Molecular Profiling for Identification of Zea mays L. True Hybrids

Maize (Zea mays L.) is the third most significant cereal crop after rice and wheat. It is a multiple aspect crop, chiefly used for forage and food (Raj et al., 2019). It also serves as a basic raw material in industries like starch, oil, protein, alcoholic beverages, food sweeteners, seasonings, fuel, etc. It is cultivated on nearly 197 million hectares globally in about 166 countries across the globe and contributes around 37% of the total global grain production (Erenstein et al., 2022). USA is the largest producer of maize which contributes 30% of the total global production (https://www.fao.org/3/cb4477en/online/cb4477en.html). All India Rabi maize production was 9.9 MT for the year 2020-21 (Maize Outlook Report, 2021). Major maize consumption states in India are Karnataka, Andhra Pradesh, Punjab, Gujarat, Haryana, Telangana, Tamil Nadu, Bihar and West Bengal (Agricultural Market Intelligence Centre, PJTSAU, 2021-22).
       
Maize has good heterotic potential for total yield, seed quality, disease resistance and uniformity (Dou et al., 2012). However, main constraints for its productivity are suboptimal plant density, inadequate fertilizer use and water supply, weed infestation, insect/pest attack and selection of unsuitable cultivars under a given set of environments. It has been noted that adoption of high yielding hybrids has not just improved grain yield and quality, but has also led to higher income per hectare as compared to conventional varieties of maize (Abbas, 2001). Further, modern maize hybrids have greater potential as compared with older hybrids (Tahir et al., 2008). Since the yield potential of existing maize varieties is deteriorating day by day, so selection of maize hybrids is essential which offer increased yield, wide adaptability and reliability in performance and quality (Ali et al., 2012).
       
To introduce heterogeneous hybrids for production of hybrid seeds, it requires two distinct homogenous inbred lines which are crossed and selfed for several generations. But during this practice, it is frequently contaminated by crossed pollens from another variety or sometimes undesired selfing occurs (Hipi et al., 2013). For dealing with this issue, plentiful strength has been directed towards achieving the largest kernel set and sophisticated genetic purity (Fonseca et al., 2002). Further, genetic purity during multiplication stages is prone to contamination due to physical admixtures, presence of pollen shedders, out crossing with foreign pollens etc. High genetic purity is an essential pre-requisite for the commercialization of hybrid seeds, which is ascertained by compulsory genetic purity test for certified seeds. Besides, success of any hybrid technology depends on the availability of quality seeds supplied in time at reasonable cost.
       
Genetic purity testing of seeds (i.e. the percentage of contamination by seeds or genetic material of other varieties or species) contributes to overall seed quality (Dou et al., 2012). One of the challenges faced during such testing is rapid and accurate assessment of hybrid seeds before they are supplied to the farmers. If low genetic quality hybrid seeds are sown then it results in loss of productivity (Hipi et al., 2013). It was estimated that for every single per cent impurity in the hybrid seeds, the yield reduction is 100 kg per hectare (Bora et al., 2016).
       
Conventional purity assessment in fields is conducted based on morphological characters. Morphological identification by grow-out test (GOT) at field level wastes so much time, limits to resources and can affect the data by different environmental conditions (Hipi et al., 2013). Compared with morphological variations, molecular and biochemical polymorphisms are generally considered more informative. However, same limitations apply for isozyme analysis also, where different conditions of environment may vary the accuracy of results. Further, selection of isozyme markers must be precise (Lucchese et al., 1999).
       
Nowadays, use of molecular markers-which reveals polymorphism at the DNA level-has been playing an increasing part in plant biotechnology and genetic studies (Kumar, 2013). Characterization by specific markers which can identify the male and female parents from selfed parental and outcrossed lines in F₁ can be effectively used for distinguishing the true hybrids (Mohan et al., 2013). Amongst molecular markers, polymerase chain reaction (PCR) based simple sequence repeats (SSRs), also known as microsatellites have been most widely used, due to high degree of information provided by their large number of alleles per locus (Asma et al., 2016). Further, SSRs are co-dominant, can discriminate between closely related individuals efficiently and can verify hybrids also (Babaei et al., 2007; Iqbal et al., 2010). As SSRs are conserved between closely related species, therefore they could provide a marker database for cultivar identification (Zhang, 2005). The aim of the present study was to obtain SSR markers specific for male and female parents of maize hybrids which can help in convenient distinction of hybrids.

Plant material
 
Seven maize hybrids viz. HM-5, HM-8, HM-9, HM-10, HM-11, HM-12 and HM-13 which are commercially cultivated in various parts of India along with their parental lines were used in the present study (Table 1). All these hybrids along with their parental lines were obtained from Maize Section, Department of Genetics and Plant Breeding, Regional Research Station, Uchani, Chaudhary Charan Singh Haryana Agricultural University, India. The seeds of the respective hybrids along with parents were grown in greenhouse of the Department of Molecular Biology and Biotechnology, College of Biotechnology, CCS HAU, Hisar during 2021-22.
 

 
DNA isolation and quantitative analysis
 
The young and fresh leaves from maize seedlings were used for extraction of DNA using cetyltrimethylammonium bromide (CTAB) method (Saghai-Maroof et al., 1984) with some modifications. Quality and quantity of the isolated genomic DNA was estimated by 0.8 percent agarose gel electrophoresis. An uncut lambda DNA (50 ng/µl) was run as standard. DNA was diluted as 1:10 on the basis of nanodrop reading. The optimum concentration of DNA was 30 ng/µl for each PCR reaction.
 
PCR amplification
 
The SSR primer pairs were designed from the sequence information available in published data base (http://www.maizegdb.org/ssr.php). A total of 45 inconsistent SSR primers which were distributed on all the 10 maize chromosomes were used for PCR amplifications (Table 2). PCR was conducted in a reaction mixture of 10 µl consisting of 1 µl of 30 ng DNA, 1X PCR Buffer, 0.25 mM of each dNTPs, 10 µM of both forward and reverse primers and 1U Taq DNA polymerase.
 

       
The cyclic parameters used for PCR amplifications included initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 45 s, amplification at 46-65°C for 40 s, extension at 72°C for 45 s and final extension step at 72°C for 10 min. Amplified products were checked on ethidium bromide (10 mg/ml) stained agarose gel (2.5%) and photographed using gel documentation system (Benchtop UVP).

With the rapid growth of population at global level, there is a high demand of maize for food and energy supplies. To tackle this increased demand, it is necessary to improve production and quality of maize through proficient breeding programs (Tester and Langridge, 2010). Hybrid maize guarantees much more yields, improve value and decrease the cost of production. Hybrid vigor also called heterosis arises on two genetically distinct inbred lines as parents are crossed to produce a hybrid (Hi bred pioneer.inc) which results in large sized, robust and sturdy plants. Therefore, instant and precise estimation of hybrid seeds purity must be done to ascertain genetic purity before the seeds are supplied to the farmers.
       
Report Dallas (1998) was the first who gave the usefulness of DNA fingerprinting for cultivar identification. For hybrid purity estimation, SSRs are the most appropriate markers which are co-dominant and distinguish homozygotes and heterozygotes so that they can easily detect heterozygosity of the hybrids. These markers are currently used for purity identification in many crops.
       
In the present study 12 SSR markers viz. pumc2246, pumc1020, pumc1040, pumc1035, pumc1071, pumc1552, pumc1228, pumc1002, pumc1023, pumc1084, pumc1115 and pumc1064 were found to be highly polymorphic. These selected markers were used for testing seed genetic purity. Among these first 3 markers (pumc2246, pumc1020 and pumc1040) were able to produce polymorphic bands and were efficient to distinguish parental lines of the seven maize hybrids at allelic size of 100 bp to 200 bp (Table 3).
 

       
It is already proved that SSRs are authentic tools for hybrid identification or hybrid purity estimation and parentage verification in many crop species (Bohra et al., 2011). Utilizing such markers, true hybrids were identified in case of peanut (Busisiwe et al., 2015), mungbean (Sorajjapinun et al., 2012), rice (Hashemi et al., 2009), cotton (Dongre et al., 2011]) sugarcane (Zhang et al., 2009), maize (Sudharani et al., 2013), sunflower (Pallavi et al., 2011), oil palm (Thawaro et al., 2009), cabbage (Liu et al., 2019) and cassava (Mohan et al., 2013). SSR markers were used for detection of true hybrids, genetic purity testing, germplasm identification, cultivar fingerprinting, heterotic pattern in hybrids and parentage confirmation of hybrids (Darvhankar et al., 2019).
       
In the present investigation, marker pumc1040 showed the amplified product at 140 bp for male parents of hybrids HM-5 and HM-12. Also, this primer resolved the band position of amplicon at 100 bp and 110 bp for their female parents, respectively (Table 4). This concludes that the hybrid HM-5 was raised from the cross pollination of Hki1344 and Hki1348-6-2 while the hybrid HM-12 was raised from the cross pollination of Hki1344 and Hki1378 i.e. heterozygosity was confirmed from both the parents. SSR marker pumc2246 was able to distinguish hybrids HM-10, HM-11 and HM-13 where allelic sizes varied between 120 bp to 170 bp. On the other hand, pumc1020 could distinguish hybrids HM-5, HM-8, HM-9, HM-10, HM-11, HM-12 and HM-13 at allelic sizes between 120 bp to 200 bp (Fig 1, 2). SSR marker pumc1040 could distinguish hybrids HM-5 and HM-12 at allelic sizes between 100 bp to 140 bp.
 

 

 

       
Kovincic et al., (2023) also suggested SSR marker use for more accurate and time-efficient maize hybrids and parental lines genetic purity testing.

This study illustrated that SSR markers are rapid, valuable and results are mostly reproducible when compared with morphological data analysis presented by field survey. The identified SSR markers can be used for testing of hybrids for their genetic purity in regular means. This study will be helpful for hybrid maize industry to pick out the suitable combinations of markers for development of SSR markers meant for genetic purity evaluation. They are very convenient for identification of true hybrid seedlings at the early stage of growth and for precise genotyping of the progenies developed for gene mapping studies. The markers can also be utilized for optimizing genomic enabled prediction for maize hybrid breeding programs.

The authors thank Head of the Department, Department of Molecular Biology, Biotechnology and Bioinformatics; and Dean, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar for providing facilities to conduct this research work.