Research Progress of Molecular Markers in Genetic Diversity of Rapeseed

Guanghuan YANG, Jibin NAN, Suping GUO, Yan YUAN, Ba DAN

Key Laboratory of Plateau Crop Molecular Breeding, Tibet Agricultural and Animal Husbandry College, Nyinchi 860000, China

AbstractIn recent years, with the continuous improvement and development of molecular technology in the application process, different types of DNA molecular markers have made rapid progress in the study of genetic diversity of rapeseed. The study of genetic diversity is conducive to the correct formulation of the strategy of collecting and in situ preservation of genetic resources of rapeseed, and it is the genetic basis for the improvement of rapeseed varieties. This article mainly starts with the three DNA molecular markers (SSR, RAPD, AFLP) widely used in the study of genetic diversity of rapeseed. By introducing the application principles and characteristics of SSR, RFPD and AFLP molecular markers, research progress of these three marker technologies in genetic diversity of rapeseed is briefly described.

Key wordsSSR molecular marker, RAPD molecular marker, AFLP molecular marker, Genetic diversity of rapeseed, Research progress

1 Introduction

Rapeseed is one of the world’s four major oil crops, rapeseed oil contains more than 10 kinds of fatty acids and multivitamins, especially high in vitamin E and rich in nutrients. The content of rapeseed protein is as high as 36% to 40%, and after pressing the oil, we can get about 60% of the cake, the cakes on the left and right sides are similar in composition to soybean cakes and are considered to be good concentrates and organic fertilizers[1]. Rapeseed oil and other vegetable oils also have a tendency to replace some petroleum products for energy use. In Europe like Germany and other countries, research has begun on the use of vegetable oil as a raw material for biodiesel. Rapeseed oil has always been China’s main edible vegetable oil. At present, China’s rapeseed cultivation area and total output account for 1/4 of the world’s rapeseed, accounting for about 2/5 of China’s oil crop planting area, ranking first in oil crops[2]. The breeding objectives of rapeseed crops usually include increasing oil content, changing fatty acid composition, improving nutrition and processing quality, and improving by-product quality. After long-term artificial selection, cultivation and domestication, a number of high-quality rapeseed varieties with low erucic acid and low thioglucoside have been selected in China. Because the breeding target of rapeseed is mostly concentrated on seed quality traits (low erucic acid, low thioglucoside), its genetic basis is narrow, and its genetic diversity is considered to be relatively low. In terms of the concept of genetic diversity, different scholars gave different definitions. McNealyetal.[3]believed that genetic diversity is the sum of genetic information, which is contained in the genes of plants, animals and micro-organisms on the earth. When it comes to ecosystem diversity and species diversity, it also contains its own genetic diversity. Some scholars pointed out that diversity refers to the sum of genetic variation among different groups within a group or between individuals within a group, which can be understood as genetic diversity in a narrow sense[4]. Genetic diversity analysis of species gene pools makes sense for both plant breeding and germplasm management. At present, rapeseed breeding is mostly hybrid breeding. Because the hybridization advantage must be based on genetic differences between parents, the genetic diversity of rapeseed is receiving new attention. Revealing the genetic variation between varieties at the molecular level has received global attention and has important practical significance. Molecular markers can effectively improve the breeding efficiency of oil crops. The DNA molecular markers (SSR, RAPD, AFLP) developed in the 1980s are genetic markers based on DNA or mRNA polymorphisms, and have been widely used and studied in the genetic diversity of rapeseed, it provides an unprecedented development opportunity for the development of new rapeseed varieties, improvement of rapeseed yield, improvement of rapeseed quality and enhancement of rapeseed resistance[5].

2 SSR molecular marker

SSR (Simple Sequence Repeat) molecular marker is a class of molecular marker which is based on PCR technology, also known as microsatellite marker[6], refers to a segment of DNA in the genome consisting of 1-6 nucleotides of the basic unit repeated multiple times, widely distributed in different locations of the genome, and the length is generally below 200 bp.

2.1 Principles and characteristics of SSR molecular marker technology

2.1.1Principle of SSR molecular marker technology. According to the conservatism of the sequences at both ends, primers can be synthesized and PCR amplification reaction can be carried out to amplify a single microsatellite locus, and then electrophoresis separation and staining for detection and analysis satellite sequence polymorphism, the sample DNA sequence and the regulation and difference in genetic traits, and finally achieve the purpose of successful identification[7].

2.1.2Characteristic of SSR technology. The DNA sequence is usually a series of repeats of several motifs consisting of 1-6 bases. The presence of microsatellite DNA can be detected at different sites of the whole genome. The difference in the number of series is that the main source of high polymorphism. The SSR molecular marker has the characteristics of multiple alleles, is inherited by Mendelian method, exhibits codominant markers, provides a high amount of information, and the polymorphism of the revealed DNA bands is also high, and each locus is composed of the sequence of the designed primers is determined, so that different experimenters can exchange and cooperate with each other to develop primers.

2.2 Application of SSR in genetic diversity of rapeSSR markers are widely used in the study of genetic diversity of rapeseed because of their co-dominant inheritance, good polymorphism, good reproducibility and abundant markers. Fang Yan and Sun Wancang[8]identified nine pairs of SSR and 36 pairs of InDeL from 252 pairs of markers, and analyzed the genetic diversity of main representative species of winter rape and spring rape in northern China, and discussed each the kinship between the varieties. Lei Jianming[9]and other scholars used SSR markers to analyze the genetic diversity of 51 winter cabbage-type rapeseed cultivars in Gansu, Shaanxi, Shanxi and other places. The results showed that the winter cabbage-type rapeseed inheritance originated from Gansu Province. The difference is large, which may be due to the vast territory of Gansu Province, the geographical and ecological differences between regions, and the formation of a variety of winter cabbage-type rapeseed genetic resources under the long-term natural environment selection. At the same time, it was found that the genetic differences between the varieties from the same region were small, and the genetic differences among different regions were large, indicating that the similarity of winter cabbage-type rapeseed varieties is related to the geographical and ecological environment of their origin, and it can be inferred that geography and ecology. The environment is the main factor affecting the genetic difference of winter cabbage type rapeseed. Guo Yimingetal.[10]selected 51 pairs of primers to study 187 copies ofBrassicanapusfrom 43 countries and five other crops of Brassica. The genetic diversity of the crops was studied by using POPGENE and PRIMEP6 software. Cluster analysis of varieties and variability, and the distribution of SSR-specific alleles of Chinese and non-Chinese materials and their distribution in the genome were statistically analyzed. Wang Jinxiongetal.[11]used SSR molecular markers to analyze the genetic diversity of 169 Chinese cabbages from different regions of Tibet. Studies have shown that The greater the genetic difference between the parents, the more significant the heterosis, so fully understand the genetic diversity between the parents, the parent material is divided into different heterotic groups, which helps to guide the selection of strong dominant hybrid combinations, so as to quickly and effectively utilize the heterosis and cultivate more ideal rape varieties. Danba[12]and others used SSR molecular marker technology to analyze the genetic diversity and genetic relationship of 22 Chinese (two cultivars and 20 wild rapeseeds) Tibetan cabbage rapeseed, which provided basic data for selecting excellent hybrid combinations.

3 RAPD molecular marker

The RAPD molecular marker technology was invented and developed in 1990[13]. it is a molecular technique based on PCR that can perform polymorphism analysis on the genome of the entire unknown sequence. Using genomic DNA as a template, a single artificially synthesized random polymorphic nucleotide sequence is a primer, usually 10 base pairs, which is subjected to PCR amplification under the action of a thermostable DNA polymerase. The amplified product was separated by agarose or polyacrylamide electrophoresis and stained with ethidium bromide, and the polymorphism was detected on an ultraviolet spectrometer.

3.1 Principles and characteristics of RAPD molecular marker technology

3.1.1Principle of RAPD molecular marker technology. RAPD is also known as any primer PCR. Its application is based on the principle that for different template DNAs, amplification with the same primer may result in the same band spectrum or different band spectra. A band appearing only in a particular template can be used as a molecular marker for the template[14]. In theory, there are always differences in different genomic DNA. Under certain amplification conditions, the number of bands amplified depends on the complexity of the genome. For a particular primer, the higher the complexity of the genome, the greater the number of amplified bands produced.

3.1.2Characteristics of RAPD molecular marker technology. RAPD technology is not only simple and easy to implement, saves time and effort, and does not require special design of amplification reaction primers, and does not need to know the nucleotide sequence of the biological genome to be studied in advance, primers can be randomly synthesized and randomly selected[15]. In each RAPD reaction, amplification can be achieved by random pairing of primers and template DNA with only a single primer added, and amplification is not specific. The PCR amplification reaction has a relatively low annealing temperature, generally 36℃, this temperature ensures stable binding of the nucleotide primer to the template, while allowing proper mismatching to expand the randomness of the primer pairing in the genomic DNA, making the RAPD Have a higher detection rate. In addition, the amount of DNA sample required for RAPD analysis is very small, only 1/1 000-1/200 of RFLP, which is very advantageous for early identification of organisms or limited DNA.

3.2 Application of RAPD in genetic diversity of rapeSince its inception, RAPD technology has rapidly penetrated into all areas of genomic research, especially in rapeseed, with its simplicity, economy, speed, accuracy, sensitivity, and without prior knowledge of the molecular and molecular information of species. A huge breakthrough has been made in terms of sex. He Yutang and Tu Jinxing[16]used RAPD markers and UPGMA cluster analysis to analyze the genetic diversity of 172 cabbage-type rapeseed resources in 23 provinces and cities in China. The results showed that the genetic diversity of Chinese cabbage-type rapeseed was The geographical distribution is closely related, and the diversity level of Chinese cabbage spring rape is higher than that of winter rape. Xu Kunetal.[17]combined the agronomic traits with RAPD to study the genetic diversity and correlation of 89 Chinese cabbage varieties in Zhejiang Province and Hubei Province in the middle and lower reaches of the Yangtze River in China, and comprehensively analyzed the complexity of varieties. The genetic diversity and evolutionary process show that the local varieties in the lower reaches of the Yangtze River also show genetic relevance on the basis of maintaining certain geographical uniqueness. Wang Jianlin and Danba[18]explored the genetic differentiation relationship between Tibetan cabbage-type rapeseed cultivars by using RAPD analysis of 107 10bp random primers from 107 Chinese cabbage-type rapeseed germplasm resources. The results showed that: The genetic diversity of cabbage rapeseed in Tibet Plateau was 88.98%, which was slightly higher than that of Chinese cabbage rape (87.18%)[19]. At the same time, it is found that the varieties from the same region or climate similar areas often gather together, indicating that the similarity of Chinese cabbage varieties is closely related to the geographical and climatic background of their origin, which is basically consistent with Julie’s research results[20]. Wang Lulu[21]in order to understand the genetic diversity of local rapeseed germplasm resources in Guizhou, 136 Guizhou cabbage rape were selected and genetic diversity analysis was carried out by RAPD molecular markers. The results showed that Guizhou local germplasm resources were abundant. The RAPD genetic diversity has a polymorphic rate of 61.6%. Huang Xianqun and Ma Rongcai[22]used RAPD technology to study the genetic diversity of 65 Chinese cabbage varieties in Guizhou, and evaluated the genetic and specificity of resources at the molecular level. The protection and utilization of resources and screening were particularly good. It lays a good foundation for trait genes, improving the breeding level of rapeseed and speeding up the breeding process. Chen Biyun[23]used 20 random primers to analyze the genetic diversity of 55 Chinese cabbage-type rapeseed, and obtained 322 RAPD markers, of which 220 were polymorphic and the polymorphism rate was 68.3%. The results showed that the cultivars with close kinship were geographically close, which may be related to the gene exchange between cabbage varieties in a certain geographical range. This polymorphism is more abundant when amplified using a series of different random primers. The polymorphism of this amplified product reflects the genetic diversity of the material being tested. Because RAPD can reflect the DNA polymorphism of the whole genome, it has been widely used in the study of genetic diversity of many animals and plants. Since the day of its birth, RAPD technology has shown great potential and advantage in genetic diversity research.

4 AFLP molecular marker

This technique was discovered by the Dutch scientist Zabeau and developed by Vos (Vos, 1995; Zabeau and Vos, 1992). AFLP is a complex multiplex PCR system that produces a large number of polymorphic sites. AFLP markers combined with the high reproducibility of RFLP markers and RAPD markers do not require prior knowledge of genomic background, rich polymorphism,etc., overcoming the shortcomings of RFLP detection sites and RAPD instability[24].

4.1 Principles and characteristics of AFLP molecular marker technology

4.1.1Principle of AFLP molecular marker technology. Genomic DNA restriction fragments are amplified based on PCR technology. The genomic DNA is first cleaved with restriction endonucleases, and random restriction fragments of different sizes are formed, and the restriction fragments are ligated at both ends of the restriction fragments. The specific linker is paired with a site, and then specific primers complementary to the linker are used for specific PCR amplification, electrophoresis, and autoradiography to display DNA fingerprints[25]. In order to selectively amplify certain restriction fragments, the specific primers also add 1-10 random deoxynucleotides of different lengths at the 3’ end of the restriction enzyme sequence, only those fragments that are strictly paired with the 3’ end.

4.1.2Characteristics of AFLP molecular marker technology. Amplification can be obtained, thereby modulating the specificity and quantity of the AFLP product band. The restriction fragments are produced by cleavage with two enzymes, one is a rare cleavage enzyme and the other is a common cleavage enzyme. The advantage of AFLP technology is to avoid the complicated process of molecular hybridization, high sensitivity and polymorphism, rapidity, less DNA usage compared with RAPD, its reproducibility is greatly improved, and a large number DNA polymorphism fragment can be observed in one experiment. The shortcomings are long operation time and many steps, which require high precision of experimental skills and equipment.

4.2 Application of AFLP in genetic diversity of rapeAFLP technology can provide a large amount of information in a short period of time, so this technology has become an important tool in the study of genetic diversity and genetic relationship of Brassica crops. Liu Bingetal.[26]showed that Dire used AFLP technology to cluster 83 varieties of cabbage rapeseed and divided them into three types, namely spring rapeseed group, winter rapeseed group, and Chinese one rapeseed group, the classification results roughly match the known pedigree. Meng Jinlingetal.[27]also used this technique to study the genetic diversity of rapeseed, and similar results were obtained. Wang Jianlinetal.[28]used 24 pairs of AFLP random primer combinations to compare the genetic diversity of 14 Tibetan rapeseed and 29 wild rapeseed (divided into cabbage and mustard), and the results showed that: no matter cabbage-type rapeseed or mustard-type rapeseed, wild species have more genetic diversity than cultivars, and the polymorphism between wild species and cultivars reaches a very significant level. Danbaetal.[29]analyzed the genetic diversity of 43 wild-type canola germplasms from Tibet using 10 pairs of AFLP primers and 13 pairs of SSR primers, the results showed that the genetic diversity of wild type rapeseed germplasm resources in Tibet was very rich, this is consistent with the previous observations on the genetic diversity of cultivated rapeseed and the phenotypic traits such as He Yan[30]. Yao Yanmei[31]used AFLP technology to analyze the genetic diversity of 41 mustard-type rapeseed cultivars (lines). A total of 234 polymorphic bands were detected by 18 pairs of AFLP primers, of which polymorphic sites accounted for the total amplification position, among them, the polymorphic loci accounted for 52.8% of the total amplification sites. The results showed that the spring mustard-type rapeseed and the southern mustard-type canola in the south were clustered in two different groups, indicating that the spring mustard-type rapeseed and the southern winter mustard-type rapeseed had Larger genetic differences are consistent with the findings of An Xianhui[32], Liu Haihengetal.[33], Li Yugangetal.[34]and Xu Aizhenetal.[35]. In the opinion of Roman[36], AFLP markers detected genetic diversity among rapeseed cultivars, which were more abundant than RAPD and SSR markers.

5 Conclusions

AFLP and SSR markers are widely used in plant molecular biology research because they are suitable for genomic DNA detection of different tissues and sizes, and have high polymorphism, high reproducibility and large amount of information[37]. RAPD inherits the advantages of high efficiency, low sample size (only a small amount of DNA sample), high sensitivity, easy detection,etc., and because of its simple technology, low cost, and no need for DNA probes, no primers are designed. Sequence information needs to be known, and primers are short (typically 9-20 bp), which allows RAPD technology to perform DNA polymorphism analysis even without any molecular biology studies of the species[38]. However, the biggest limitation of RAPD is that the stability is poor and the repeatability is poor. Because RAPD is PCR-based, it is extremely sensitive to the reaction conditions, and slight changes in reaction conditions will cause some changes in the amplification products. As a widely used molecular marker technology, AFLP and SSR are not perfect. For example, the use of isotopes in AFLP technology poses a certain threat to the environment and the health of the tester, and the test cost is also high, the SSR markers are mostly located in the non-expression area of the genome, making the detected polymorphic markers are non-uniformly distributed, and the SSR molecular markers have the advantages of large amount of information, stable and reliable results, and good reproducibility. However, because of this method must know the sequence information at both ends of the repeat sequence, the primers are determined accordingly. sequence. The high cost of screening repeat sequences and primer design in the early stage limits the practical application of SSR technology. Once the specific primers are determined, the cost is greatly reduced and the operation is simple. The establishment and development of the RAPD marker technology system has made great progress in the study of species genetic diversity. In addition, the in-depth study of DNA fingerprinting, identification of seed purity, construction of molecular marker linkage maps, gene mapping and the selection of quantitative traits, population and germline genetic analysis, and the construction of genetic linkage maps provide a new approach. AFLP and SSR differ in principles and applications, the application in rapeseed is mainly focused on researching of genetic diversity, genetic map construction, cultivar identification, gene mapping and molecular marker-assisted breeding, but the extension and application of these technologies in the molecular field of species will certainly deepen people’s understanding of the genetic laws on animals and plants.

With the continuous improvement and development of the molecular level, some corresponding improvements can be made for the shortcomings of the three molecular marker technologies of SSR, AFLP and RAPD. For example, using non-isotope or silver staining techniques instead of isotopes, simplifying the procedure, or combining AFLP with SSR techniques and DNA sequence analysis to achieve better results and will be an important technology and methods for molecular biology researchers. For the shortcomings of RAPD, the most important thing is to improve the stability of the reaction, and it is recommended to make following improvements, such as implementing standard operation, increasing the resolution of amplified fragments or converting RAPD marker into SCAR marker, followed by routine PCR analysis, this will improve the stability and reliability of the reaction.