Application of Molecular Marker Technologies in Stress Resistance Breeding of Rapeseed

Suping GUO, Yuan YAN, Ba DAN

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

Abstract Rapeseed is the largest source of China’s edible vegetable oil and plays a pivotal role in China’s edible oil market. With global warming and frequent occurrence of various extreme climates, traditional hybrid breeding has been unable to meet the production requirements for rape yield and stress resistance. In recent years, the rapid development and maturity of molecular marker technologies have opened up new ways for stress resistance breeding in rape. This article mainly discusses several types of molecular marker technologies commonly used in rapeseed breeding, such as RFLP, SSR, and SNP, the advantages of molecular marker technologies over conventional breeding and other genetic marker technologies, as well as the application of molecular marker technologies in rapeseed disease resistance, drought resistance, cold resistance and lodging resistance breeding, the current problems and development prospects in breeding.

Key words Rapeseed, Breeding, Stress resistance, Molecular markers

1 Introduction

Rapeseed (Brassicaceae:Brassica) is one of the world’s major oil crops. It is also the best choice to guarantee and improve the self-sufficiency rate of oil crops in China[1], with good development prospects. In recent years, due to global warming and the frequent occurrence of various extreme weathers, traditional breeding, which mainly depends on the selection of plant phenotypes, has been unable to meet the requirements for rapeseed yield and stress resistance in production. With the rapid development of molecular biology and genomics, the application of molecular marker technology was born, and it is becoming more and more mature, creating a new way for rapeseed resistance breeding[2].

2 Overview of molecular marker technologies

Molecular markers are a class of genetic markers based on the polymorphism of biological macromolecules, especially nucleic acids. With the development of genetics, molecular markers have become the most reliable labeling technology after morphological, cytological, and biochemical markers. There are broad and narrow senses. Broad molecular markers refer to DNA sequences or proteins that can be inherited and detected. Narrow molecular markers refer to specific DNA fragments that can reflect certain differences in the genomes of individual organisms or populations. In practice, molecular markers are generally referred to as this meaning[3].

2.1 Commonly used molecular marker technologiesMolecular marker technologies can be divided into two categories based on technical characteristics. The first category is based on Southern hybridization technology, which mainly includes restriction fragment length polymorphism (RFLP) and DNA fingerprinting techniques. The second category is based on PCR technology, which mainly includes amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR), and randomly amplified polymorphic DNA (RAPD) and microsatellites (SSR),etc.[4]. At present, RFLP, SSR, SNP, and SCoT are the most commonly used marker technologies in rapeseed resistance breeding.

2.1.1RFLP. RFLP (restriction fragment length poly-morphism) is the earliest molecular tagging technology[5]. RFLP refers to the difference in the length of restriction fragments between genotypes, due to differences in restriction sites in different species, or changes in nucleotides at restriction sites due to recombination, mutation,etc., and differences in the length of DNA fragments of different sizes and digested fragments that are homologous to the probe sequence[6]. RFLP tags have the following two advantages. The first is that the RFLP marker is a codominant marker, which can well distinguish and identify heterozygotes and homozygotes. The second is that it has high reproducibility, no epistatic effect, and is not affected by the stage of plant development and external natural conditions. However, the marker has high requirements for DNA, and complicated operation, and is too dependent on the use of endonucleases, which is time-consuming and expensive, and is not suitable for large-scale molecular breeding. These have affected the development of the technology in the breeding field[7-9].

2.1.2SSR. SSR (simple sequence repeat) is also called microsatellite labeling. The key to its labeling is to design specific primers based on specific short sequences at both ends of the microsatellite repeat sequence, and then amplify the microsatellite fragments by PCR. Detection of polymorphisms[10]. SSR is a co-dominant marker with good data reproducibility, rich polymorphisms, and mature technical means. It has been widely used in crop breeding[11].

2.1.3SNP. SNP (single nucleotide polymorphisms) is a third-generation molecular marker that appears after RFLP and SSR. SNP is the highest known polymorphism marker technology[12], and compared to other marker technologies, it has significant advantages. In addition, SNP can exist inside the locus, and the markers produced by other labeling methods are at most closely linked to the locus, which is suitable for large-scale gene screening and has promising applications. In addition, there are molecular marker technologies such as RAP, TRAP and ScoT. SRAP and TRAP marker technologies have the advantages of simplicity, efficiency, high co-dominance, repeatability, easy sequencing, and low cost[13], but they are far less widely used than SSR marker technologies. SCoT is the target initiation codon polymorphism marker. It is a newly developed molecular marker of the target gene[14],with simple operation steps, high sensitivity, good stability, high band polymorphism, and low cost[15-16]. Compared with SSR molecular markers, SCoT molecular markers are more widely used in species diversity analysis, germplasm identification and genetic mapping.

2.2 Advantages of molecular marker technologiesConventional breeding requires a lot of manpower and material resources, and it is susceptible to the external environment. The breeding years are long, and the breeding efficiency is low[17]. Molecular marker technologies are used to identify the differences in genes or genotypes of individual organisms with high accuracy and fewer linkage burdens. They can obtain desired individuals[18], and can be identified at any growth stage of the crop, which is not affected by the external environment and effectively shortens the breeding time to improve breeding efficiency[19]. Compared with other genetic markers, molecular marker technologies are simple and easy to operate; most markers are codominant and can be used to identify homozygous and heterozygous genotypes[20]; genomic variation is extremely rich and there are many markers, with high resolution; marker analysis can be performed at different stages of crop growth and development, and there are no problems such as expression or not, not limited by the natural environment, and the detection procedures for all crops are basically the same and easy to grasp[21].

3 Application of molecular markers in resistance breeding of rapeseed

3.1 Disease resistanceSclerotiniasclerotiorumis caused by the fungusSclerotiniasclerotiorum(Lib.) De Bary. It is a broad-spectrum disease that seriously damages the yield and quality of rapeseed[22]. It can cause huge yield and economic losses. In China, the annual rapeseed output reduction caused by sclerotinia can reach 10% to 70%[23-24].There are currently only a few partially resistant varieties, and there are no highly resistant or fully resistant varieties[24]. Although there are chemical agents on the market that can be used to prevent sclerotinia, chemical agents have certain limitations and can cause environmental pollution and higher costs. Therefore, breeding new rapeseed varieties with high resistance toS.sclerotiorumis an efficient, environmentally friendly and low-cost best method. Zhou[25]used two materials, H100 (resistant) and D126 (sensitive), which have obvious differences in resistance toS.sclerotiorum, to construct a DH population, and to detect resistance toS.sclerotiorumin rapeseed stalks at the plant stage. QTLs related to sclerotinia resistance can explain phenotypic variation from 8.00% to 13.8%. Ma[26]used SSR and SRAP marker technologies to map the antibacterial nuclear disease QTL ofBrassicanapusand detected 3 QTLs, which could explain the phenotypic variation of 10.51%,14.52%, and 21.80%. A total of two resistant QTLs were detected using the adult toothpick stalk puncture inoculation method, which could explain phenotypic variation of 16.79% and 14.58%, respectively. Liu[27]used rapeseed 085 (resistant) and Xiangyou 13 to construct F2 generation as identification materials. Through RAPD marker mapping, three RAPD markers related to resistance to sclerotinia disease were detected, which could explain 15.3%, 13.8% and 29.2% phenotypic variation. Wuetal.[28]detected three QTLs related toS.sclerotiorumby using isolated leaves at the seedling stage and 10 QTLs related to S. sclerosis by using stems from the adult stage. Two main QTLs were detected in all environments, explaining 29.01% to 32.61% and 8.54% to 15.86% of the phenotypic variation. Zhangetal.[29]used SCoT, SSR and SRAP molecular markers to study the genetic diversity of 43 parental materials of rapeseed breeding with antibacterial nucleosis. ZHANGetal.[30]used SSR and SPAR markers to analyze the genetic diversity of 43 materials with high resistance to sclerotinia disease screened by the Agricultural Science Institute of Hanzhong, Shaanxi Province.

Club root is a soil-borne disease caused byPlasmodiophorabrassicaeWor.[31]. It mainly harms the roots of rapeseed, and rapeseed will be harmed during the whole growth period, and it is easy to cause rot with other saprophytic fungus with a stench[32]. Zhanetal.[33]used the disease-resistant turnip ECD04 (Brassicarapa, AA, 2n=20) as the donor, combined with backcrossing and molecular marker-assisted selection, the position of ECD04-resistant rhizobium physiological race 4 Point Pb Ba8.1 was transferred to the genetic background ofB.napus(AACC, 2n=38), a conventional rape variety. By testing the obtained new disease-resistant strains in multiple field natural nurseries, the ZHE-226 new disease-resistant strains were bred. Peng[34]used molecular marker A08-300 closely linked to PbBa8.1, a physiological race site of resistance 4, to conduct molecular detection of 30 rape varieties. The results showed that the disease index of 30 varieties was 47 or above, all of which belonged to susceptible or highly susceptible varieties. In addition, the molecular test results showed that none of the 30 varieties contained molecular markers of Rhizobium resistance physiological race 4.

Black shank and downy mildew are also important diseases affecting rape growth. Strussetal.[35]found 4 RFLP markers and 3 AFLP markers, and found STS markers located between RFLP and AFLP, and obtained the EST for defense response to black shank disease. Plieskeetal.[36]mapped the gene for resistance to black shank toB.napusLG6 through RFLP markers, and pointed out that resistance to black shank can be obtained through interspecific hybridization. Wangetal.[37]obtained the F2 segregated population by crossing rapeseed with downy mildew resistant and susceptible parent rape and F1 self-bred, and using BSA and SLAF(specific length amplified fragment sequencing) technology to obtain two rapeseed frosts with cabbage mildew resistance tightly linked SNPs.

3.2 Drought resistanceAs drought problem restricts the development of the world’s agriculture, its impact on the reduction of food production is far greater than the sum of other adverse effects[38-39]. In recent years, due to global warming, the problem of drought has become increasingly serious. Not only does rape planting area decrease and yield decrease, but it also severely affects rape quality[40-41], so it is particularly important to cultivate new resistant rape varieties. Li[42]used rapeseed rape DH population as material, and detected 50 QTLs related to drought tolerance in rapeseed, including 31 under drought stress and 28 under normal irrigation, which were common under the two conditions. Nine were detected, and 19 comprehensive drought tolerance indicators, the drought tolerance coefficient, were detected. Wangetal.[43]used the recombinant inbred line F2∶4 family as the material to perform QTL analysis on 5 drought-resistance-related traits and drought-resistance coefficients. A total of 8 QTLs were detected, and a single locus could explain the phenotype of 6.6%-12.4% variation. Huangetal.[44-45]used the drought-resistant line QY8-1 and the drought-sensitive variety Huyou 16 to construct a F2∶3 population containing 183 families as research materials. The drought stress treatment was carried out from the bud to the tillering stage, and normal irrigation was used as a control. A1661.9 cM genetic map was constructed using 335 SRAP markers. A total of 28 QTLs were detected, which could explain phenotypic variation from 1.1% to 36.6%. In addition, rape-related drought resistance genes were screened. Using RNA-Seq sequencing technology, a total of 144 131 SNP markers and 100 534 gene expression markers were developed in related populations.

3.3 Cold resistanceFrost damage to plants caused by low temperatures has always been one of the important reasons affecting crop yield and quality worldwide[46]. The optimum temperature for the growth and development of rapeseed is 0-23 ℃. If it is lower than 4 ℃, cold damage will occur and the growth will stop. In recent years, extreme temperatures have frequently appeared in various regions, causing rape to be severely stressed by low temperatures. In January 2008, the low temperature of snow and ice caused a severe impact on winter rapeseed production in China. According to the survey, the affected area accounted for about 77.8% of the China’s winter rapeseed area. In order to reduce the frost damage of rapeseed, agronomic measures such as heat preservation and temperature increase, removal of frozen stems and leaves, topdressing of potassium fertilizer, and clearing and drainage of ditch are generally used in production, but they will consume a lot of manpower and material resources. Therefore, the most effective way to solve the frost damage is to breed new drought-resistant varieties[47]. Zhu[48]used the SSR marker method to select 6 pairs of primers with polymorphic bands from 29 pairs of primers, and selected 4 pairs of primers to construct fingerprints of 8 cabbage-type rape varieties (lines). Zhang[49]used the Chinese cabbage winter rape variety Longyou 6 with strong cold resistance and Tianyou 8 with weak cold resistance to conduct F1 and F2 generations. Using SSR molecular marker primers to analyze 508 F2 generation plants,7 molecular markers closely linked to cold resistance Rf of winter rapeseed were obtained. Zhang[50]used strong cold-tolerant winter rape variety (Longyou 6) and weak cold-tolerant winter rape variety (Tianyou 4) as parents to construct an F2 population, using 504 SSR primers and 144 pairs. The parents were selected by AFLP primers. There were 47 pairs of SSR primers and 97 pairs of AFLP primers, all of which showed differences between the parents. The polymorphism rates were 9% and 67.4%, respectively. These polymorphic primers were used to amplify the F2 population. The results showed that 42 pairs of SSR primers and 94 pairs of AFLP primers could display polymorphisms in the F2 population, amplifying 48 and 162 sites, respectively. Zhao[51]used the F2 population and F2∶3 family formed by the strong cold-resistantB.napusGZ Hui and weak cold-resistant 10B, and GZ Hui×10B as research materials, and carried out QTLs for controlling the frost damage of the progeny population through SSR markers the study. The results showed that a total of 20 QTLs related to cold resistance inB.napuswere obtained, and QTL hotspots were detected in A7, A8 and A10 chromosomes. Wuetal.[52]used the F2 population constructed by Chinese cabbage winter rape varieties Longyou 7 and Longyou 9 with different cold resistance as materials to map QTL physiological indexes related to cold resistance analysis. Twenty-four QTL loci with 6 traits of CAT activity, POD activity, SOD activity, MDA content, soluble protein content, and free proline content were mapped on 10 chromosomes ofB.napus, which could explain 11.178 3% to 81.175 3% phenotypic variation.

3.4 Lodging resistanceIn recent years, China’s rapeseed production model has gradually changed from an artificial planting model to a mechanized production model, with the aim of improving rapeseed production efficiency. However, in the production practice, it is difficult to achieve mechanized harvest of the fallen rape. Lodging not only significantly reduces rape production, but also greatly affects rape quality, resulting in a decline in harvest index and increased production costs. Therefore, improving the lodging resistance of rapeseed is also an important goal of rapeseed breeding[53-54]. Gu[55]constructed a total of 189 F2 strains fromB.napusZheping 1×04Pb11(I) and Ning 1243×04Pb11(II) as materials, and obtained a linkage genetic map containing 24 SSR markers and 137 SRAP markers. Furthermore, this map was used to locate QTLs controlling lodging resistance inB.napus, and three QTLs related to lodging resistance indexes were obtained, which could explain the phenotypic variation of 42.38%, 7.88%, and 13.52%, respectively. Lietal.[56]used a combination of the anti-lodging line Zheping 1 and the easy-lodging line Gao Jie 1 to recombine inbred lines (RIL) families. After two years of two-point tests, F2∶6 and F2∶7 QTL analysis of stress resistance traits was performed, and QTL analysis of corneal stratum traits of F2∶6 families in this population was performed using two points of data for one year. The results showed that a total of 11 pedigree anti-stress QTLs were detected in 4 experiments, which could explain the phenotypic variation of 4.41%-52.64%. Zhang[57]performed lodging phenotypic identification on multiple separated generation populations of two combinations (Hua Shuang 5×A7005, Zhong Shuang 9×A6104), and performed QTL mapping analysis on one of them. A linkage group consisting of 20 chromosomes was constructed using 448 pairs of SSR markers, with a total map length of 3 497.58 cM and an average marker spacing of 12.27cM. QTL mapping of lodging-related traits was found. A total of 3 QTLs with high reliability were found. On chromosomes 4, 13, and 16 of the linkage map, 11.9%,12.1%,and 19.8% of the phenotypic variations can be explained.

4 Prospects

As a class of emerging and effective breeding methods, molecular marker technologies have been widely used in the field of crop genetics and breeding, and play an important role in creating new genetic variations, controlling genetic expression of traits, especially genetic expression of quantitative traits[58]. However, the current molecular marker technologies have only reached the more basic stages of marker identification, localization, and mapping. It is proficient in the selection and application of several simple genes, and there are certain limitations in molecular markers for complex polygene-controlled traits, such as quality traits and yield traits, which need further research and development. It is believed that with the rapid development of molecular biology and genetics, molecular marker technologies will further advance the process of crop breeding and make greater contributions to agricultural production.