Research Progress in Spinach (Spinacia oleracea L.) Germplasm Resources

Jiawen DONG Shuliang CHEN Chengling E Hongbo GUAN Xishan WANG Xiaohua SHI Jing XIAO

Abstract Spinach is one of the main vegetable species. The study of spinach germplasm resources is beneficial to discovering excellent genes and accelerating the breeding efficiency, thereby further promoting the development of the spinach industry. In this study, the origin, propagation, collection, preservation, genetic diversity and breeding status of spinach were reviewed, and the problems and prospects of spinach germplasm resources were put forward. This paper provides reference for the research and breeding of spinach germplasm resources.

Key words Spinach; Germplasm resources; Collection and preservation; Breeding; Genetic diversity

Spinach (Spinacia oleracea L.) is an annual or biennial herb in Spinacia (Chenopodiaceae), also known as Bosicao and  Chigencai. It is originated in Persia and has a history of more than 2 000 years. S. tetandra and S. turkestanica, two diploid species of spinach in India and northeastern Nepal, are the architype of spinach. Spinach was introduced to Spain in the 11th century, extensively planted in Germany in the 13th century, and later spread to European countries. Later, with the introduction of the early colonies into the United States, spinach began to be widely planted in the United States and counted in the US Seed Catalog in 1806. According to the literatures, spinach seeds were introduced to China from Nepal as a tribute as early as more than 1 300 years ago, that is, in the 2nd year of the Tang Dynastys Zhenguan period［1］. In its propagation process, two different types of spinach varieties were formed: the thorny variety S. oleracea var. spinasa Moench and the non-thorny variety S. oleracea var. inermis Peterm, of which the thorny variety is cultivated in China with a long history and is widely distributed. It is also known as "Chinese spinach". Its main features are thin and narrow smooth leaves, slender petiole, strong cold resistance, weak heat resistance, easy bolting under long daylight in spring, being suitable for autumn sowing, wintering cultivation and autumn cultivation. However, the non-thorny variety is mostly leafy, with many wrinkles and short petiole. Compared with the thorny variety, the non-thorny variety has strong heat resistance and high yield, but weaker  cold tolerance, and is mostly used for spring and autumn cultivation［2-3］.

Spinach mainly uses green leaves and tender stems as edible organs, and is an excellent source of vitamin B and vitamin C in human diet. Due to its adaptability and diverse cultivation methods, it can achieve annual supply. It is widely cultivated in various countries as a fresh-eating and processed variety, and thus has become one of the vegetables most widely distributed and most widely consumed by the masses in China, as well as one of the main vegetables earning foreign exchange through exports in  China［4］. According to statistics from the Food and Agriculture Organization of the United Nations (FAO) in 2008, Chinas annual spinach production is about 25 million t, accounting for 89.2% of the worlds total spinach production. China is the worlds largest spinach growing and consuming country. In China, about 6 800 t of conventional species and about 1 800 t of hybrids are used for spinach production. The hybrids currently used in the market are mainly from companies such as Seminis, Syngenta, Sakata and Takii, which occupy most of the seed market in China. The market is already foreign-owned. However, there are few Chinas self-cultivated spinach F1 hybrids, resulting in limited promotion［5］. This study introduced the research progress of spinach germplasm resources from the collection and evaluation of spinach germplasm resources, genetic diversity research and breeding, in order to provide reference for the research work of spinach germplasm  resources.

Collection and Evaluation of Spinach Germplasm Resources

Spinach has a long history of cultivation with extensive geographical distribution and abundant germplasm resources. Researchers at home and abroad have extensively collected and utilized spinach germplasm resources. According to the report of Dr. KIK from Centre for Genetic Resources, The Netherlands (CGN), the total collection of spinach germplasm resources is currently 1 938. Among them, 387 spinach germplasm resources (including wild species) were connected and preserved by the CGN; the National Crop Germplasm Bank of China collected 326 germplasms; The US Department of Agriculture (USDA) collected 301 germplasms; and researchers of other countries have also collected and preserved a large number of spinach germplasm resources［5］.

Since the 1950s, researchers of vegetables in China have started collecting and appraising spinach germplasm resources. At present, 333 spinach germplasm resources have been collected from all over the country and in other three countries［6］. In addition, spinach researchers in China have also carried out evaluation studies on the cold resistance, heat resistance, disease resistance and nutrition content of spinach germplasm resources.

Sun et al.［7］identified the cold resistance and disease resistance in different spinach germplasms, and found that the cold resistance and disease resistance of spinach germplasm resources are closely related to their origins. The cold resistance and disease resistance of spinach varieties originating in the northern regions are stronger than those of southern varieties. Zhang［8］determined a number of indexes of spinach varieties with different cold tolerance under low temperature stress treatment, developed a comprehensive evaluation system for cold tolerance of spinach varieties, and selected from 24 spinach germplasm materials, 2 extremely cold-tolerant varieties, 3 cold-tolerant varieties, 3 slightly cold-tolerant varieties, 15 susceptible varieties and 1 extremely cold-susceptible variety. Shen et al.［9-10］identified the heat tolerance of 14 spinach varieties from different regions common on the market at the germination and seedling stages, and used the comprehensive  evaluation method to screen out 5 varieties tolerant to heat at the germination stage and 3 varieties tolerant to heat at the seedling stage. The test results also showed that there was no significant correlation between the heat resistance at the seed germination stage and the heat resistance at the seedling stage. Li et al.［11］ evaluated the nutrition contents of 42 spinach germplasm resources in Shennongjia and the Three Gorges area, and selected 5 varieties with high vitamin content, 11 varieties with high crude protein content and 7 varieties with low oxalic acid content. He［12］selected low-nitrate large-leaf spinach varieties under low nitrogen level and low-nitrate round-leaf spinach variety GZ under high nitrogen level by pot culture and salicylic acid method from 40 spinach varieties. Qi et al.［13-14］measured the water, vitamin C, soluble sugar, crude fiber, oxalic acid, tannin and nitrate contents of 34 spinach germplasms, and comprehensively analyzed their nutritional quality. The results showed that the quality of different spinach varieties varied greatly. Among the 34 spinach materials, Shengxianfeng from the Europe had the best comprehensive nutritional quality, while among all the varieties, the highest vitamin C content was determined in domestic "Jingbo 1", which also has the lowest nitrate content.

Research on Genetic Diversity

Genetic diversity in a broad sense refers to the sum of the genetic information carried by all living things on the earth, while genetic diversity in the narrow sense refers to the genetic diversity within the species, that is, the sum of the inheritable variations of individuals within a species or within a population［15］. The study on genetic diversity of spinach can be applied to identify the abundance of genetic variations to facilitate the selection of distantly related materials for hybridization, increase the genetic variations of offsprings, and breed varieties with strong heterosis and comprehensive traits, to thereby improve the breeding efficiency［16］.

With the deepening of biological research and the advancement of science and technology, the detection methods of biodiversity have been continuously improved, from the initial morphological level to the current molecular level. These methods have also been applied in the study of spinach genetic diversity. However, each research method has its own advantages and limitations.

Detection of genetic diversity at morphological level

Morphological markers are studied from the phenotypic traits of the organism (plant height, plant width and leaf straightness in Fig. 1). Because morphological or phenotypic trait detection is simple, easy, and intuitive, it is the most direct and simple method for genetic diversity research［17］.

Wu et al.［18］analyzed 6 quantitative traits and 11 quality traits of 33 spinach germplasm resources from different regions, and divided the 33 spinach germplasms by cluster analysis into two groups, which were further divided into 6 subgroups, each of which had certain morphological characteristics. Moreover, the coefficients of variation of quantitative traits among the 33 spinach germplasms ranged from 29.07% to 78.97%. Among them, the coefficient of variation of leaf length was the smallest, while the coefficient of variation of single plant weight was the largest. Various traits exhibited different degrees of diversity among different germplasm materials. Ebadi-Segheloo et al.［19］, Yao et al.［20］also identified the genetic diversity of spinach germplasm resources  using morphological markers.

However, morphological identification can be influenced by environmental conditions, human factors, measurement tools and genetic dominance and recessiveness, and the genetic expression is unstable. Therefore, morphological identification has certain limitations in the study of plant genetic diversity［21］. At present, genetic diversity research is mostly carried out by combining morphological identification with molecular marker technology.

Genetic diversity detection at the DNA level

DNA markers are genetic diversity at the DNA level, referred to as molecular markers. Compared with the detection of genetic diversity at the morphological level, molecular markers have obvious advantages［9］. At present, the molecular marker methods used in the study of genetic diversity of spinach germplasm resources are RAPD, AFLP, SSR and SRAP.

RAPD (random amplified polymorphic DNA), i.e., random amplified polymorphic DNA marker, is a relatively simple DNA polymorphism detection technique invented by Williams［22］in 1990. Altemimi et al.［23］applied RAPD markers to evaluate the antibacterial activity of spinach leaf extract; and Wang et al.［24］extracted mitochondrial DNA from spinach leaves and performed RAPD analysis. However, RAPD technology is rarely applied in genetic diversity research. Zhang［8］applied RAPD markers to analyze 24 spinach germplasm resources. The results showed that the 24 germplasms were divided into 5 groups, of which the 5th group can be further divided into 4 subgroups. The results are basically consistent with the morphological markers.

AFLP (amplified fragment length polymorphism), a new method for detecting DNA polymorphism, was invented in 1993 by Zargar et al.［25］from the netherlands. Meng et al.［26］established the AFLP reaction system of spinach, which provided a new method for the genetic correlation analysis and further research in the field of genetic breeding of spinach. Jeon et al.［27］analyzed the genetic diversity of 55 spinach germplasm resources using AFLP markers. The results showed that the genetic difference between New Zealand spinach was 29%, and these germplasms were clustered into 7 genetic diversity groups. In addition, Wu［28］, Mei  et al.［29］and Meng［30］also applied AFLP markers to analyze the genetic diversity of spinach germplasm resources.

SSRs (simple sequence repeats), also known as microsatellite technology, are widely used in the study of genetic diversity of germplasm resources. Kuwahara et al.［31］applied SSR markers to analyze 250 spinach germplasm collected from geographically diverse regions (West Asia, East Asia, Japan, Europe, and the  United States) and found that these germplasms had high genetic variability. Among them, West Asia and Canada had the highest genetic diversity, followed by East Asian germplasms. These results further confirmed that spinach was originated in West Asia. In addition, the results also showed that there was low genetic differentiation between East Asian and Japanese germplasm resources, both of which showed high genetic differentiation with European germplasm resources. The differences between East Asian and European gene banks may be attributed to the initiator effect associated with crop propagation, as well as selection and genetic drift that occur during breeding. GL et al.［32］identified 48 spinach germplasms using SSR markers. GL et al.［32］identified 48 spinach germplasms using SSR markers. The results showed that the SSR markers were suitable for assessing the genetic diversity and population structures of spinach germplasms. The results also showed that the germplasms based on geographical origins were significantly separated from the Far East planting area and had the highest genetic diversity compared with the Persian, Turkish, European and American germplasms.

SRAP (sequence-related amplified polymorphism), proposed by Li et al.［33］from the Department of Vegetable Crops, University of California, in 2001, has great prospects in plant genetic map construction, genetic diversity, gene mapping, comparative genomics and heterosis prediction［34-38］. Avsar［39］used the SRAP marker system to analyze the genetic diversity of 95 Turkish spinach germplasms. For 19 SRAP markers, 123 bands were amplified, including 67 polymorphic ones. These polymorphic bands were used to construct a tree diagram of the spinach varieties and determine the genetic distances. Tree analysis was performed by the UPGMA method using the DICE matrix, and the genetic similarity ranged from 0.30 to 0.95. Fahim et al.［40］applied SRAP markers to study the genetic diversity of Iranian spinach germplasms. They found that the combinations of 8 SRAP primers produced 88 bands of 50-1 000 bp, of which 73 were polymorphic, and the average polymorphism information content was 0.35. The results of cluster analysis divided the spinach germplasms into two groups, indicating that the SRAP markers can be used to assess the genetic diversity among spinach germplasms. The different molecular markers for the study of spinach genetic diversity［41-43］are shown in Table 1.

Jiawen DONGG et al. Research Progress in Spinach (Spinacia oleracea L.) Germplasm Resources

Spinach Breeding

As one of the most important vegetable cultivars, the cultivation of new spinach varieties is particularly important. Chinas spinach breeders have carried out extensive and in-depth research based on actual production needs, and have achieved great results in the introduction of spinach, the purification and rejuvenation of local varieties, and crossbreeding.

Introduction

As a common method of spinach breeding, introduction has the advantages of simplicity and quick effect. In the 1980s, spinach cultivars in a large part of China were excellent varieties or slightly improved varieties introduced from abroad, such as "Japan Quanneng", "Chunxia Bocai", "Chunqiu Daye Bocai", "Chunqiu Bocai", "Siji Bocai"［44］. In addition, in 1995, China introduced a new spinach variety "Gaogan Bocai" from Ukraine, which has high yield, high quality, cold resistance, drought resistance and salt and alkali resistance［45］. In 1996, Anyang City, Henan Province introduced a new variety "Hebo" from the Scarpaster Horticulture Company of the Netherlands, which has wide adaptability, late bolting, long supply period and high yield［46］. China also introduced a new high-quality cold-resistant spinach "Mobao" from Japan［47］.

Variety rejuvenation

In the 1980s and 1990s, Chinese spinach breeders carried out genetic improvement and purification and rejuvenation work on excellent local conventional varieties. For example, the local varieties of the Inner Mongolia Autonomous Region, "Yuanye Bocai" "Jianye Bocai " and "Inner Mongolia Erhun Bocai", were approved by the Inner Mongolia Autonomous Region Crop Variety Examination and Approval Committee in 1989; Vegetable Research Institute, the Inner Mongolia Academy of Agricultural Sciences, selected from local fine varieties a new spinach variety "Neibo Yuanye  86-4" in 1991; Vegetable Research Institute, the Inner Mongolia Academy of Agricultural Sciences, selected a new variety "Neibo 1" in 1986, which was approved and named by the Inner Mongolia Autonomous Region Crop Variety Examination and Approval Committee in January 1994［44］; and Bayannaoer Academy of Agricultural & Animal Husbandry Sciences selected through the system breeding method, "Babo 1", which was approved by the Inner Mongolia Autonomous Region Crop Variety Examination and Approval Committee in 2011［48］.

Crossbreeding

In the 1980s, due to the urgent need for spinach production, the selection of spinach varieties became an important part of national projects and local research projects. Crossbreeding is one of the most important methods of spinach breeding. After years of efforts, Chinas spinach breeding has produced a series of excellent spinach varieties (Table 2), which have greatly promoted the development of Chinas spinach industry.

In addition, Sun et al.［49］also selected the spinach hybrids such as "Boza 9" and "Boza 15. In 1987, Lin et al.［44］bred "Lianhe 1" and "Lianhe 11". In 2004, Anyang Institute of Vegetable Science of Henan Province selected with the strong female lines directionally bred from the round-leaf spinach varieties in the Netherlands and excellent inbred lines selected from Anyang local round-leaf spinach varieties as the parents, the high-temperature high-quality fast-growing high-yield spinach hybrid "Anbo Daye"［61］.

Existing Problems and Prospect

The collection and preservation of spinach germplasm resources has been started very early in China. After the efforts of breeders, a large part of germplasm resources have been collected and preserved. Moreover, some researchers have identified and evaluated the cold resistance, heat tolerance, disease resistance, nutrition content and genetic diversity of some collected spinach germplasm resources. Some breeders have also studied the genetic diversity of some germplasm resources, and the basic research on spinach germplasm resources has achieved some results.

However, the lack of systematic collation and identification of the spinach germplasm resources collected, especially the biological confounding, makes the genetic traits of the excellent germplasms unstable and incapable of being effectively utilized in spinach breeding. However, the market demand for spinachs varietal characters continues to increase. The spinach germplasm materials that can be used in breeding research in China are extremely insufficient, which increases the difficulty of application of excellent resources in the breeding process. Furthermore, the emerging technological means are rarely applied in the research of spinach germplasm resources, which restricts the breeding process of excellent varieties.

Therefore, extensively collecting domestic and international fine spinach female materials and spinach germplasm resources with excellent commercial traits and disease resistance, systematically sorting and identifying the germplasm resources that have been collected and preserved and conducting comprehensive evaluation are the primary work of current research on spinach germplasm resources.

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