the DNA polymorphisms between the studied individuals. The more the individuals are related, the more their fingerprint will match. The level of polymorphisms in a group of individuals reveals the genetic diversity within this group [5].

There are different types of molecular markers. Molecular markers can be divided into two groups (a) biochemical markers, which detect variation at the gene product level such as changes in proteins and amino acids and (b) molecular markers, which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion, and/or insertion. DNA markers are based on hybridization or PCR (amplification of DNA) [3]. The multiplex ratio is defined as the amount of markers generated with one single reaction (e.g., one PCR, one hybridization reaction). The obtained pattern can be simple (one or a few bands, low multiplex ratio) or complex (a high number of bands, high multiplex ratio).

Molecular marker methods are either dominant or co-dominant. Using co-dominant marker techniques, the different genotype combinations can be distinguished from each other at the study locus (or multiple loci). This means that homozygous (two identical alleles at a certain DNA locus) and heterozygous (two different alleles at a certain DNA locus) individuals will be identifiable. In the case of a dominant technique, it is not possible to detect the alleles that are present at a certain locus/loci, so homozygotes are not distinguishable from heterozygotes [6, 7].

The high conservation of microRNA sequences provided an opportunity to develop miRNA-based marker system referred to as stable, polymorphic, functional, and transferable genotyping technique.

#### **2. The role of miRNAs in plant genome response to abiotic stress**

The plant organism has to cope with the environmental stress in natural and agricultural conditions. The genetic background of the plant organism allows it to adapt and defend itself through different mechanisms at a molecular level.

RNA interference represents the plant immune and defense system. It is a conserved mechanism induced by double-stranded RNA (dsRNA) or hairpinstructured RNA (hpRNA). One of the modules of RNA interference is provided by the microRNA (miRNA) molecules [8–10], which are capable to form doublestranded hairpin-like structures referred to as pre-miRNA. These small molecules have significant regulatory potential in the genetic and epigenetic control of gene expression. They are one of the key players in plant genome response to abiotic and biotic stress factor(s). Especially, deeply conserved miRNA families are integral components of many regulatory networks in plant organism [11, 12].

In general, the function of miRNAs molecules in plant organisms is defined as regulatory in the following processes:

**73**

*MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application…*

Plant adaptation mechanism requires complex modifications of gene expression machinery at the transcriptional and posttranscriptional level. Detailed studies of posttranscriptional gene regulation allow identifying stress-responsive miRNAs, which are differentially regulated under various stress factors. In plants, various abiotic stress-regulated miRNAs have been identified and characterized [13–17]. Certain families of miRNAs are either under or over-expressed, or new types of miRNAs can be synthesized under stress [13, 18]. Regulation of target genes expression by miRNA molecules is mediated by hybridization between miRNA sequences and their nascent reverse complementary sequences of mRNAs, which leads to their degradation or translational repression [19–21]. Because of their mode of action, they are generally referred to as negative regulators of gene

Plant adaptation mechanism to environmental conditions includes minimization of their growth rates and reorganizing their resources. The primary focus of the adjustment is cell cycle, cell division, and cell wall constitution [22]; it means

Genomic conservation of miRNA sequences and especially the stem-loop region of precursor molecules of miRNA (pre-miRNA) provided an opportunity to

MicroRNA-based genotyping technique as a novel type of marker system was published in 2013 by authors Fu et al. [23]. Since then, this system has been applied to genotyping applications of *Setaria italica* and in related grass species [24]. The structure analysis of miRNA genes revealed that repetitive sequences are part of them, which led to development of miRNA-based microsatellite markers in *Oryza sativa* [25, 26] and *Medicago truncatula* and related legume species [27]. Given the origin of sequences of this type of markers, they can be considered as functional

developmental processes regulated by conserved families of microRNAs.

The attributes of miRNA-based markers [23, 24] are as follows:

• improved reproducibility and sequence specificity due to high annealing temperature (more than 60°C) and the use of "touchdown" PCR approach

• relatively high polymorphism because of possible random combinations of

• putative functionality due to their polymorphism nature and the ability to

• cross-genera transferability potential because of the conservation level of miRNAs between species and the way of deriving markers from the consensus

Following subsection provides the approach of microRNA-based marker assay applied in our laboratory. As referred in **Table 1,** the following procedure has been

• good stability due to a direct PCR-based marker system

applied in several plant species for different research purposes.

predict phenotypes controlled by miRNAs

*DOI: http://dx.doi.org/10.5772/intechopen.88064*

expression.

**3. MicroRNA-based markers**

develop a novel type of molecular markers.

markers at the DNA levels [23, 24, 28].

primers

sequences of miRNAs.

**3.1 MicroRNA-based marker assay**

*MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application… DOI: http://dx.doi.org/10.5772/intechopen.88064*

Plant adaptation mechanism requires complex modifications of gene expression machinery at the transcriptional and posttranscriptional level. Detailed studies of posttranscriptional gene regulation allow identifying stress-responsive miRNAs, which are differentially regulated under various stress factors. In plants, various abiotic stress-regulated miRNAs have been identified and characterized [13–17].

Certain families of miRNAs are either under or over-expressed, or new types of miRNAs can be synthesized under stress [13, 18]. Regulation of target genes expression by miRNA molecules is mediated by hybridization between miRNA sequences and their nascent reverse complementary sequences of mRNAs, which leads to their degradation or translational repression [19–21]. Because of their mode of action, they are generally referred to as negative regulators of gene expression.

Plant adaptation mechanism to environmental conditions includes minimization of their growth rates and reorganizing their resources. The primary focus of the adjustment is cell cycle, cell division, and cell wall constitution [22]; it means developmental processes regulated by conserved families of microRNAs.

#### **3. MicroRNA-based markers**

*Non-Coding RNAs*

this group [5].

These patterns reveal the DNA polymorphisms between the studied individuals. The more the individuals are related, the more their fingerprint will match. The level of polymorphisms in a group of individuals reveals the genetic diversity within

There are different types of molecular markers. Molecular markers can be divided into two groups (a) biochemical markers, which detect variation at the gene product level such as changes in proteins and amino acids and (b) molecular markers, which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion, and/or insertion. DNA markers are based on hybridization or PCR (amplification of DNA) [3]. The multiplex ratio is defined as the amount of markers generated with one single reaction (e.g., one PCR, one hybridization reaction). The obtained pattern can be simple (one or a few bands, low multiplex ratio)

Molecular marker methods are either dominant or co-dominant. Using co-dominant

marker techniques, the different genotype combinations can be distinguished from each other at the study locus (or multiple loci). This means that homozygous (two identical alleles at a certain DNA locus) and heterozygous (two different alleles at a certain DNA locus) individuals will be identifiable. In the case of a dominant technique, it is not possible to detect the alleles that are present at a certain locus/loci, so homozygotes are

The high conservation of microRNA sequences provided an opportunity to develop miRNA-based marker system referred to as stable, polymorphic, func-

The plant organism has to cope with the environmental stress in natural and agricultural conditions. The genetic background of the plant organism allows it to

In general, the function of miRNAs molecules in plant organisms is defined as

**2. The role of miRNAs in plant genome response to abiotic stress**

adapt and defend itself through different mechanisms at a molecular level. RNA interference represents the plant immune and defense system. It is a conserved mechanism induced by double-stranded RNA (dsRNA) or hairpinstructured RNA (hpRNA). One of the modules of RNA interference is provided by the microRNA (miRNA) molecules [8–10], which are capable to form doublestranded hairpin-like structures referred to as pre-miRNA. These small molecules have significant regulatory potential in the genetic and epigenetic control of gene expression. They are one of the key players in plant genome response to abiotic and biotic stress factor(s). Especially, deeply conserved miRNA families are integral

components of many regulatory networks in plant organism [11, 12].

• processes of transition from embryogenic to vegetative phase

• flowering time, formation of flower organs, and reproduction

• defense mechanisms through transferring of signaling molecules.

or complex (a high number of bands, high multiplex ratio).

not distinguishable from heterozygotes [6, 7].

tional, and transferable genotyping technique.

regulatory in the following processes:

• plant growth and development

• root formation

• leaf morphology and plant polarity

**72**

Genomic conservation of miRNA sequences and especially the stem-loop region of precursor molecules of miRNA (pre-miRNA) provided an opportunity to develop a novel type of molecular markers.

MicroRNA-based genotyping technique as a novel type of marker system was published in 2013 by authors Fu et al. [23]. Since then, this system has been applied to genotyping applications of *Setaria italica* and in related grass species [24]. The structure analysis of miRNA genes revealed that repetitive sequences are part of them, which led to development of miRNA-based microsatellite markers in *Oryza sativa* [25, 26] and *Medicago truncatula* and related legume species [27]. Given the origin of sequences of this type of markers, they can be considered as functional markers at the DNA levels [23, 24, 28].

The attributes of miRNA-based markers [23, 24] are as follows:

#### **3.1 MicroRNA-based marker assay**

Following subsection provides the approach of microRNA-based marker assay applied in our laboratory. As referred in **Table 1,** the following procedure has been applied in several plant species for different research purposes.

The genomic DNA isolation protocol is based on the type of plant biological material (in terms of secondary metabolite content or other aspects). The primers for the miRNA-based markers are designed according to the mature or precursors

#### **Table 1.**

*Protocol of miRNA-based marker assay.*

#### **Table 2.**

*Amplification protocol of miRNA-based markers.*

**75**

made.

*MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application…*

sequences (pre-miRNAs) available on the miRBase database (http://www.mirbase. org, version 22), taking into account the primer design approach of published methodology [23, 24]. The primers are combined as follows: (a) forward and reverse primers of the same type, (b) forward and reverse primers in random combinations

The amplification protocol has originated from methodologies [23, 24] and was modified [30] (**Tables 1** and **2**). The total volume of PCR was 20 μl and the DNA

polyacrylamide (PAGE) gels, running in 1× TBE running buffer at constant power 180 V, 30 mA for 90 min. The gels are stained with PAGE GelRed™ Nucleic Acid Gel stain and are visualized on G-Box Syngene electrophoresis documentation system. For the recording of loci number and their position, as well as the identification of unique fragments, the gels are analyzed by GeneTools software (Syngene) (**Figure 1**).

**4. Contribution of miRNA-based markers on plant genome response to**

Genomic polymorphism of plants is the basis of their survival and ability to different climatic conditions. The cognition and mapping of plant genome variability using molecular markers is a prerequisite for extending the genetic base of crops to reduce their susceptibility to adverse environmental conditions [26]. An ideal molecular marker should be polymorphic, stable, reproducible, providing sufficient resolution, fast, and with fairly low cost [31]. The miRNA-based marker system is characterized by relatively high polymorphism, reproducibility, transferability across species, and ease of use with putative functionality [23]. The high level of transferability demonstrates the usability of miRNA-based markers for comparative

Recognizing relationships between species or within species help to focus more closely on a wide range of human interests, from basic description and disaggregation, through efficient resource genetic management to the production of quality

Within our research, we focused on the use of miRNA-based markers to highlight their broad spectrum of regulatory impact activities in different plant species of nutritional and pharmaceutical uses (**Table 3**): flax (*Linum usitatissimum* L.), medlar (*Messpilus germanica* L.), milk thistle (*Silybum marianum* (L.) Gaertn.), ginkgo (*Ginkgo biloba* L.), common ivy (*Hedera helix* L.), avocado (*Persea americana* Mill), and ribwort plantain (*Plantago lanceolata* L.). A total of 13 miRNA-based markers were applied of which 28 primer combinations were

Useful molecular markers produce fragments between 150 and 500 bp in length, as this size of fragments can easily be distinguished using agarose or PAGE gels [23]. In our experiments, this size of fragments varied predominantly from 40 to 300 bp

This range is due to variability of stem-loop structure of which the mature miRNA sequences are part of and the length of stem-loop structure ranges from less than 100 to over 900 nt. The primers' design based on miRNAs sequences can be linked to different places of the same stem-loop structure. Another possibility is

. Amplification products are separated on 15% TBE-urea

or (c) specific forward primer and universal reverse primer [29].

−1

**abiotic stress and for genotyping applications**

genome mapping and phylogenetic studies [24].

and safe food, whether plant or animal origin [32].

and could be clearly identified on agarose gels (**Figure 2**).

that primers amplify regions between neighboring miRNAs [23].

**5. MiRNA-based markers in genotyping applications**

*DOI: http://dx.doi.org/10.5772/intechopen.88064*

concentration was 70 ng μl

*MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application… DOI: http://dx.doi.org/10.5772/intechopen.88064*

sequences (pre-miRNAs) available on the miRBase database (http://www.mirbase. org, version 22), taking into account the primer design approach of published methodology [23, 24]. The primers are combined as follows: (a) forward and reverse primers of the same type, (b) forward and reverse primers in random combinations or (c) specific forward primer and universal reverse primer [29].

The amplification protocol has originated from methodologies [23, 24] and was modified [30] (**Tables 1** and **2**). The total volume of PCR was 20 μl and the DNA concentration was 70 ng μl −1 . Amplification products are separated on 15% TBE-urea polyacrylamide (PAGE) gels, running in 1× TBE running buffer at constant power 180 V, 30 mA for 90 min. The gels are stained with PAGE GelRed™ Nucleic Acid Gel stain and are visualized on G-Box Syngene electrophoresis documentation system. For the recording of loci number and their position, as well as the identification of unique fragments, the gels are analyzed by GeneTools software (Syngene) (**Figure 1**).

#### **4. Contribution of miRNA-based markers on plant genome response to abiotic stress and for genotyping applications**

Genomic polymorphism of plants is the basis of their survival and ability to different climatic conditions. The cognition and mapping of plant genome variability using molecular markers is a prerequisite for extending the genetic base of crops to reduce their susceptibility to adverse environmental conditions [26]. An ideal molecular marker should be polymorphic, stable, reproducible, providing sufficient resolution, fast, and with fairly low cost [31]. The miRNA-based marker system is characterized by relatively high polymorphism, reproducibility, transferability across species, and ease of use with putative functionality [23]. The high level of transferability demonstrates the usability of miRNA-based markers for comparative genome mapping and phylogenetic studies [24].

#### **5. MiRNA-based markers in genotyping applications**

Recognizing relationships between species or within species help to focus more closely on a wide range of human interests, from basic description and disaggregation, through efficient resource genetic management to the production of quality and safe food, whether plant or animal origin [32].

Within our research, we focused on the use of miRNA-based markers to highlight their broad spectrum of regulatory impact activities in different plant species of nutritional and pharmaceutical uses (**Table 3**): flax (*Linum usitatissimum* L.), medlar (*Messpilus germanica* L.), milk thistle (*Silybum marianum* (L.) Gaertn.), ginkgo (*Ginkgo biloba* L.), common ivy (*Hedera helix* L.), avocado (*Persea americana* Mill), and ribwort plantain (*Plantago lanceolata* L.). A total of 13 miRNA-based markers were applied of which 28 primer combinations were made.

Useful molecular markers produce fragments between 150 and 500 bp in length, as this size of fragments can easily be distinguished using agarose or PAGE gels [23]. In our experiments, this size of fragments varied predominantly from 40 to 300 bp and could be clearly identified on agarose gels (**Figure 2**).

This range is due to variability of stem-loop structure of which the mature miRNA sequences are part of and the length of stem-loop structure ranges from less than 100 to over 900 nt. The primers' design based on miRNAs sequences can be linked to different places of the same stem-loop structure. Another possibility is that primers amplify regions between neighboring miRNAs [23].

*Non-Coding RNAs*

**Amplification process**

*Protocol of miRNA-based marker assay.*

**Table 1.**

**Table 2.**

*Amplification protocol of miRNA-based markers.*

PCR buffer KCl, (NH4)2SO4, 20 mmol × dm<sup>−</sup><sup>3</sup>

The genomic DNA isolation protocol is based on the type of plant biological material (in terms of secondary metabolite content or other aspects). The primers for the miRNA-based markers are designed according to the mature or precursors

**PCR component Concentration Final concentration**

dNTP mix 2.5 mmol × dm<sup>−</sup><sup>3</sup> 0.8 mmol × dm<sup>−</sup><sup>3</sup> Primer forward 100 μmol × dm<sup>−</sup><sup>3</sup> 10 pmol × dm<sup>−</sup><sup>3</sup> Primer reverse 100 μmol × dm<sup>−</sup><sup>3</sup> 10 pmol × dm<sup>−</sup><sup>3</sup> *Taq* polymerase 5 U 2 U

Initial denaturation 94°C 5 min 1 cycle Denaturation 94°C 30 s 5 cycles

cycle)

Denaturation 94°C 30 s 30 cycles

Annealing 64°C (with temperature reduction of 1°C per

Polymerization 72°C 60 s

Annealing 60°C 45 s Polymerization 72°C 60 s Final polymerization 72°C 10 min

*Representative profile of amplified miRNA loci analyzed by GeneTool software (Syngene).*

**Temperature Time**

MgCl2 10× 1×

**period**

45 s

**Number of cycles**

**74**

**Figure 1.**

#### **Table 3.**

*The list of realized miRNA-based markers experiments.*

#### **Figure 2.**

*Representative gel showing amplification profile of miRNA-based markers of Plantago lanceolata (L.). 1–19 combinations of primers. F—forward, R—reverse. 1: lus-miR-R + lus-miR168F, 2: lus-miR-R + gm-miR156bF, 3: lus-miR-R + hyp-miR414F, 4: lus-miR-R + gm-miR171aF, 5: lus-miR-R + lus-miR156aF, 6: gm-miR-R + lusmiR168F, 7: gm-miR-R + gm-miR156bF, 8: gm-miR-R + hyp-miR414F, 9: gm-miR-R + gm-miR171aF, 10: gm-miR-R + lus-miR156aF, 11: miR-R + lus-miR168F, 12: miR-R + gm-miR156bF, 13: miR-R + hyp-miR414F, 14: miR-R + lus-miR171aF, 15: miR-R + lsa-miR156aF, 16: lsa-miR169aF + lsa-miR169aR, 17: lusmiR156aF + lus-miR156aR, 18: hvu-miR827F + hvu-miR827R, 19: lsa-miR396aF + lsa-miR396aR.*

The level of polymorphism varied from 70 to 90%. Mature miRNAs are expressed as small 21–24 nt endogenous molecules. As a result of different transcriptional activity among *MIRNA* genes, the miRNAs abundance in the cell varies greatly in dependence of miRNA family [11]. It should be noted that the level of polymorphism depended on the effectiveness of primer combination as well as the level of marker transferability.

The results of the studies have repeatedly confirmed the following:

**77**

*MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application…*

• MicroRNA-based markers provide genotype-specific profile of miRNA loci.

**5.1 MiRNA-based plant genome response to abiotic stress conditions**

stress-regulated genes are found to be regulated by miRNAs.

Different mechanism of stress response contributes to stress tolerance or resistance at different morphological, biochemical, and molecular level [13]. Many

• The abundance of selective miRNA-based markers is tissue specific and devel-

In the flax study, we applied nutritional stress factor under *in vitro* conditions [41]. The genome response of the flax genotype CDC Bethune was analyzed under five variants (including control variant) of Murashige-Skoog [42] medium by two miRNA-based markers, lus-miR395 and lus-miR399 [16, 17]. The results show that flax genome responds to the nutritional stress stimulus. Our results have supported the capability of miRNA-based molecules as potential biomarkers of abiotic stress

Another study was conducted in order to test the ultrasound-induced oxidative stress in lettuce (*Lactuca sativa* L.) tissues by miRNA-based markers [43]. We have confirmed that reactive oxygen species (ROS), caused by sonication treatment, induced the polymorphism at the molecular level detected by miRNA-based stress markers. We have observed the statistically significant differences (p ≤ 0.01) in miRNA markers ability to detect this polymorphism. The response of miR168 marker was statistically more sensitive in comparison with miR156 marker as a

The aim of research into the impact of soil compaction was to identify the barley (*Hordeum vulgare* L.) genome response by stress-responsive miRNA-based markers. A prerequisite for the research was that the plants are exposed to a lack of soil moisture and nutrients due to soil compaction. The effect of soil compaction was analyzed by four different miRNA-based markers (hvu-miR156, hvu-miR399, hvumiR408, and hvu-miR827), within the leaf, stem, and root tissues of barley plants. We can state that due to soil compaction, the barley plants were exposed to the lack of moisture which subsequently affected the intake and utilization of nutrients from the soil and showed lower plant growth parameters and reduced the yields. Moreover, this genome response was tissue specific. The roots were most affected by dehydration, and the nutrient deficiency was the most pronounced on leaves. The number of amplified miRNA loci was statistically significantly dependent on the

We have conducted experiments in connection with research on the drought resistance of wheat (*Triticum aestivum* L.). Genomes of susceptible and droughtresistant genotypes were screened by stress-sensitive miRNA-based markers

(hvu-miR408 and hvu-miR827). Genotypes were tested under *in vitro* conditions on Murashige-Skoog culture medium with different concentrations (0, 5, 10, 15, and 20%) of polyethylene glycol (PEG 6000) to induce dehydration stress. Droughtresistant wheat genotypes responded to dehydration stress, by significantly higher activity of hvu-miR408 biomarker in comparison with susceptible genotypes. This response points to a better genome adaptation ability of the resistant genotypes to abiotic stress. By using the conserved type marker hvu-miR156, which is involved in the regulation of plant growth and development processes, a reduced activity of this type of marker was observed, both in susceptible and resistant genotypes, indicating that the adaptation mechanism of plants to cope with stress conditions is

*DOI: http://dx.doi.org/10.5772/intechopen.88064*

result of their specific regulatory nature.

stress-sensitive marker applied.

implemented at the expense of growth processes.

opmental specific.

factors.

*MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application… DOI: http://dx.doi.org/10.5772/intechopen.88064*

#### **5.1 MiRNA-based plant genome response to abiotic stress conditions**

Different mechanism of stress response contributes to stress tolerance or resistance at different morphological, biochemical, and molecular level [13]. Many stress-regulated genes are found to be regulated by miRNAs.

In the flax study, we applied nutritional stress factor under *in vitro* conditions [41]. The genome response of the flax genotype CDC Bethune was analyzed under five variants (including control variant) of Murashige-Skoog [42] medium by two miRNA-based markers, lus-miR395 and lus-miR399 [16, 17]. The results show that flax genome responds to the nutritional stress stimulus. Our results have supported the capability of miRNA-based molecules as potential biomarkers of abiotic stress factors.

Another study was conducted in order to test the ultrasound-induced oxidative stress in lettuce (*Lactuca sativa* L.) tissues by miRNA-based markers [43]. We have confirmed that reactive oxygen species (ROS), caused by sonication treatment, induced the polymorphism at the molecular level detected by miRNA-based stress markers. We have observed the statistically significant differences (p ≤ 0.01) in miRNA markers ability to detect this polymorphism. The response of miR168 marker was statistically more sensitive in comparison with miR156 marker as a result of their specific regulatory nature.

The aim of research into the impact of soil compaction was to identify the barley (*Hordeum vulgare* L.) genome response by stress-responsive miRNA-based markers. A prerequisite for the research was that the plants are exposed to a lack of soil moisture and nutrients due to soil compaction. The effect of soil compaction was analyzed by four different miRNA-based markers (hvu-miR156, hvu-miR399, hvumiR408, and hvu-miR827), within the leaf, stem, and root tissues of barley plants. We can state that due to soil compaction, the barley plants were exposed to the lack of moisture which subsequently affected the intake and utilization of nutrients from the soil and showed lower plant growth parameters and reduced the yields. Moreover, this genome response was tissue specific. The roots were most affected by dehydration, and the nutrient deficiency was the most pronounced on leaves. The number of amplified miRNA loci was statistically significantly dependent on the stress-sensitive marker applied.

We have conducted experiments in connection with research on the drought resistance of wheat (*Triticum aestivum* L.). Genomes of susceptible and droughtresistant genotypes were screened by stress-sensitive miRNA-based markers (hvu-miR408 and hvu-miR827). Genotypes were tested under *in vitro* conditions on Murashige-Skoog culture medium with different concentrations (0, 5, 10, 15, and 20%) of polyethylene glycol (PEG 6000) to induce dehydration stress. Droughtresistant wheat genotypes responded to dehydration stress, by significantly higher activity of hvu-miR408 biomarker in comparison with susceptible genotypes. This response points to a better genome adaptation ability of the resistant genotypes to abiotic stress. By using the conserved type marker hvu-miR156, which is involved in the regulation of plant growth and development processes, a reduced activity of this type of marker was observed, both in susceptible and resistant genotypes, indicating that the adaptation mechanism of plants to cope with stress conditions is implemented at the expense of growth processes.

*Non-Coding RNAs*

**76**

**Figure 2.**

**Table 3.**

*The list of realized miRNA-based markers experiments.*

level of marker transferability.

The level of polymorphism varied from 70 to 90%. Mature miRNAs are expressed as small 21–24 nt endogenous molecules. As a result of different transcriptional activity among *MIRNA* genes, the miRNAs abundance in the cell varies greatly in dependence of miRNA family [11]. It should be noted that the level of polymorphism depended on the effectiveness of primer combination as well as the

*14: miR-R + lus-miR171aF, 15: miR-R + lsa-miR156aF, 16: lsa-miR169aF + lsa-miR169aR, 17: lusmiR156aF + lus-miR156aR, 18: hvu-miR827F + hvu-miR827R, 19: lsa-miR396aF + lsa-miR396aR.*

*Representative gel showing amplification profile of miRNA-based markers of Plantago lanceolata (L.). 1–19 combinations of primers. F—forward, R—reverse. 1: lus-miR-R + lus-miR168F, 2: lus-miR-R + gm-miR156bF, 3: lus-miR-R + hyp-miR414F, 4: lus-miR-R + gm-miR171aF, 5: lus-miR-R + lus-miR156aF, 6: gm-miR-R + lusmiR168F, 7: gm-miR-R + gm-miR156bF, 8: gm-miR-R + hyp-miR414F, 9: gm-miR-R + gm-miR171aF, 10: gm-miR-R + lus-miR156aF, 11: miR-R + lus-miR168F, 12: miR-R + gm-miR156bF, 13: miR-R + hyp-miR414F,*

**Research purpose Plant species Reference** Genome profiling with regard to genotype origin *Linum usitatissimum* [30] Spatial and temporal abundance of individual miRNA markers *Linum usitatissimum* [33, 34] Functional markers of commercial type of the crop *Linum usitatissimum* [35, 36] Genotyping *Mespilus germanica* [36] Genomic authentication of varieties *Silybum marianum* [37] Genotyping *Ginkgo biloba* [38, 39] Genotyping *Hedera helix* [40] Genotyping *Persea americana* — Genotyping *Plantago lanceolata* —

The results of the studies have repeatedly confirmed the following:

• MicroRNA-based markers show the cross-genera transferability potential.

• MicroRNA-based markers display sufficient level of polymorphism in analyzed genotypes and are suitable to differentiate within genotypes of one specimen.