Screening of Bush Type French Bean (Phaseolus vulgaris L.) Accessions for Micronutrient Variations and Characterization of Selected Genotypes using Micronutrient Content Linked Markers

Common bean (Phaseolus vulgaris L.) is a major herbaceous diploid (2n=2x=22) annual, grown worldwide for its edible dry seeds (Beans/Rajma) and tender pods. It is a member of legume family Fabaceae and genus Phaseolus, which has over fifty species and Rajma is one among them. Bean exhibits wide genetic variation with respect to growth habits. Two general types of beans grown, such as bush and pole type beans. Bush beans are most popular because of early maturity and trellising is not required (Dass et al., 2014).
       
Common bean is highly nutritious with twice the protein levels of cereals, lesser fats than soybean/peanut. It has higher amount of lysine, potassium, phosphorus, iron, zinc, magnesium, copper and calcium than cereals and rich in certain vitamins like thiamin and folic acid. Individuals between 25-50 years, the daily required micronutrients in human diet as estimated by Food and Agriculture Organization (FAO) and Word Health Organization (WHO) includes 10-15 mg iron, 12-15 mg zinc, 5.5 mg manganese and 1-5 mg copper (Welch and Graham, 2004). Iron plays role in carrying oxygen throughout body, assisting energy metabolism, supporting immune function, production of neurotransmitters and chemicals that carry messages between nerve cells and development of nervous system. Zinc is critical for normal growth and sexual maturation. It has a role in immune system and functioning of at least 70 enzymes. Copper helps in transporting iron in blood. Manganese is primer to many enzyme-mediated chemical reactions including enzymes involved in synthesis of cartilage (Shenkin, 2006).
       
Micronutrient deficiencies affect one in three humans of all age groups worldwide and they required supplements. Crop improvement programmes through bio-fortification is one of the solution to tackle the micronutrient deficiency (Achom et al., 2016). Beans are recalcitrant to regeneration from cell cultures and there are no efficient transgenic solutions. Therefore, the only viable option available for bean improvement is genetic approach by utilization of marker- assisted selection (MAS) program (Prakash et al., 2015). Genetics of seed Fe and Zn content have known to be complex; hence, the present study is an attempt to determine the variability of seed micronutrient accumulation in Indian bean accessions using micronutrient content linked markers.

Experimental condition
 
The study has been conducted at Department of Plant Biotechnology, UAS, GKVK, Bengaluru. The field plots with randomized complete block design (RCBD) measuring 2 M ×1.5 M with three replications were prepared during kharif 2015-16. Twenty Indian bean accessions were collected from NBPGR, New Delhi and four-bush type commercial bean varieties (Arka Anoop, Arka Komal, Arka Sharadh and Arka Suvidha) were used as checks in the study. Package of practices were followed as and when required.
 
Observations recorded
 
Twelve morphological characters were studied such as number of days taken for pod initiation, number of pods per plant, pod length, pod width, pod colour, dry pod weight per plant, number of seeds per pod, seed colour, seed shape, seed length, seed breadth and total pod yield per plot, seed yield per plot and crop duration were observed and recorded.
 
Micronutrient estimation by AAS
 
Estimation of iron, zinc, manganese and copper content in bean samples carried out by AAS (Chakraborthi et al., 2011). One gram of seeds powdered in grinder and digested in tri-acids (HNO₃+HCl₄+H₂SO₄) mixture (10:4:1) in micro-oven digester. Samples cooled for 30 minutes and volume made up to 50ml with double distilled water. A known quantity of aliquot used for subsequent analysis. Suitable blanks run simultaneously to account for contamination from reagents.
 
DNA extraction and microsatellite amplification
 
Genomic DNA was extracted (Amelia et al., 2015) from twenty-four accessions by Cetyl Trimethyl Ammonium Bromide (CTAB) method. Leaves free from pest and disease were collected from 30-45 days old plants and grounded in liquid nitrogen for DNA extraction. DNA suspended in TE buffer and quality evaluated on 0.8% agarose gels followed by quantification with Nano Drop™ spectrophotometer. DNA diluted to 50 ng/µl for further experiments (L’taiefet_al2008). Each 20 µL amplification reaction consisted of 1x PCR buffer, 1.88 mM MgCl, 200 µM of each deoxyribonucleotide triphosphate, 1 µM of primer, 1 unit of Taqpolymerase and 50 ng of template DNA. Amplification performed in a 96-well BioRad thermal cycler.
       
Fourteen SSR primers used were BMd9, BMd16, BMd27, BMd33, BMd47, BMd54, BM139, BM154, BM170, BM175, BM181, BM164, BM160 and BM211 markers, recently developed by (Blair et al., 2011) (Table 4). PCR protocol consisted of hot start for 3 min at 95^oC, denaturation at 94^oC for 3 min, followed by 35 cycles of 95^oC for 20s annealing temperature from 55 to 58^oC for 45 s and extension at 72^oC for 2 min, 1 cycle of final extension for 72^oC for 10 min. In all cases, amplified products separated on 2.5% agarose gel with 80 V and run for 2 hours.
 
Statistical analysis
 
Data for phenotypic correlations analyzed using statistical tool SPSS 16.0 version. Seed micronutrients content data were subjected to analysis of variance (ANOVA). After gel electrophoresis of PCR products, the presence or absence of each single fragment was coded by 1 or 0 respectively. A dendrogram was plotted using software STATISTICA version 13.2 based on scored data to identify the diversity of genotypes.

Pod characters
 
Pod initiation was significantly early in released variety Arka Sharadh (31.66 days), followed by local variety Selection (33.33 days), EC530843 (33.66 days) and IC540876 (37.33 days) than other varieties. Average days taken for pod initiation were 38.73 days. Pod length was significantly higher in Arka Komal, EC559574 and pod width found significantly higher in IC540876 and EC530842 than other accessions. Average dry pod weight per plant was 40.47 g. Higher dry pod weight per plant was observed in IC342273 (58.94 g) than released variety Arka Komal (52.64 g). Higher number of pods per plant were found both in IC342273 (40.33) and IC540876 (33.33) is higher than average number of 22.34 pods (Table 1). Thus, accessions viz., EC530843, IC540876, EC559574, EC530842 and IC342273 can screened for pod characteristics as they were on par with released varieties. Rana et al., (2015) reported similar results.


 
Seed characters
 
Average number of seeds per pod was 4.81, however, in accessions EC530843 and IC405546 found significantly higher number of seeds per pod (6.33). Seed breadth and length was higher in accessions IC11676 and IC405546, which were on par with Arka Sharadh and Arka Suvidha. Yield parameters viz., total pod and seed yield per plot was found to be significantly higher in the accessions IC342273, EC531078 and IC328655 than other accessions. Crop duration was more in released variety Arka Suvidha followed by EC530819 and EC304657 (Table 2). Dass et al., (2014) and Rana et al., (2015) reports similar results. Further exploitation of the accessions with higher seed and pod yield traits will results in identifying the good quality of varieties.
 

 
Estimation of seed micronutrient (Fe, Zn, Mn and Cu) contents
 
Among twenty-four bean accessions analysed, the mean Fe content was 159.25 ppm. The accession IC538420 (177.31 ppm) showed significantly higher level of Fe content followed by released variety Arka Suvidha (175.98 ppm), EC530886 (173.67 ppm) and IC342273 (170.02 ppm). Lower level of Fe content was recorded in accessions IC328655 (139.90 ppm), Arka Anoop (143.86 ppm), IC329154 (148.30 ppm) and IC311676 (149.95 ppm) (Fig 1). Ability of genotypes to absorb and accumulate Fe depends on genetic potential, soil and agro climatic condition of place where the genotypes are grown. Beebe et al., (2000) have made similar reports in Andean and Mesoamerican gene pools of bean. Similarly, in present study Fe content ranged from 139.90-177.31 ppm, where wild accessions and released varieties exhibited a difference of 20-25 ppm of Fe accumulation.
 

       
The evaluation of twenty bean accessions and four released varieties for Zn content revealed a range of 20.23-26.91 ppm with an average value of 23.73 ppm. Accessions with significantly higher Zn content were found in EC500226 (26.91 ppm), EC530843 (26.85 ppm), EC530819 (26.49 ppm), released variety Arka Sharadh showed 25.03 ppm and lower level of Zn content in accessions EC559576 (20.23 ppm), IC329154 (21.71ppm) and IC342273 (21.90 ppm) (Fig 2). Similar reports were made by Beebe et al., (2000); Blair et al., (2009); Golam et al., (2011).
 

       
Manganese content varied significantly, with a range of 5.23-15.31 ppm and mean Mn content was 8.46 ppm. Accession with significantly higher Mn content were found in EC500226 (15.31 ppm), IC538420 (11.83 ppm), EC530819 (10.78 ppm), EC530842 (10.25 ppm), released varieties Arka Sharadh (8.77 ppm), Arka Komal (8.65 ppm) (Fig 2) and lower level of Mn content in accessions IC329154 (5.23 ppm) and IC311676 (5.98 ppm). Similar reports were made by Beebe et al., (2000); Brigide et al., (2014).
       
Copper content ranged from 3.71-8.55 ppm with an average of 6.17ppm. The accession with recorded higher Cu content were EC500226 (8.55 ppm), EC531078 (8.40 ppm), EC304657 (7.98 ppm) and among released varieties, Arka Sharadh showed significantly higher Cu content (7.19 ppm). Whereas released varieties Arka Komal (3.71 ppm), Arka Anoop (4.30 ppm), Arka Suvidha (4.76 ppm) and accession IC538430 (4.26 ppm) had found lower Cu content compared to other released varieties accessions (Fig 2). Present study revealed higher concentration of Cu in accessions compared to released varieties. Similar reports were made by Beebe et al., (2000); Giuseppa et al., (2016).
       
Micronutrient contents are higher in the wild types compared to released varieties, due to the ability of genotypes to absorb and assimilate micronutrients into seeds, also the factors like soil and environments plays a major role (Beebe et al., 2000).
 
Molecular screening of French bean accessions using SSR markers linked to Fe and Zn content
 
Among fourteen SSR markers, twelve SSR primers have given good scorable loci out of which genomic SSR marker BM154 has detected polymorphic loci in eleven accessions (EC500226, EC530819, EC530842, EC530843, EC530886, EC559574, EC559576, IC342273, IC538420, IC540876 and Arka Komal) with expected amplified product size of 218 bp and present on linkage group b09 amplified. These eleven accessions having green pods and brown seeds have Polymorphism Information Content (PIC) of 0.18.
       
Another genomic SSR marker BM211 depicted high polymorphism among the twenty-four bean accessions studied and is considered for Fe content present on linkage group b08. The ten polymorphic loci has detected in ten accessions (EC500226, EC530819, EC530842, EC530843, EC530886, EC559574, EC559576, IC342273, IC538420 and Arka Komal) with expected amplified product size of 186 bp (Fig 3). These accessions had green pods and brown seeds with PIC of 0.37 as PIC analysis used to evaluate the markers; it helps in selecting the most appropriate one for genetic mapping, phylogenetic analysis or association genetics (Anderson et al., 1993). Average number of alleles and PIC detected were 109 and 0.16 respectively. The Genomic SSR markers showed good polymorphism than gene based SSR markers. Blair et al., (2011) reports similar results as they revealed that the gene based microsatellites were frequently bi-allelic or tri-allelic along with spotting the difference between various gene pools. Whereas most of the genomic microsatellites detected, reveled more than three alleles and were able to resolve within gene pool variations (Schloss et al., 2002).
 

 
Dendrogram for Fe and Zn diversity in French bean accessions
 
To understand the genetic relationship among bean accessions and cultivars, heirarchial cluster analysis with Unweighted Pair group-average (UPGMA) clustering method following Sequential Agglomerative Hierarchial Nested (SAHN) cluster analysis module was performed using software NTSYS 2.20. Based on the seed Fe and Zn accumulation, cluster analysis separated the twenty-four accessions into two interrelated clusters namely Major Cluster-I with one accession (EC531078) and Major Cluster-II with remaining twenty-three accessions and varieties (Fig 4). Further major cluster-II was sub divided as sub cluster-I with nine accessions and three released varieties (EC500226, EC500474, IC538430, Selection, IC328655, IC311676, IC405546, IC540876, IC329154, Arka Sharadh, Arka Suvidha and Arka Anoop) and sub cluster-II with ten accessions and one released variety (EC304657, EC512812, EC530843, EC530819, EC530842, EC530886, EC559574, IC342273, EC559576, IC538420 and Arka Komal). It has also revealed that three released varieties (Arka Anoop, Arka Sharadh and Arka Suvidha) showing close linkage with many accessionsin sub cluster-I of Major Cluster-II. Similarly, Arka Komal variety is showing close relation to many accessions in sub cluster-II of Major Cluster-II. It was found that either there was no correlation between morphological characteristics or micronutrient content with accessions grouped in sub clusters-I and II. Blair et al., (2009) made similar kind of observations.
 

Higher pod and seed yield was recorded in accessions IC342273, EC531078 and IC328655 compared to released varieties. Early crop duration has been recorded in two accessions viz., EC500226, EC500474 and whereas lower yield potential was observed in these two accessions compared to other accessions and released varieties. Higher Fe content recorded in two accessions IC538420 and EC30886 and they are on par with released variety Arka Suvidha. The accessions EC500226, IC538420, IC342273, EC530842 and EC530843 showed higher concentration of Mn, Zn, Cu and Fe. Between fourteen SSR markers used to screen the bean accessions for micronutrients, twelve SSR primers amplified and two genomic SSR markers (BM154 and BM211) exhibited higher polymorphism. These variations grouped as separate gene pools in turn can exploited for crossing programmes to develop elite lines with enhanced Fe and Zn content (Beebe et al., 2000; Achom et al., 2016). The above results will help in selection of accessions for further improvement of micronutrient content in French beans for commercial cultivation. Hence, the study helps in accomplishing the goal of micronutrient enhancement, which leads to sustainable solution for malnutrition and food security (Rathnayake et al., 2012).