Identification Cold Tolerance of Pineapple Germplasms at Seedling Stage

Pineapple plant was always injured due to low temperature. Breeding varieties with high cold tolerance was the supreme way to resolve this problem and identifying cold tolerance of germplasms collected was necessary. In this study, SSR locus around homologous sequences of cold-tolerant genes was searched and screened using MD-2 (cold-tolerant variety) and Tainong 17 (cold-sensitive variety). Seventy three pairs of primers of which PCR results were different between these two varieties were gotten. PCRs were performed using these as primers and genomic DNA of germplasms collected as tem-plates. Six pairs of primers were found that their PCR results were in good consistent with cold tolerance. Using GLM (General linear model) association mapping analysis, two SSR markers linked to cold tolerance were found. Their efficiency was verified further using pineapple germplasms with high cold tolerance which had been validated in field by cold wave. They will be used in cold-tolerant breeding of pineapple in the future.


Introduction
Pineapple plants were sensitive to low temperature. Only when temperature was higher than 14˚C, pineapple plants begin to grow [1]. Most cultivars are injured during winter periods, especially in sub-tropical regions [2]. Almost all pineapple varieties are injured after exposure to 4˚C for 24 hours [2]. In 2021, the lowest temperature in Fujian province, China was only 2˚C. Many pineapple plants died because of low temperature. Although suspending sheets or plastic over pineapple plants can provide protection in a certain extent, they increase cost, re-duce profit and are not sufficiently effective [2]. Cultivating new varieties with high tolerance to cold was the supreme way for resolving this problem.
Collecting germ plasm resource and identifying their characteristics was the first step for pineapple breeding [3]. The direct way for identifying cold tolerance was planting the materials in field and observing their cold tolerance in winter. However, this method needs much labor and time. Furthermore, people cannot be sure whether cold wave passes experimental field or not at a certain stage. If molecular markers related with cold tolerance can be developed, cold tolerance of many germ plasm resources can be identified in short time.
Using two rice genes analog (RGA) primers, the genetic variability and relationships among 265 rice germplasm accessions with different resistance to rice Tungro virus were analyzed, and 48 polymorphic bands in the germplasm samples with an average of 28.6 bands per accessions were gotten [4]. Using similar method, 14 pairs of primers related with glucosinolates (GS) biosynthesis in Chinese cabbage were amplified in 75 accessions and produced 26 SSR alleles [5]. Using GLM (General linear model) association mapping analysis, four SSR markers linked with GS were gotten [5]. These demonstrated that molecular markers always located near to the genes with similar functions. Molecular markers can be found around these genes. Developing molecular markers related with certain genes was feasible.
There were many cold-tolerant genes in plant. In this study, homologous sequences of these cold tolerant genes were found. Simple sequence repeat (SSR) locus was screened in 5-kb upstream and downstream around these sequences. Certain cold-tolerant variety and cold-sensitive variety were used to screen these SSR primers. PCRs were performed using those primers of which the PCR results were different between cold-tolerant variety and cold-sensitive variety and genomic DNA of pineapple germplasms collected as template. Comparing with the cold tolerance of these pineapple germplasms, two SSR primers and two markers linked to cold tolerance were gotten. Their efficiency was verified further using pineapple germplasms with high cold tolerance which had been validated in field by cold wave. They will be used in cold-tolerant breeding of pineapple in the future.

Materials and Methods
Plant materials MD-2 was the most tolerant variety to cold [6]. Tainong 17 was the most sensitive variety to cold [6]. Pineapple varieties Tainong  Cold tolerance determination for pineapple varieties Plants growth were determined according the paper published [7]. Tissue-culture seedlings were grown in pot in which gardening soil was put. When the fifth leaf appeared, the seedlings were put in 7˚C chamber for 5 days. And then, the seedings were returned to 26˚C for 15 days. On the 16 th day, area of the second leaf of the plant was measured. After seedlings were cultured on MSO medium for two months, they were transferred into 4˚C chamber for 7 days. And then, they were returned to 26˚C for 15 days. On the 16 th day, scorched area of the fourth leaf versus the area of the whole leaf (SCFA) was measured.
Determination of the target genes and searching SSR locus According to the papers published, plant cold-resistant genes were determined (Table 1). Their homologous sequences were blasted using "Search annotation" and in pineapple genome (http://pineapple.angiosperms.org/pineapple/html/annotation.php). According to the sequence 5-kb-upstream, the gene itself and 5-kb-downstream, using the software FASTPCR 6.0, gene-specific SSR were found. The shortest length of the sequence searched was 12 bp. Single nucleotide and double nucleotides were repeated 6 times at least. Trinucleotides, tetrnucleotides, and pentnucleotides were repeated 5 times at least.
Designation of SSR primers According to the sequence of SSR locus, 500-bp-upstream sequence and 500-bp-downstream sequence were selected. Primers were designed using the software Primer 3.0 (http://Frodo.wi.mit.edu/cgibin/primer3/primer3-WWW.cgi). Two hundreds and seventy six pairs of primers were designed and synthesized by Sangon Biotech (Shanghai, China).
Analysis for marker polymorphism Electrophoresis data was counted using digital matrix. The result without band was recorded as 0. The result with band was recorded as 1. Bands amplified by each SSR primer were counted. Total bands corresponding to each pair of primers were counted. The average bands for each pair of primers were calculated. The varieties without band, with 1 band or more than 1 band were counted. C-repeat binding factor CBF PeCBF4a Populus euphratica [9] Basic helix-loop-helix bHLH DNA-binding superfamily protein gene bHLH
Association analysis General linear model (GLM) in software TASSEL3.0 was used for association analysis. Q data of each variety was used as covariant. Regression analysis was performed between SSR markers and data about cold tolerance identification. The equation of regression analysis was trait = population structure + marker effect + residual.

1) Cold tolerance of pineapple varieties
Tissue-culture seedlings of pineapple varieties were grown in soil. When the fifth leaf appeared, the seedlings were transferred into 4˚C chamber. After 7 days, the seedlings were returned to 26˚C for 15 days. Results showed that after pineapple seedlings were treated with low temperature, MD-2 grew the most fast among the varieties. Tainong17 and Peral grew the slowest. When scorched area of the fourth leaf from the apex (SCFA) was measured, it was found that SCFA value of MD-2 was the least, while that values of Tainong17 was the most (Figure 1 and Figure 2). These demonstrated that MD-2 was the most cold-tolerant variety among the materials. Tainong 17 was the most cold-sensitive variety.
2) Cold-tolerant genes in pineapple genome and SSR locus The homologous sequences of Bhlh, COR, SAMDC, SWEET, LEA, HSP, GPAT, FAD were found in pineapple genome. The homologous sequences of other cold-tolerant genes, such as SAD, CBF, CRT/DRE, AFP, A/NINV, SiCSD, HY5, SiDhn2 had not been found in pineapple genome. One hundred and sixty nine cold-tolerant genes were found in pineapple genome, including 4 FAD genes, 20 GPAT genes, 112 bHLH genes, 5 COR genes, 3 SAMDC genes, 1 SWEET gene, 23 LEA genes, 1 HSP gene (Figure 3). Three hundreds and two locus were found arouns these genes (Figure 4). No locus was found around 36 genes. One SSR locus has been found around 43 genes. Two SSR loci were found around 31 genes.
Three SSR loci were found around 22 genes. Four SSR loci were found around 10 genes. Five SSR loci were found around 5 genes. Six SSR loci were found around 7 genes. Seven SSR loci were found around 1 gene. Eight SSR loci were found around 1 gene. Aco020569.1, a basic helix-loop-helix bHLH DNA-binding superfamily protein had 8 SSR loci, which had the most.   chrosome 5,2,7,17,6,12,10,3,15,11,1,21,14,9,21,13,25,22,4,18 respectively. Chrosome 3 had four SSRs of which PCR results were different between MD-2 and Tainong17. Chrosome 2 had 10 SSRs of which PCR results were different, which was the most among the chrosomes. Chrosome 5 had 8 SSR loci of which PCR results were different. Chrosome 15 had 7 SSRs of which PCR results were different. These SSRs can be used for identifying MD-2 and Tainong 17. SSRs related with cold tolerance might among them. 4) SSRs markers identification using pineapple varieties collected The SSRs of which PCR results were different between MD-2 and Tainong 17 were used to identify the polymorphysium of the germplasms collected. Comparing with the cold tolerance identification of the seedlings, seven pairs of primers had good correlationship between PCR results and cold-tolerant identification ( Figure 6). They might be used as molecular markers for predicting  . SSR markers were identified using cold-tolerant germplasms. A showed the primer S15158-2. B showed the primer S15158-3. (a) S15158-3; (b) S5354-1.

5) SSRs markers identification using pineapple varieties with high cold tolerance
To identify the SSR markers related with cold tolerance, pineapple materials with high cold tolerance were used. Twenty two pineapple plantlines were collected in field when cold wave passed. When these materials were used for identifying SSR markers, results showed that S15158-3 and S5354-1 had good consistence with cold-tolerance characters (Figure 7). This demonstrated that these two markers can be used for identifying pineapple cold-tolerant character.

Discussion
Identifying the cold tolerance of gerplasms was the necessary step for breeding pineapple cold-tolerant varieties. Molecular marker was an important method for identifying characters of germplasm. Sanewski (2020) identified molecular markers associated with spiny-tip margin character using genome-wide association study (GWAS) and quantitative trait loci (QTL) analysis [26]. The gene responsible for pineapple spiny-tip margin was positioned at 14,355,639 and 14,341,745 bp respectively, on or very near, a zeaxanthin epoxidase (ZEP) gene [26]. Using DarTseq method, DArTseq molecular markers for resistance to Phytophthora cinnamomi in pineapple (Ananas comosus L.) were found in a single broad locus on chromosome five covering the positions 4.6 -5.5 Mb and two separate scaffolds [27]. Thirteen putative resistance/susceptibility proteins were identified in these positions and might be responsible for resistance to Phytophthora cinnamomi [27].
Simple Sequence Repeat (SSR) markers or microsatellites are a powerful technique for quantifying levels of inter-species genetic variation. According to the genomic sequence information of Arabidopsis thaliana and Chinese Cabbage, gene sequences related to glucosinolates (GS) biosynthesis were alignment and analysis, finding 102 candidate genes associated with GS content distributed in 10 chromosomes [5]. The homologous genes for GS biosynthensis were detected in Chinese cabbage. By analyzing sequences of candidate genes and their up/down franking 5 kb nucleotides with software FASTPCR 6.0, 237 SSRs were developed in 102 genes [5]. Seventy seven SSR specific primers linked to GS synthesis genes could be designed from 86 genes with SSR locus [5]. Eleven pairs of primers were amplified in all accessions and produced 26 SSR alleles [5]. Using similar method, we found 169 homologous sequences of cold-tolerant genes in pineapple genome in this study. Three hundreds and two loci were found around these genes. After all of the primers were screened with MD-2 and Tainong 17, PCR results of 73 pairs of primers were found different. These SSRs located in chrosome 5,2,7,17,6,12,10,3,15,11,1,21,14,9,21,13,25,22,4,18 respectively. These SSRs were screened among pineapple varieties. Results showed that seven pairs of primers had good consistence with cold-tolerant characters. When these SSRs markers were screened with cold-tolerance-identified plantlines, it was found that S15158-3 and S5354-1 had good consistence with cold-tolerant characters. This demonstrated that these two markers can be used for identifying pineapple cold-tolerant character and screening cold-tolerant germplasm.