Lathyrus 2020 Paper

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Development of SSR markers and association studies of markers with

phenology and yield-related traits in grass pea (Lathyrus sativus).
Article in Crop and Pasture Science · July 2020
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Crop & Pasture Science, 2020, 71, 768–775
https://doi.org/10.1071/CP19557
Development of SSR markers and association studies of

markers with phenology and yield-related traits in grass pea
(Lathyrus sativus)
Khela Ram Soren A,D, Aravind Kumar Konda A, Priyanka Gangwar B, Vijay A. Tiwari C,
P. S. Shanmugavadivel A, Ashok Kumar Parihar A,D, Girish Prasad Dixit A,
and Narendra Pratap Singh A
A
ICAR-Indian Institute of Pulses Research, Kanpur, UP 208024, India.
B
Amity Institute of Biotechnology, Amity University, Gomti Nagar Extension, Lucknow, UP 226028, India.
C
Department of Biotechnology, Chatrapati Shahu Ji Maharajah University, Kanpur, UP 208024, India.
D
Corresponding authors. Email: sorenars@gmail.com; ashoka.parihar@gmail.com
Abstract. Grass pea (Lathyrus sativus L.) is an important food crop cultivated in dryland agricultural ecosystem. It is
an important source of dietary protein to millions of people living in low-income countries in South-east Asia and
Africa. The present study emphasises the development of genomic resources and their application in marker–trait
association for plant phenology and yield-related traits in lathyrus. In silico mining of nucleotide sequences identified
203 simple sequence repeat (SSR) motifs, of which trimer repeats (62%) were most abundant followed by tetramer
(19%), hexamer (10%), pentamer (6%) and dimer (3%) nucleotide repeats. Of 150 SSR markers screened, 60 markers
were amplified 75 alleles from 50 germplasm lines with 2–3 alleles per locus and the polymorphic information content
of 0.45 was observed. We report 6 significant marker–trait associations using the developed SSR markers for plant
phenology and yield-related traits following mixed linear model (Q+K) analysis. Gene ontology search of trait linked
markers revealed marker regions encoding genes related to homeobox-leucine zipper protein ATHB-6-like, rubredoxin
family protein, and cationic peroxidise. Understanding the association of novel alleles in trait expression will play a
significant role in future lathyrus crop improvement programmes.
Additional keywords: gene flow, genetic improvements, PIC, population structure, putative functions.
Received 31 December 2019, accepted 4 July 2020, published online 31 July 2020
Introduction (28–30%); this suggests promise as a good alternative protein

Grass pea (Lathyrus sativus L.) is a diploid species (2n = 14; source for malnourished populations. Furthermore, the crop is
genome size ~8.2 Gb) (Leitch et al. 2019) in the genus rich in sulfur-containing amino acids, w-3 unsaturated fats,
Lathyrus (Fabaceae), which includes >187 annual and dietary fibre, non-starch polysaccharides, and phytochemicals
perennial species. Grass pea is cultivated over marginal such as phytosterols, saponins, phytoestrogens, isoflavones
land in India, Bangladesh, Pakistan, Nepal and Ethiopia for and antioxidants (Narasinga Rao 2002). With these intrinsic
food and fodder purposes. This crop is highly resilient to valuable traits, the crop is ideal for mining of candidate gene(s)
climatic extremes and performs well under drought, heat, involved in overcoming various abiotic and biotic stresses
waterlogging, salinity, and poor soil conditions; it is (Lambein et al. 2019).
therefore suitable to fit into various cropping ecosystems Next-generation sequencing technology and bioinformatics
(Dixit et al. 2016). However, grass pea seeds contain tools have revolutionised genomic research. Now researchers
the neuro-degenerative compound ODAP (b-N-oxalyl- can rapidly access and generate data for whole-genome
a,b-diaminopropionic acid), and excessive consumption sequencing and re-sequencing of various plant, animal and
causes lathyrism, a neurodegenerative disorder in humans. microorganism species within short period and at affordable
This has resulted in bans being imposed on cultivation of cost. This technology has helped in developing large-scale
grass pea. The crop has therefore lagged behind cereals and enriched DNA libraries through simple sequence repeat (SSR)
other major pulses in term of the genetic and genomic resource mining from expressed sequence tags (ESTs), allele-specific
improvements essential for deploying advanced breeding PCR (KASP) assay, Illumina’s GoldenGate and Infinium
technologies. Nutritionally, grass pea is rich in protein and assays, and development of the Axiom SNP Array,
minerals and provides an excellent and cheap source of protein resulting in improved precision of breeding programs and
Journal compilation CSIRO 2020 www.publish.csiro.au/journals/cp

Association mapping in grass pea Crop & Pasture Science 769
accelerated genetic gain (Varshney et al. 2019). These whisker plot analysis, and to determine the strength of the
landmark achievements have been made in most of the relationship between variables via correlation analysis using
major pulse crops such as chickpea (Cicer arietinum L.) R software (The R Foundation, Vienna).
(Varshney et al.2013), pigeonpea (Cajanus cajan (L.)
Millsp.) (Varshney et al. 2012) and mungbean (Vigna Retrieval of transcriptome assembly and mining of markers
radiata (L.) R.Wilczek) (Kang et al. 2014). The retrieved transcriptome assembly of grass pea
Lathyrus represents a large gap in availability of genomic (PRJAN258356) from the National Center for Biotechnology
resources. Efforts have been made, especially in last 5 years, Information (NCBI) database was examined using CD-HIT
toward enhancing the genomic resource repository, and as a Software (Huang et al. 2010) for the purpose of eliminating
result, thousands of ESTs and pyrosequencing-based duplicate sequences, with a threshold parameter of 95%
microsatellite markers have been developed (Ponnaiah et al. identity for clustering. Resulting non-redundant EST
2011; Sun et al. 2012; Yang et al. 2014; Iquebal et al. 2017). sequences were searched for repeat motifs (>20 bp) for
However, the average polymorphism (14–26%) reported for the designing primer pairs with the web-based program
total markers available is very limited. BatchPrimer 3.0 (You et al. 2008), using defined
Basic studies of genetic diversity among 176 accessions of parameters: melting temperature, 55–578C; GC content,
L. sativus, using EST-SSRs, explained the existence of two 50–61%; secondary structure, 0.0; and PCR product range,
subpopulations with predicted horizontal gene flow among 100–300 bp (optimum 200 bp).
accessions (Soren et al. 2015). However, there is dearth of
genetic resources (mutant populations, bi-parental and multi- Functional annotation
parent populations) for trait discovery. In such situations, Functional annotation of the sequence possessing SSRs motifs
association mapping, which relies on the principle of linkage was performed with Blast2GO (Conesa et al. 2005). Mapping
disequilibrium, may help in the rapid identification of and InterPro functions of Blast2GO were used for extracting
quantitative trait loci (QTLs) from a set of germplasm lines or GO (gene ontology) terms associated with annotations
random populations (Gupta et al. 2014). Therefore, the present obtained from the BLAST result. The output result was
study aimed to increase the extent of genomic resources for categorised to molecular function, biological process and
Lathyrus spp. and establish marker–trait association (MTA) cellular components based on GO function.
studies for yield-related traits, which would accelerate the
Lathyrus improvement program worldwide. Genotyping the panel with SSRs
Amplification via PCR was performed in a G-40402
Material and methods thermocycler (G-Storm; Gene Technologies, Braintree, UK)
with the following conditions: initial denaturation step at
Plant materials and DNA extraction
948C for 3 min, followed by 35 cycles (948C for 1 min,
Fifty geographically diverse accessions of Lathyrus spp. from annealing at 50–608C for 30 s, elongation at 728C for 2 min
India (30), Syria (10), Ethiopia (5), France (2), Italy (1), and final extension of 10 min at 728C). Initially, 150 SSR
Bangladesh (1) and Korea (1) were used as the association markers were tested, and primer pairs producing clear,
mapping panel for establishing the MTA study. The unambiguous and reproducible amplification were resolved
association mapping panel consisted of 41 accessions of in 4% agarose gel for data scoring. Details of the 143 SSR
L. sativus, six accessions of L. cicera L. and three accessions markers are provided in Supplementary Materials table S1
of wild relatives of L. aphaca L. Basic information on (available at the journal’s website).
accessions is provided in Table 1. Each accession was
grown in an augmented design with spacing of 30 cm Diversity and population structure analysis
between rows and 10 cm between plants in rows. Farmyard Genetic diversity and population structure were assessed by
manure at 4–5 t ha–1 was applied during field preparation. using an allelic dataset developed from 60 polymorphic SSR
Genomic DNA was isolated from 20–25-day-old young leaf markers. The number of alleles (Na), gene diversity per locus,
tissues of plants, as per the manufacturer’s instructions observed heterozygosity (Ho), and polymorphic information
(DNASure 96 Plant Kit; Genetix Biotech, New Delhi: content (PIC) were analysed in the software PowerMarker
http://www.genetixbiotech.com/manuals/ version 3.25 (Liu and Muse 2005). DARwin version 6.0.13
DNA_sure_96_PLANT.pdf). software (Perrier and Jacquemoud-Collet 2006) was used for
generating genetic distance following a distance-based
Phenotyping and data analyses unweighted neighbour-joining tree. The Bayesian model-
Accessions were phenotyped under two environments at based program Structure 2.3.4 was used to find the number
Kanpur Nagar (268200 22.88400 N, 808170 34.58400 E) and of subgroups (Q matrix) in the given set of genotypes
Kanpur Dehat (268320 13.27200 N, 798570 50.716800 E) during (Pritchard et al. 2000). The software package was used with
November–April 2015-16 and 2016-17. Data were recorded the admixture model and the number of subpopulations (K) set
for days to flowering (DTF), days to maturity (DTM), plant as 1–10, with the length of the burn-in period set as 100 000
height, number of primary branches, pods per plant (PPP), and numbers of MCMC (Markov chain Monte Carlo) draws
number of seeds per plant, 100-seed weight, yield per plant after a burn-in period set as 200 000. The optimum number of
(YPP), biomass yield (BY) and harvest index (HI). These data subpopulations (K) was determined using Structure Harvester
were used to plot phenotypic distribution following box and v0.6.94 (Earl and vonHoldt 2012) based on the ad hoc statistic
770 Crop & Pasture Science K. R. Soren et al.
Table 1. Detailed information on place of origin, species and geographical locations of Lathyrus accessions used in marker–trait association studies
Serial no. Accession no. Source Latitude Longitude Species

0 00 0 00
1 EC 315525 Syria 34848 7.47 N 38859 48.533 E L. sativus
2 EC 315527 Syria 348480 7.4700 N 388590 48.53300 E L. sativus
3 EC 208952 Rome 418540 10.01800 N 128290 46.91800 E L. sativus
4 EC 200322 France 468130 39.49600 N 028120 49.49600 E L. sativus
5 EC 200324 France 468130 39.49600 N 028120 49.49600 E L. sativus
6 EC 203330 Bangladesh 238410 5.97800 N 908210 22.79100 E L. sativus
7 Sel. 527 Ethiopia 098080 4200 N 408290 22.82300 E L. sativus
12 DL 247 NBPGR, India 288420 14.61200 N 778060 8.96400 E L. sativus
23 Sel. 505 IARI, New Delhi 288420 14.61200 N 778060 8.96400 E L. sativus
24 VKG 28/94 Bihar, India 258050 45.86600 N 858180 47.228400 E L. sativus
25 VKG 28/104 Bihar, India 258050 45.86600 N 858180 47.228400 E L. sativus
26 IC 296745 NBPGR, India 288420 14.61200 N 778060 8.96400 E L. sativus
27 BioR 202 IARI, New Delhi 288380 14.53600 N 778090 46.36400 E L. sativus
30 LSD 3A IARI, New Delhi 288380 14.53600 N 778090 46.36400 E L. sativus
31 LSD 3C IARI, New Delhi 288380 14.53600 N 778090 46.36400 E L. sativus
32 PUSA 534 IARI, New Delhi 288380 14.53600 N 778090 46.36400 E L. sativus
33 RLK 854 Madhya Pradesh, India 228580 24.32300 N 7880390 24.81800 E L. sativus
36 AKP-AA/5/58 Jharkhand, India 238200 38.7600 N 858180 34.42300 E L. sativus
37 AKP-KR/5/29 Jharkhand, India 238200 38.7600 N 858180 34.42300 E L. sativus
38 PBJ/SSC-2/62 Gujarat, India 228150 31.14700 N 718110 32.57200 E L. sativus
39 PBJ/SSC-2/54 Bihar, India 258050 45.86600 N 858180 47.22800 E L. sativus
40 PBJ/SSC-2/66 Gujarat, India 228150 31.14700 N 718110 32.57200 E L. sativus
41 VKG-21/187 Bihar, India 228150 31.14700 N 718110 32.57200 E L. sativus
42 EC 540936 Syria 348480 07.4700 N 388590 48.5342 00 E L. aphaca
43 VKG 15/391 Korea 378390 50.39300 N 1278580 42.44800 E L. aphaca
44 EC 539013 Syria 348480 07.4700 N 388590 48.53400 E L. aphaca
45 EC 541913 Syria 348480 07.4700 N 388590 48.53400 E L. cicera
delta K (Evanno et al. 2005). MTA analysis using TASSEL software, were used for GLM and MLM analyses. The number
v2.0.1 (Bradbury et al. 2007) was performed to evaluate MTAs of permutation runs in GLM was set to 10 000 to obtain a
by following a general linear model (GLM with population marker significance value of P 0.001. MLM with the Q + K
structure (Q) matrix) and mixed linear model (MLM with Q + matrix was analysed following default run parameters,
K matrix). The Q matrix derived from the Structure program, i.e. convergence criterion of 1.0 10–4 and the maximum
and the relative kinship (K) matrix calculated by TASSEL number of iterations set to 200. Significant MTAs were
declared at a = 0.1 and P 0.001 with relative magnitude method with information on 60 SSR markers. The
represented by the R2 value as the proportion of variation maximum delta K was reached at K = 4, indicating the
explained by the marker. presence of four distinct subgroups (Fig. 3). Of 50
genotypes, 16 belonged to Subgroup 1 (red bars in Fig. 3),
Results nine to Subgroup 2 (green bars), 10 to Subgroup 3 (blue bars),
and the remaining 15 to Subgroup 4 (yellow bars). The
Mining of simple sequence repeats (SSRs)
population showed no clear discontinuities in overall
In total, 122 994 sequence tags (PRJNA258356) were retrieved structure, even in the neighbour-joining dendrogram
from the NCBI database and subsequently reduced to 71 602 (Fig. 4). A high proportion (23 of 50 genotypes) of
after elimination of duplicated sequences. Analysis of the non- admixtures (Q <0.8) in the population was observed.
redundant sequences using BatchPrimer 3 (https://wheat.pw.
usda.gov/demos/BatchPrimer3/http://batchprimer3.bioinformatics.
ucdavis.edu/cgi-bin/batchprimer3/batchprimer3.cgi) identified Di Tri Tetra Penta hexa
203 SSRs, wherein 152 sequences were observed with a
unique motif and 21 sequences had more than one SSR
motif (Table 2). Of the total sequence tags, six sequences 3%
(3%) were found to be dinucleotide repeat (AG/TC, CT/GA, 10%
TA/AT) motifs; 135 (62%) trinucleotide repeat (TTA/AAT) 6%
motifs; 42 (19%) tetranucleotide repeat (TGGT/ACCA)
motifs; 13 (6%) pentanucleotide repeat (ACACA/TGTGT)
motifs; and 22 (10%) hexanucleotide repeat (GCCATT/
CGGTAA) motifs (Fig. 1). In addition, 80% of the SSR 19%
motifs emerged to be trinucleotide and tetranucleotide
repeats positioned 150 bp toward the 50 -terminus (54.5%) 62%
and 120 bp toward the 30 -terminus of the transcribed
sequences (45.4%) (Fig. 2).
Population structure assessment

Genetic relationships among the 50 Lathyrus accessions were
Fig. 1. Percentage distribution of major classes of microsatellites
investigated by using a model-based Bayesian clustering
(dinucleotide to hexanucleotide) recorded in Lathyrus spp. transcriptome
sequences.
Table 2. Summary table on retrieved Lathyrus transcriptome
assembly information and related information generated on total
number of identified SSRs and novel primers developed
Hexa
Category
Penta
Total no. of sequences retrieved 122 994
Total no. of sequences examined 71 602 Tetra 5' UTR Region
Date of data submission to NCBI 27 October 2014
3' UTR Region
Total no. of identified SSRs 203 Tri
No. of SSR-containing sequences 152
No. of sequences containing more than one SSR 21 Di
Total no. of synthesised primers 150
No. of amplified primers 125 0 50 100 150
No. of monomorphic primers 65
No. of polymorphic primers 60 Fig. 2. Distribution of major classes of microsatellites repeats within
close proximity to the 50 and 30 ends of the transcript in Lathyrus spp.
1.00
0.80
0.60
0.40
0.20
0.00
40 15 30 16 11 13 38 41 23 24 24 21 18 33 34 35 8 7 44 1 3 5 6 4 47
29 39 31 27 12 10 48 28 20 42 17 26 36 32 37 46 9 22 49 14 2 19 43 45 50
Fig. 3. Estimated population structure of 50 Lathyrus accessions collected across the world (K = 4). Each individual is represented by a single vertical
column broken into K segments, with lengths proportional to each of the K inferred clusters. Numbers on x-axis correspond to serial numbers in Table 1.
r ia b
Se m
e
Ro
c ce
2448 an
Fr
34 17
ria
Se 32
23 43
29
ia
op
33
18 Eth
19 20
30 13
14
31
35
37
36
38
3 1
6
4
28 10
8 5
27 26 2
9 11 7
ia
S er
21
d Ko
re
a 39 25
h 22 16 12
es 15
d a
a
gla 40
di
n 44
In
Ba
42
4946 41
50 4547
0 0.2 L. aphaca L. cicera L. satiya
Fig. 4. Elucidation of the genetic relationship in Lathyrus accessions and their wild relatives.
Maximum admixtures were found in Subgroups 1 and 2. This produced 75 alleles (average 1.25 alleles per locus) with an
outcome closely concurred with an outcome from neighbour- average of 1.16 alleles per locus. The gene diversity and PIC
joining dendrogram investigation, whereby exotic genotypes values of markers were 0.35–0.66 and 0.29–0.59, respectively,
are mainly grouped in Clusters a, c and d, and most of with average values of 0.54 and 0.54. Number of observed
the Indian genotypes/varieties are grouped in Clusters b alleles (Na) extended from 1 to 2, observed heterozygosity
(Fig. 4). (Ho) ranged from 0 to 0.50, and Shannon’s information index
(I) ranged from 0.0 to 0.69 (table S2). Hexamer repeats motif
Phenotypic correlation study (GGTTTT)18 exhibited the highest polymorphism among all
analysed repeats, followed by trimer and pentamer repeat
Genotypes were screened for two consecutive years during
motifs. On the basis of allele-sorting results, L. sativus
November–April (2015–16 and 2016–17) for 10 yield-related
grouped in Clusters a and b independent of their
agronomic traits. Outliers were identified for five traits,
geographical origins (India, Ethiopia, France, Syria, Rome
i.e. number of primary branches, 100-seed weight, YPP, BY
and Bangladesh), whereas the wild relatives of L. cicera and
and HI. The analysis revealed positive correlation between PPP
L. aphaca were grouped in Clusters c and d.
and YPP (0.81), BY and YPP (0.76), BY and PPP (0.63), HI and
PPP (0.55), number of seeds per plant and PPP (0.46), and HI and
YPP (0.64). Negative correlation was observed between DTF Association analysis
and number of primary branches (–0.39), DTF and PPP (–0.40),
Association mapping considered 10 phenotypic traits following
and DTF and YPP (–0.33) (Fig. 5).
GLM (Q) and MLM (Q + K) models, which detected six MTAs
representing two QTLs having P 0.001 for DTF and DTM
Marker validation and genetic diversity assessment (Table 3). Two markers (IPL-102 and IPL-65) showed
Among 150 markers tested, 125 (86%) produced expected association with DTF, and four markers (IPL-102, IPL-65,
clear bands; however, 14 primers (9.33%) did not amplify, and IPL-66 and IPL-103) showed association with DTM in both
11 (7.33%) produced non-specific amplification. For the GLM (Q) and MLM (Q + K) models. Two marker positions
genotyping study, 60 SSR markers were used, which (IPL-102 and IPL-65) therefore showed association with both
DTF and DTM. Phenotypic variance explained by these MTAs process, and cellular components. BLASTX analysis of
varied from 4.55% to 32.66% in the entire population. unigenes containing EST-SSRs showed a match with at least
one important protein in the NCBI Protein database, namely
Putative candidate gene identification for important markers DNA-binding transcription factor, auxin response factor, metal-
BLASTX analysis of unigenes containing EST-SSRs in Lathyrus binding protein, transporter proteins, nitrate-assimilating
showed the best match with the following members of the protein, and proteins involved in abiotic tolerance mechanism
Fabaceae family: Medicago truncatula (74%), Trifolium (glutaredoxin (GRX) family, peroxidase 42, universal stress A,
subterraneum (24%), Pisum sativum (21%), and Cicer indole-3-acetic acid-induced ARG2, and Y2K4 dehydrin)
arietinum (21%). However, the lowest match (5%) was (table S3).
observed with Lathyrus sativus because of its poor genomic
repository in the NCBI database. Of the total BLAST sequences, Discussion
~90% were mapped with the reference sequences and showed
BLAST hit, InterPro and GO annotation results describing This study represents an effort to enrich genomic resources in
important ontology categories: molecular function, biological grass pea. The development of SSR markers is a valuable
contribution to the genomic repository for this crop and paves
(a) 120 135 6 10 16 2.5 3.5 5 15 20 50
the way for genetic diversity and MTA studies. The potential for
−0.25 −0.39 −0.40 −0.21 −0.33 −0.25 −0.24
90 marker-discrimination efficiency was tested by establishing
0.31 0.28 0.34 0.15 0.21 0.35 0.22 0.30
80 genetic relationships and transferability across different
120
0.42 0.13 0.34 0.044 0.49
species. Cross-species transferability among closely related
30
14
0.31 0.28 0.43
species reported to be more than across genus, tribe and
8
0.46 0.15 0.81 0.63 0.55
100 subfamily levels (Mathithumilan et al. 2013). The newly
0.37 0.43 0.42 0.28
20
developed markers showed a high level of polymorphism
2.5
0.32 0.29 0.21
9 (37.28%) and cross-transferability (100%) in Lathyrus spp.;
5
20
0.76 0.64 notably, L. sativus was grouped in mega Clusters a and b
5
80
independent of geographical origin of species (India, Ethiopia,
20
France, Syria, Rome and Bangladesh), whereas two wild
20
60 75 90 30 60 20 60 100 5 7 9 20 40 60
relatives, L. cicera and L. aphaca, were grouped in Clusters c
and d, respectively. The closer the ancestral relationship
HSW
PPP
SPP
YPP
DM
PH
DF
PB
BY
HI
(b) 1.0
among the cultivated accessions regardless of geographic
DF distance and a high average genetic similarity can be
0.8
DM attributed to 100% cross-transferability of markers. This
0.6 high cross-transferability enables a marker system for
PH
0.4 comparative genomics and genetic diversity studies of
PB underutilised pulse crops.
0.2
PPP On basis of Lathyrus transcriptome sequence analysis, we
0
SPP found that trimer (TTA/AAT) (62.0%) and tetramer (TGGT/
−0.2 ACCA) (19.0%) repeat motifs are the most abundant in the
HSW
−0.4 genome. Tri- and dinucleotide repeats have been reported to be
YPP the most abundant SSR motifs in legume species including
−0.6
BY adzuki bean (Vigna angularis (Willd.) Ohwi & Ohashi)
−0.8
HI
(Dikshit et al. 2016), cowpea (Vigna unguiculata (L.)
−1.0 Walp.) (Gupta and Gopalakrishna 2010; Chen et al. 2017),
Fig. 5. Phenotypic distribution of yield-related traits and their significant
and chickpea (Choudhary et al. 2009). The presence of
and non-significant correlation in Lathyrus spp. DF, Days to flowering; microsatellites intrinsic to the transcribed sequences is well
DM, days to maturity; PH, plant height; PB, primary branches; PPP, pods established, making them amenable to direct gene tagging for
per plant; SPP, seeds per plant; HSW, 100-seed weight; YPP, yield per QTL mapping of targeted traits. However, confinement of the
plant; BY, biomass yield; HI, harvest index. repeat motifs to the flanking or untranslated regions subjects
Table 3. Significant marker–trait association at a = 0.1 and P # 0.001 for days to flowering (DTF) and days to maturity (DTM) in Lathyrus spp.
GLM, General linear model; MLM, mixed linear model; PVE, phenotypic variation explained
Trait name Marker name F marker P marker PVE % (R2)

GLM MLM GLM MLM GLM MLM
DTF IPL-102 8.0292 8.0535 0.001 0.001 26.03 6.1845
IPL-65 8.0292 8.0535 0.001 0.001 26.03 6.1845
DTM IPL-102 – 8.3474 – 0.001 – 4.5832
IPL-65 – 8.3474 – 0.001 – 4.5832
IPL-66 10.2253 – 0.0009 – 32.66 –
IPL-103 10.2253 – 0.0009 – 32.66 –
them to lower selection pressure and allows the accumulation Conflicts of interest
of more mutations than their counterparts located internal to The authors declare no conflicts of interest.
the coding sequences (Subramanian and Kumar 2003).
Furthermore, such markers have been shown to possess a Acknowledgements
higher transferability rate across species than genomic
SSRs. Therefore, the markers reported in the analysis This work was supported by ICAR Indian Institute of Pulses Research,
Kanpur, India. The authors thank the Indian Council of Agricultural
possess a conserved genome sequence and provide a high
Research, Ministry of Agriculture and Farmer’s Welfare, Government of
propensity for developing functional markers and cross- India, for their consistent support.
transferability in Lathyrus spp.
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Development of SSR markers and association studies of markers with

Article in Crop and Pasture Science · July 2020

Aravind Kumar Priyanka Gangwar

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Development of SSR markers and association studies of

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Population structure assessment

Trait name Marker name F marker P marker PVE % (R2)

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